- Professor, Cellular and Molecular Medicine
- Co-Program Leader, Cancer Biology Research Program
- Professor, BIO5 Institute
- Chair, Cancer Biology - GIDP
- Member of the Graduate Faculty
- Ph.D. Biochemistry
- Harvard Medical School, Boston, Massachusetts, United States
- How Signaling Pathways Regulate c-fos Transcription
- M.A. Microbiology
- Colorado State University, Fort Collins, Colorado, United States
- Characterization of Temperature Sensitive Mutants of Japanese Encephalitis Virus
- B.A. Microbiology
- Southern Illinois University, Carbondale, Illinois, United States
- University of Arizona, Tucson, Arizona (2016 - Ongoing)
- Van Andel Research Institute (2000 - 2016)
- Harvard Medical School, Boston, Massachusetts (1995 - 2000)
- Emily Frederick DiMaggio Endowed Lecture
- Dana Farber Cancer Institute, Fall 2018
- University of Arizona Cancer Center Hero Award
- UACC, Winter 2017
- SWIU/SBUR Excellence in Research Award
- Society for Basic Urologic Research, Fall 2017 (Award Nominee)
- Outstanding Graduate Advisor Award
- Michigan State University, Spring 2010
- American Cancer Society Research Scholar Award
- American Cancer Society, Fall 2005
No activities entered.
ResearchCBIO 900 (Spring 2023)
Research ConferenceCBIO 695A (Spring 2023)
CBIO GIDP Seminar SeriesCBIO 596H (Fall 2022)
Cancer BiologyCBIO 552 (Fall 2022)
Directed RsrchMCB 492 (Fall 2022)
DissertationCBIO 920 (Fall 2022)
ResearchCBIO 900 (Fall 2022)
Research ConferenceCBIO 695A (Fall 2022)
ThesisCMM 910 (Fall 2022)
Cancer Bio Clin ExprnceCBIO 561 (Spring 2022)
DissertationCBIO 920 (Spring 2022)
Research ConferenceCBIO 695A (Spring 2022)
CBIO GIDP Seminar SeriesCBIO 596H (Fall 2021)
Cancer BiologyCBIO 552 (Fall 2021)
Directed ResearchMCB 792 (Fall 2021)
DissertationCBIO 920 (Fall 2021)
Research ConferenceCBIO 695A (Fall 2021)
Adv Topics in Cancer BiologyCBIO 553 (Spring 2021)
DissertationCBIO 920 (Spring 2021)
Research ConferenceCBIO 695A (Spring 2021)
CBIO GIDP Seminar SeriesCBIO 596H (Fall 2020)
Cancer BiologyCBIO 552 (Fall 2020)
DissertationCBIO 920 (Fall 2020)
ResearchCBIO 900 (Fall 2020)
Research ConferenceCBIO 695A (Fall 2020)
ResearchCBIO 900 (Spring 2020)
Research ConferenceCBIO 695A (Spring 2020)
Cancer BiologyCBIO 552 (Fall 2019)
ResearchCBIO 900 (Fall 2019)
Research ConferenceCBIO 695A (Fall 2019)
ThesisCMM 910 (Fall 2019)
Adv Topics in Cancer BiologyCBIO 553 (Spring 2019)
ResearchCBIO 900 (Spring 2019)
ResearchCMM 900 (Spring 2019)
Research ConferenceCBIO 695A (Spring 2019)
ThesisCMM 910 (Spring 2019)
Cancer BiologyCBIO 552 (Fall 2018)
ResearchCBIO 900 (Fall 2018)
Research ConferenceCBIO 695A (Fall 2018)
ThesisCMM 910 (Summer I 2018)
Adv Topics in Cancer BiologyCBIO 553 (Spring 2018)
Introduction to ResearchMCB 795A (Spring 2018)
ThesisCMM 910 (Spring 2018)
Introduction to ResearchMCB 795A (Fall 2017)
ThesisCMM 910 (Fall 2017)
Adv Topics in Cancer BiologyCBIO 553 (Spring 2017)
Introduction to ResearchMCB 795A (Fall 2016)
- Miranti, C., Bergsma, A., & van Spriel, A. (2014). Tetraspanins as master organizers of the plasma membrane. In Cell Membrane Nanodomains: from Biochemistry to Nanoscopy(pp p59-86). Boca Raton: CRC Press.More infoMiranti CK, Bergsma A, and van Spriel AB. 2014. Chapter 4: Tetraspanins as master organizers of the plasma membrane. In Cell Membrane Nanodomains: from Biochemistry to Nanoscopy, Alessandra Cambi and Diane Lidke, eds. Boca Raton:CRC Press, p59-86. ISBN 978-1-4822-0989-1
- Miranti, C., & Nollet, E. A. (2013). Integrin and adhesion regulation of autophagy and mitophagy. In Autophagy – A Double-Edged Sword – Cell Survival or Death?(pp p465-85). New York: InTech.More infoNollet EA and Miranti CK. 2013. Chapter 21: Integrin and adhesion regulation of autophagy and mitophagy. In Autophagy – A Double-Edged Sword – Cell Survival or Death?, Yannick Bailly, ed. New York:InTech. p465-85. ISBN 978-953-51-1062-0.
- Casillas, A. L., Chauhan, S. S., Toth, R. K., Sainz, A. G., Clements, A. N., Jensen, C. C., Langlais, P. R., Miranti, C. K., Cress, A. E., & Warfel, N. A. (2021). Direct phosphorylation and stabilization of HIF-1α by PIM1 kinase drives angiogenesis in solid tumors. Oncogene, 40(32), 5142-5152.More infoAngiogenesis is essential for the sustained growth of solid tumors. Hypoxia-inducible factor 1 (HIF-1) is a master regulator of angiogenesis and constitutive activation of HIF-1 is frequently observed in human cancers. Therefore, understanding the mechanisms governing the activation of HIF-1 is critical for successful therapeutic targeting of tumor angiogenesis. Herein, we establish a new regulatory mechanism responsible for the constitutive activation of HIF-1α in cancer, irrespective of oxygen tension. PIM1 kinase directly phosphorylates HIF-1α at threonine 455, a previously uncharacterized site within its oxygen-dependent degradation domain. This phosphorylation event disrupts the ability of prolyl hydroxylases to bind and hydroxylate HIF-1α, interrupting its canonical degradation pathway and promoting constitutive transcription of HIF-1 target genes. Moreover, phosphorylation of the analogous site in HIF-2α (S435) stabilizes the protein through the same mechanism, indicating post-translational modification within the oxygen-dependent degradation domain as a mechanism of regulating the HIF-α subunits. In vitro and in vivo models demonstrate that expression of PIM1 is sufficient to stabilize HIF-1α and HIF-2α in normoxia and stimulate angiogenesis in a HIF-1-dependent manner. CRISPR mutants of HIF-1α (Thr455D) promoted increased tumor growth, proliferation, and angiogenesis. Moreover, HIF-1α-T455D xenograft tumors were refractory to the anti-angiogenic and cytotoxic effects of PIM inhibitors. These data identify a new signaling axis responsible for hypoxia-independent activation of HIF-1 and expand our understanding of the tumorigenic role of PIM1 in solid tumors.
- Staquicini, D. I., Barbu, E. M., Zemans, R. L., Dray, B. K., Staquicini, F. I., Dogra, P., Cardó-Vila, M., Miranti, C. K., Baze, W. B., Villa, L. L., Kalil, J., Sharma, G., Prossnitz, E. R., Wang, Z., Cristini, V., Sidman, R. L., Berman, A. R., Panettieri, R. A., Tuder, R. M., , Pasqualini, R., et al. (2021). Targeted Phage Display-based Pulmonary Vaccination in Mice and Non-human Primates. Med (New York, N.Y.), 2(3), 321-342.More infoThe extensive alveolar capillary network of the lungs is an attractive route for administration of several agents. One key functional attribute is the rapid onset of systemic action due to the absence of first-pass metabolism.
- Watson, M. J., Berger, P. L., Banerjee, K., Frank, S. B., Tang, L., Ganguly, S. S., Hostetter, G., Winn, M., & Miranti, C. K. (2021). Aberrant CREB1 activation in prostate cancer disrupts normal prostate luminal cell differentiation. Oncogene, 40(18), 3260-3272.More infoThe molecular mechanisms of luminal cell differentiation are not understood well enough to determine how differentiation goes awry during oncogenesis. Using RNA-Seq analysis, we discovered that CREB1 plays a central role in maintaining new luminal cell survival and that oncogenesis dramatically changes the CREB1-induced transcriptome. CREB1 is active in luminal cells, but not basal cells. We identified ING4 and its E3 ligase, JFK, as CREB1 transcriptional targets in luminal cells. During luminal cell differentiation, transient induction of ING4 expression is followed by a peak in CREB1 activity, while JFK increases concomitantly with CREB1 activation. Transient expression of ING4 is required for luminal cell induction; however, failure to properly down-regulate ING4 leads to luminal cell death. Consequently, blocking CREB1 increased ING4 expression, suppressed JFK, and led to luminal cell death. Thus, CREB1 is responsible for the suppression of ING4 required for luminal cell survival and maintenance. Oncogenic transformation by suppressing PTEN resulted in constitutive activation of CREB1. However, the tumor cells could no longer fully differentiate into luminal cells, failed to express ING4, and displayed a unique CREB1 transcriptome. Blocking CREB1 in tumorigenic cells suppressed tumor growth in vivo, rescued ING4 expression, and restored luminal cell formation, but ultimately induced luminal cell death. IHC of primary prostate tumors demonstrated a strong correlation between loss of ING4 and loss of PTEN. This is the first study to define a molecular mechanism whereby oncogenic loss of PTEN, leading to aberrant CREB1 activation, suppresses ING4 expression causing disruption of luminal cell differentiation.
- Dodla, P., Bhoopalan, V., Khoo, S. K., Miranti, C., & Sridhar, S. (2020). Gene expression analysis of human prostate cell lines with and without tumor metastasis suppressor CD82. BMC cancer, 20(1), 1211.More infoTetraspanin CD82 is a tumor metastasis suppressor that is known to down regulate in various metastatic cancers. However, the exact mechanism by which CD82 prevents cancer metastasis is unclear. This study aims to identify genes that are regulated by CD82 in human prostate cell lines.
- Ganguly, S. S., Hostetter, G., Tang, L., Frank, S. B., Saboda, K., Mehra, R., Wang, L., Li, X., Keller, E. T., & Miranti, C. K. (2020). Notch3 promotes prostate cancer-induced bone lesion development via MMP-3.. Oncogene, 39(1), 204-218. doi:10.1038/s41388-019-0977-1More infoProstate cancer metastases primarily localize in the bone where they induce a unique osteoblastic response. Elevated Notch activity is associated with high-grade disease and metastasis. To address how Notch affects prostate cancer bone lesions, we manipulated Notch expression in mouse tibia xenografts and monitored tumor growth, lesion phenotype, and the bone microenvironment. Prostate cancer cell lines that induce mixed osteoblastic lesions in bone expressed 5-6 times more Notch3, than tumor cells that produce osteolytic lesions. Expression of active Notch3 (NICD3) in osteolytic tumors reduced osteolytic lesion area and enhanced osteoblastogenesis, while loss of Notch3 in osteoblastic tumors enhanced osteolytic lesion area and decreased osteoblastogensis. This was accompanied by a respective decrease and increase in the number of active osteoclasts and osteoblasts at the tumor-bone interface, without any effect on tumor proliferation. Conditioned medium from NICD3-expressing cells enhanced osteoblast differentiation and proliferation in vitro, while simultaneously inhibiting osteoclastogenesis. MMP-3 was specifically elevated and secreted by NICD3-expressing tumors, and inhibition of MMP-3 rescued the NICD3-induced osteoblastic phenotypes. Clinical osteoblastic bone metastasis samples had higher levels of Notch3 and MMP-3 compared with patient matched visceral metastases or osteolytic metastasis samples. We identified a Notch3-MMP-3 axis in human prostate cancer bone metastases that contributes to osteoblastic lesion formation by blocking osteoclast differentiation, while also contributing to osteoblastogenesis. These studies define a new role for Notch3 in manipulating the tumor microenvironment in bone metastases.
- Hall, A., Fontelonga, T., Wright, A., Bugda Gwilt, K., Widrick, J., Pasut, A., Villa, F., Miranti, C. K., Gibbs, D., Jiang, E., Meng, H., Lawlor, M. W., & Gussoni, E. (2020). Tetraspanin CD82 is necessary for muscle stem cell activation and supports dystrophic muscle function. Skeletal muscle, 10(1), 34.More infoTetraspanins are a family of proteins known to assemble protein complexes at the cell membrane. They are thought to play diverse cellular functions in tissues by modifying protein-binding partners, thus bringing complexity and diversity in their regulatory networks. Previously, we identified the tetraspanin KAI/CD82 as a prospective marker for human muscle stem cells. CD82 expression appeared decreased in human Duchenne muscular dystrophy (DMD) muscle, suggesting a functional link to muscular dystrophy, yet whether this decrease is a consequence of dystrophic pathology or a compensatory mechanism in an attempt to rescue muscle from degeneration is currently unknown.
- Miranti, C. K., Moore, S., Kim, Y., Chappeta, V. R., Wu, K., De, B., Gokhale, V., Hurley, L. H., & Reyes-Reyes, E. M. (2020). Nucleolin represses transcription of the androgen receptor gene through a G-quadruplex. Oncotarget, 11(19), 1758-1776.More infoThe androgen receptor (AR) is a major driver of prostate cancer development and progression. Men who develop advanced prostate cancer often have long-term cancer control when treated with androgen-deprivation therapies (ADT). Still, their disease inevitably becomes resistant to ADT and progresses to castration-resistant prostate cancer (CRPC). ADT involves potent competitive AR antagonists and androgen synthesis inhibitors. Resistance to these types of treatments emerges, primarily through the maintenance of AR signaling by ligand-independent activation mechanisms. There is a need to find better ways to block AR to overcome CRPC. In the findings reported here, we demonstrate that the nuclear scaffold protein, nucleolin (NCL), suppresses the expression of AR. NCL binds to a G-rich region in the AR promoter that forms a G-quadruplex (G4) structure. Binding of NCL to this G4-element is required for NCL to suppress AR expression, specifically in AR-expressing tumor cells. Compounds that stabilize G4 structures require NCL to associate with the G4-element of the promoter in order to decrease AR expression. A newly discovered G4 compound that suppresses AR expression demonstrates selective killing of AR-expressing tumor cells, including CRPC lines. Our findings raise the significant possibility that G4-stabilizing drugs can be used to increase NCL transcriptional repressor activity to block AR expression in prostate cancer. Our studies contribute to a clearer understanding of the mechanisms that control AR expression, which could be exploited to overcome CRPC.
- Nollet, E. A., Cardo-Vila, M., Ganguly, S. S., Tran, J. D., Schulz, V. V., Cress, A., Corey, E., & Miranti, C. K. (2020). Androgen receptor-induced integrin α6β1 and Bnip3 promote survival and resistance to PI3K inhibitors in castration-resistant prostate cancer. Oncogene, 39(31), 5390-5404.More infoThe androgen receptor (AR) is the major driver of prostate cancer growth and survival. However, almost all patients relapse with castration-resistant disease (CRPC) when treated with anti-androgen therapy. In CRPC, AR is often aberrantly activated independent of androgen. Targeting survival pathways downstream of AR could be a viable strategy to overcome CRPC. Surprisingly, little is known about how AR drives prostate cancer survival. Furthermore, CRPC tumors in which Pten is lost are also resistant to eradication by PI3K inhibitors. We sought to identify the mechanism by which AR drives tumor survival in CRPC to identify ways to overcome resistance to PI3K inhibition. We found that integrins α6β1 and Bnip3 are selectively elevated in CRPC downstream of AR. While integrin α6 promotes survival and is a direct transcriptional target of AR, the ability of AR to induce Bnip3 is dependent on adhesion to laminin and integrin α6β1-dependent nuclear translocation of HIF1α. Integrins α6β1 and Bnip3 were found to promote survival of CRPC cells selectively on laminin through the induction of autophagy and mitophagy. Furthermore, blocking Bnip3 or integrin α6β1 restored sensitivity to PI3K inhibitors in Pten-negative CRPC. We identified an AR driven pathway that cooperates with laminin and hypoxia to drive resistance to PI3K inhibitors. These findings can help explain in part why PI3K inhibitors have failed in clinical trials to overcome AR-dependent CRPC.
- Bergsma, A., Ganguly, S. S., Wiegand, M. E., Dick, D., Williams, B. O., & Miranti, C. K. (2019). Regulation of cytoskeleton and adhesion signaling in osteoclasts by tetraspanin CD82. Bone reports, 10, 100196.More infoWe used a myeloid-specific Cre to conditionally delete CD82 in mouse osteoclasts and their precursors. In contrast to global loss of CD82 (gKO), conditional loss of CD82 (cKO) in osteoclasts does not affect cortical bone, osteoblasts, or adipocytes. CD82 loss results in greater trabecular volume and trabecular number but reduced trabecular space in 6-month old male mice. Though this trend is present in females it did not reach significance; whereas there was an increase in osteoclast numbers and eroded surface area only in female cKO mice. , there is an increase in osteoclast fusion and defects in actin assembly in both gKO and cKO mice, irrespective of sex. This is accompanied by altered osteoclast morphology and decreased release of CTX . Integrin αvβ3 expression is reduced, while integrin β1 is increased. Signaling to Src, Syk, and Vav are also compromised. We further discovered that expression of Clec2 and its ligand, Podoplanin, molecules that also signal to Syk and Vav, are increased in differentiated osteoclasts. Loss of CD82 reduces their expression. Thus, CD82 is required for correct assembly of the cytoskeleton and to limit osteoclast fusion, both needed for normal osteoclast function.
- Ganguly, S. S., Hostetter, G., Tang, L., Frank, S. B., Saboda, K., Mehra, R., Wang, L., Li, X., Keller, E. T., & Miranti, C. K. (2019). Notch3 promotes prostate cancer-induced bone lesion development via MMP-3. Oncogene, 39(1), 204-218.More infoProstate cancer metastases primarily localize in the bone where they induce a unique osteoblastic response. Elevated Notch activity is associated with high-grade disease and metastasis. To address how Notch affects prostate cancer bone lesions, we manipulated Notch expression in mouse tibia xenografts and monitored tumor growth, lesion phenotype, and the bone microenvironment. Prostate cancer cell lines that induce mixed osteoblastic lesions in bone expressed 5-6 times more Notch3, than tumor cells that produce osteolytic lesions. Expression of active Notch3 (NICD3) in osteolytic tumors reduced osteolytic lesion area and enhanced osteoblastogenesis, while loss of Notch3 in osteoblastic tumors enhanced osteolytic lesion area and decreased osteoblastogensis. This was accompanied by a respective decrease and increase in the number of active osteoclasts and osteoblasts at the tumor-bone interface, without any effect on tumor proliferation. Conditioned medium from NICD3-expressing cells enhanced osteoblast differentiation and proliferation in vitro, while simultaneously inhibiting osteoclastogenesis. MMP-3 was specifically elevated and secreted by NICD3-expressing tumors, and inhibition of MMP-3 rescued the NICD3-induced osteoblastic phenotypes. Clinical osteoblastic bone metastasis samples had higher levels of Notch3 and MMP-3 compared with patient matched visceral metastases or osteolytic metastasis samples. We identified a Notch3-MMP-3 axis in human prostate cancer bone metastases that contributes to osteoblastic lesion formation by blocking osteoclast differentiation, while also contributing to osteoblastogenesis. These studies define a new role for Notch3 in manipulating the tumor microenvironment in bone metastases.
- Jiang, L., Ivich, F., Tahsin, S., Tran, M., Frank, S. B., Miranti, C. K., & Zohar, Y. (2019). Human stroma and epithelium co-culture in a microfluidic model of a human prostate gland. Biomicrofluidics, 13(6), 064116.More infoThe prostate is a walnut-sized gland that surrounds the urethra of males at the base of the bladder comprising a muscular portion, which controls the release of urine, and a glandular portion, which secretes fluids that nourish and protect sperms. Here, we report the development of a microfluidic-based model of a human prostate gland. The polydimethylsiloxane (PDMS) microfluidic device, consisting of two stacked microchannels separated by a polyester porous membrane, enables long-term cocultivation of human epithelial and stromal cells. The porous separation membrane provides an anchoring scaffold for long-term culturing of the two cell types on its opposite surfaces allowing paracrine signaling but not cell crossing between the two channels. The microfluidic device is transparent enabling high resolution bright-field and fluorescence imaging. Within this coculture model of a human epithelium/stroma interface, we simulated the functional development of the human prostate gland. We observed the successful differentiation of basal epithelial cells into luminal secretory cells determined biochemically by immunostaining with known differentiation biomarkers, particularly androgen receptor expression. We also observed morphological changes where glandlike mounds appeared with relatively empty centers reminiscent of prostatic glandular acini structures. This prostate-on-a-chip will facilitate the direct evaluation of paracrine and endocrine cross talk between these two cell types as well as studies associated with normal vs disease-related events such as prostate cancer.
- Khan, N. S., Lukason, D. P., Feliu, M., Ward, R. A., Lord, A. K., Reedy, J. L., Ramirez-Ortiz, Z. G., Tam, J. M., Kasperkovitz, P. V., Negoro, P. E., Vyas, T. D., Xu, S., Brinkmann, M. M., Acharaya, M., Artavanis-Tsakonas, K., Frickel, E. M., Becker, C. E., Dagher, Z., Kim, Y. M., , Latz, E., et al. (2019). CD82 controls CpG-dependent TLR9 signaling. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 33(11), 12500-12514.More infoThe tetraspanin CD82 is a potent suppressor of tumor metastasis and regulates several processes including signal transduction, cell adhesion, motility, and aggregation. However, the mechanisms by which CD82 participates in innate immunity are unknown. We report that CD82 is a key regulator of TLR9 trafficking and signaling. TLR9 recognizes unmethylated cytosine-phosphate-guanine (CpG) motifs present in viral, bacterial, and fungal DNA. We demonstrate that TLR9 and CD82 associate in macrophages, which occurs in the endoplasmic reticulum (ER) and post-ER. Moreover, CD82 is essential for TLR9-dependent myddosome formation in response to CpG stimulation. Finally, CD82 modulates TLR9-dependent NF-κB nuclear translocation, which is critical for inflammatory cytokine production. To our knowledge, this is the first time a tetraspanin has been implicated as a key regulator of TLR signaling. Collectively, our study demonstrates that CD82 is a specific regulator of TLR9 signaling, which may be critical in cancer immunotherapy approaches and coordinating the innate immune response to pathogens.-Khan, N. S., Lukason, D. P., Feliu, M., Ward, R. A., Lord, A. K., Reedy, J. L., Ramirez-Ortiz, Z. G., Tam, J. M., Kasperkovitz, P. V., Negoro, P. E., Vyas, T. D., Xu, S., Brinkmann, M. M., Acharaya, M., Artavanis-Tsakonas, K., Frickel, E.-M., Becker, C. E., Dagher, Z., Kim, Y.-M., Latz, E., Ploegh, H. L., Mansour, M. K., Miranti, C. K., Levitz, S. M., Vyas, J. M. CD82 controls CpG-dependent TLR9 signaling.
- Rubenstein, C. S., Gard, J. M., Wang, M., McGrath, J. E., Ingabire, N., Hinton, J. P., Marr, K. D., Simpson, S. J., Nagle, R. B., Miranti, C. K., Warfel, N. A., Garcia, J. G., Arif-Tiwari, H., & Cress, A. E. (2019). Gene Editing of α6 Integrin Inhibits Muscle Invasive Networks and Increases Cell-Cell Biophysical Properties in Prostate Cancer. Cancer research, 79(18), 4703-4714.More infoHuman prostate cancer confined to the gland is indolent (low-risk), but tumors outside the capsule are aggressive (high-risk). Extracapsular extension requires invasion within and through a smooth muscle-structured environment. Because integrins respond to biomechanical cues, we used a gene editing approach to determine if a specific region of laminin-binding α6β1 integrin was required for smooth muscle invasion both and . Human tissue specimens showed prostate cancer invasion through smooth muscle and tumor coexpression of α6 integrin and E-cadherin in a cell-cell location and α6 integrin in a cell-extracellular matrix (ECM) distribution. Prostate cancer cells expressing α6 integrin (DU145 α6WT) produced a 3D invasive network on laminin-containing Matrigel and invaded into smooth muscle both and . In contrast, cells without α6 integrin (DU145 α6KO) and cells expressing an integrin mutant (DU145 α6AA) did not produce invasive networks, could not invade muscle both and , and surprisingly formed 3D cohesive clusters. Using electric cell-substrate impedance testing, cohesive clusters had up to a 30-fold increase in normalized resistance at 400 Hz (cell-cell impedance) as compared with the DU145 α6WT cells. In contrast, measurements at 40,000 Hz (cell-ECM coverage) showed that DU145 α6AA cells were two-fold decreased in normalized resistance and were defective in restoring resistance after a 1 μmol/L S1P challenge as compared with the DU145 α6WT cells. The results suggest that gene editing of a specific α6 integrin extracellular region, not required for normal tissue function, can generate a new biophysical cancer phenotype unable to invade the muscle, presenting a new therapeutic strategy for metastasis prevention in prostate cancer. SIGNIFICANCE: This study shows an innovative strategy to block prostate cancer metastasis and invasion in the muscle through gene editing of a specific α6 integrin extracellular region.
- Tam, J. M., Reedy, J. L., Lukason, D. P., Kuna, S. G., Acharya, M., Khan, N. S., Negoro, P. E., Xu, S., Ward, R. A., Feldman, M. B., Dutko, R. A., Jeffery, J. B., Sokolovska, A., Wivagg, C. N., Lassen, K. G., Le Naour, F., Matzaraki, V., Garner, E. C., Xavier, R. J., , Kumar, V., et al. (2019). Tetraspanin CD82 Organizes Dectin-1 into Signaling Domains to Mediate Cellular Responses to. Journal of immunology (Baltimore, Md. : 1950), 202(11), 3256-3266.More infoTetraspanins are a family of proteins possessing four transmembrane domains that help in lateral organization of plasma membrane proteins. These proteins interact with each other as well as other receptors and signaling proteins, resulting in functional complexes called "tetraspanin microdomains." Tetraspanins, including CD82, play an essential role in the pathogenesis of fungal infections. Dectin-1, a receptor for the fungal cell wall carbohydrate β-1,3-glucan, is vital to host defense against fungal infections. The current study identifies a novel association between tetraspanin CD82 and Dectin-1 on the plasma membrane of -containing phagosomes independent of phagocytic ability. Deletion of CD82 in mice resulted in diminished fungicidal activity, increased viability within macrophages, and decreased cytokine production (TNF-α, IL-1β) at both mRNA and protein level in macrophages. Additionally, CD82 organized Dectin-1 clustering in the phagocytic cup. Deletion of CD82 modulates Dectin-1 signaling, resulting in a reduction of Src and Syk phosphorylation and reactive oxygen species production. CD82 knockout mice were more susceptible to as compared with wild-type mice. Furthermore, patient -induced cytokine production was influenced by two human CD82 single nucleotide polymorphisms, whereas an additional CD82 single nucleotide polymorphism increased the risk for candidemia independent of cytokine production. Together, these data demonstrate that CD82 organizes the proper assembly of Dectin-1 signaling machinery in response to .
- Toth, R. K., Tran, J. D., Muldong, M. T., Nollet, E. A., Schulz, V. V., Jensen, C. C., Hazlehurst, L. A., Corey, E., Durden, D., Jamieson, C., Miranti, C. K., & Warfel, N. A. (2019). Hypoxia-induced PIM kinase and laminin-activated integrin α6 mediate resistance to PI3K inhibitors in bone-metastatic CRPC. American journal of clinical and experimental urology, 7(4), 297-312.More infoBone-metastatic castration-resistant prostate cancer (CRPC) is lethal due to inherent resistance to androgen deprivation therapy, chemotherapy, and targeted therapies. Despite the fact that a majority of CRPC patients (approximately 70%) harbor a constitutively active PI3K survival pathway, targeting the PI3K/mTOR pathway has failed to increase overall survival in clinical trials. Here, we identified two separate and independent survival pathways induced by the bone tumor microenvironment that are hyperactivated in CRPC and confer resistance to PI3K inhibitors. The first pathway involves integrin α6β1-mediated adhesion to laminin and the second involves hypoxia-induced expression of PIM kinases. and models demonstrate that these pathways transduce parallel but independent signals that promote survival by reducing oxidative stress and preventing cell death. We further demonstrate that both pathways drive resistance to PI3K inhibitors in PTEN-negative tumors. These results provide preclinical evidence that combined inhibition of integrin α6β1 and PIM kinase in CRPC is required to overcome tumor microenvironment-mediated resistance to PI3K inhibitors in PTEN-negative tumors within the hypoxic and laminin-rich bone microenvironment.
- Bergsma, A., Dick, D., Williams, B. O., & Miranti, C. K. (2018). Global deletion of Tetraspanin CD82 attenuates bone growth and enhances bone marrow adipogenesis.. Bone.
- Bergsma, A., Ganguly, S. S., Dick, D., Williams, B. O., & Miranti, C. K. (2018). Global deletion of tetraspanin CD82 attenuates bone growth and enhances bone marrow adipogenesis. Bone, 113, 105-113.More infoCD82 is a widely expressed member of the tetraspanin family of transmembrane proteins known to control cell signaling, adhesion, and migration. Tetraspanin CD82 is induced over 9-fold during osteoclast differentiation in vitro; however, its role in bone homeostasis is unknown. A globally deleted CD82 mouse model was used to assess the bone phenotype. Based on microCT and 4-point bending tests, CD82-deficient bones are smaller in diameter and weaker, but display no changes in bone density. Histomorphometry shows a decrease in size, erosion perimeter, and number of osteoclasts in situ, with a corresponding increase in trabecular surface area, specifically in male mice. Male-specific alterations are observed in trabecular structure by microCT and in vitro differentiated osteoclasts are morphologically abnormal. Histomorphometry did not reveal a significant reduction in osteoblast number; however, dynamic labeling reveals a significant decrease in bone growth. Consistent with defects in OB function, OB differentiation and mineralization are defective in vitro, whereas adipogenesis is enhanced. There is a corresponding increase in bone marrow adipocytes in situ. Thus, combined defects in both osteoclasts and osteoblasts can account for the observed bone phenotypes, and suggests a role for CD82 in both bone mesenchyme and myeloid cells.
- Corey, E., Miranti, C. K., Schulz, V. V., Nollet, E. A., & Ganguly, S. S. (2018). Abstract A083: Cell adhesion to laminin, by integrin α6β1, mediates drug resistance to PI3K in castration-resistant prostate cancer through AR-dependent induction of BNIP3. Cancer Research, 78. doi:10.1158/1538-7445.prca2017-a083More infoPten loss leading to constitutive PI3K/Akt/mTor activation is a well-characterized PCa survival pathway frequently activated in castration-resistant prostate cancer (CRPC). Unfortunately, targeted mono-therapies to PI3K/Akt/mTor with or without anti-androgen therapy failed to achieve significant response in clinical trials with CRPC patients. The mechanisms that drive PI3K resistance in CRPC are unknown. The androgen receptor (AR) is also a major driver of prostate cancer (PCa) survival, even in CRPC. However, surprisingly very little is known about how AR promotes PCa survival. We previously identified an AR-dependent survival pathway in androgen-dependent tumor cells whereby AR induction of integrin α6β1 and adhesion to laminin in the tumor microenvironment activates NF-κB to induce Bcl-xL. This pathway acts in parallel to the PI3K/Akt pathway in Pten-null tumor cells such that combined inhibition of both PI3K and integrin α6β1 is required to effectively kill tumor cells and suppress tumor growth. While this drug combination was effective in androgen-responsive tumors in vivo, it poorly suppressed CRPC. Thus, we hypothesized there are additional survival mechanisms operating in CRPC. To identify CRPC survival pathways, we mined the gene expression data comparing androgen-sensitive to CRPC tumors in a series of human PDX LuCaP models. We found the hypoxia-inducible protein, BNIP3, to be preferentially elevated in CRPC tumors, and this correlated with increased integrin α6β1 expression and enhanced AR signaling. Furthermore, elevated BNIP3 predicts for poor outcome in PCa patients. We discovered that androgen induces BNIP3 expression specifically in CRPC C4-2 cell lines, but not the androgen-dependent parental LNCaP cells. BNIP3 expression is dependent on AR induction of integrin α6β1, adhesion to laminin, and nuclear translocation of HIF-1α. The CRPC C4-2 cells are highly resistant to PI3K inhibition by PX866 compared to LNCaP when adherent to laminin, and siRNA-mediated loss of integrin α6 or BNIP3 sensitizes these cells to PI3K inhibition. Blocking Bnip3 expression in C4-2 cells injected orthotopically into mice reduced tumor growth and induced apoptosis. BNIP3 can promote survival by activating autophagy and/or mitophagy. Even though autophagic flux was specifically enhanced in laminin-adherent CRPC C4-2 cells, but not in LNCaP cells, this was not dependent on BNIP3. WT BNIP3 can restore PI3K drug resistance in shBNIP3-expressing cells, but the BNIP3 ΔLIR mutant, which is unable to target mitochondria to autophagosomes, cannot restore drug resistance. We have identified an AR-dependent survival pathway in CRPC involving BNIP3-induced mitophagy that is dependent on and mediated by integrin α6β1 adhesion to laminin that promotes resistance to PI3K inhibition. Laminin is abundant in prostate tissue, lymph nodes, and bone—the primary sites for PCa growth. Thus, laminin within the tumor microenvironment of both primary and metastatic PCa drives drug resistance in CRPC. Citation Format: Eric Nollet, Sourik Ganguly, Veronique Schulz, Eva Corey, Cindy K. Miranti. Cell adhesion to laminin, by integrin α6β1, mediates drug resistance to PI3K in castration-resistant prostate cancer through AR-dependent induction of BNIP3 [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A083.
- Das, L., Gard, J. M., Prekeris, R., Nagle, R. B., Morrissey, C., Knudsen, B. S., Miranti, C. K., & Cress, A. E. (2018). Novel Regulation of Integrin Trafficking by Rab11-FIP5 in Aggressive Prostate Cancer. Molecular cancer research : MCR, 16(8), 1319-1331.More infoThe laminin-binding integrins, α3β1 and α6β1, are needed for tumor metastasis and their surface expression is regulated by endocytic recycling. β1 integrins share the Rab11 recycling machinery, but the trafficking of α3β1 and α6β1 are distinct by an unknown mechanism. Using a mouse PDX tumor model containing human metastatic prostate cancer, Rab11 family interacting protein 5 (Rab11-FIP5) was identified as a lead candidate for α6β1 trafficking. Rab11-FIP5 and its membrane-binding domain were required for α6β1 recycling, without affecting the other laminin-binding integrin (i.e., α3β1) or unrelated membrane receptors like CD44, transferrin receptor, or E-cadherin. Depletion of Rab11-FIP5 resulted in the intracellular accumulation of α6β1 in the Rab11 recycling compartment, loss of cell migration on laminin, and an unexpected loss of α6β1 recycling in cell-cell locations. Taken together, these data demonstrate that α6β1 is distinct from α3β1 via Rab11-FIP5 recycling and recycles in an unexpected cell-cell location. Rab11-FIP5-dependent α6β1 integrin recycling may be selectively targeted to limit migration of prostate cancer cells into laminin-rich tissues. .
- Das, L., Prekeris, R., Gard, J., Nagle, R. B., Morrissey, C., Knudsen, B. S., Miranti, C. K., & Cress, A. E. (2018). Novel regulation of α6β1 integrin recycling by Rab11-FIP5 in aggressive prostate cancer. Molecular Cancer Research.
- Frank, S. B., Berger, P. L., Ljungman, M., & Miranti, C. K. (2017). Human prostate luminal cell differentiation requires NOTCH3 induction by p38-MAPK and MYC. Journal of cell science, 130(11), 1952-1964.More infoMany pathways dysregulated in prostate cancer are also involved in epithelial differentiation. To better understand prostate tumor initiation, we sought to investigate specific genes and mechanisms required for normal basal to luminal cell differentiation. Utilizing human prostate basal epithelial cells and an in vitro differentiation model, we tested the hypothesis that regulation of NOTCH3 by the p38 MAPK family (hereafter p38-MAPK), via MYC, is required for luminal differentiation. Inhibition (SB202190 and BIRB796) or knockdown of p38α (also known as MAPK14) and/or p38δ (also known as MAPK13) prevented proper differentiation. Additionally, treatment with a γ-secretase inhibitor (RO4929097) or knockdown of NOTCH1 and/or NOTCH3 greatly impaired differentiation and caused luminal cell death. Constitutive p38-MAPK activation through MKK6(CA) increased NOTCH3 (but not NOTCH1) mRNA and protein levels, which was diminished upon MYC inhibition (10058-F4 and JQ1) or knockdown. Furthermore, we validated two NOTCH3 enhancer elements through a combination of enhancer (e)RNA detection (BruUV-seq) and luciferase reporter assays. Finally, we found that the NOTCH3 mRNA half-life increased during differentiation or upon acute p38-MAPK activation. These results reveal a new connection between p38-MAPK, MYC and NOTCH signaling, demonstrate two mechanisms of NOTCH3 regulation and provide evidence for NOTCH3 involvement in prostate luminal cell differentiation.
- Frank, S. B., Schulz, V. V., & Miranti, C. K. (2017). A streamlined method for the design and cloning of shRNAs into an optimized Dox-inducible lentiviral vector. BMC biotechnology, 17(1), 24.More infoShort hairpin RNA (shRNA) is an established and effective tool for stable knock down of gene expression. Lentiviral vectors can be used to deliver shRNAs, thereby providing the ability to infect most mammalian cell types with high efficiency, regardless of proliferation state. Furthermore, the use of inducible promoters to drive shRNA expression allows for more thorough investigations into the specific timing of gene function in a variety of cellular processes. Moreover, inducible knockdown allows the investigation of genes that would be lethal or otherwise poorly tolerated if constitutively knocked down. Lentiviral inducible shRNA vectors are readily available, but unfortunately the process of cloning, screening, and testing shRNAs can be time-consuming and expensive. Therefore, we sought to refine a popular vector (Tet-pLKO-Puro) and streamline the cloning process with efficient protocols so that researchers can more efficiently utilize this powerful tool. METHODS: First, we modified the Tet-pLKO-Puro vector to make it easy ("EZ") for molecular cloning (EZ-Tet-pLKO-Puro). Our primary modification was to shrink the stuffer region, which allows vector purification via polyethylene glycol precipitation thereby avoiding the need to purify DNA through agarose. In addition, we generated EZ-Tet-pLKO vectors with hygromycin or blasticidin resistance to provide greater flexibility in cell line engineering. Furthermore, we provide a detailed guide for utilizing these vectors, including shRNA design strategy and simplified screening methods.
- Krishnan, V., Mitchell, K., Miska, J., Shaw, P., Chekmareva, M., Becker, L., Parkash, V., Seewaldt, V. L., Rinker-schaeffer, C. W., Miranti, C. K., Lesniak, M. S., & George, S. H. (2016). Abstract A82: Ovarian cancer cells hijack immune functions of omental milky spots for metastatic colonization.. Clinical Cancer Research, 22. doi:10.1158/1557-3265.ovca15-a82More infoIntroduction: The omentum is the primary site of metastasis in both ovarian cancer models and clinical disease. It is composed predominantly of adipose studded with lymphoreticular organs (milky spots), distinguishing it from other peritoneal adipose. Milky spots are specialized for immune cell trafficking and peritoneal surveillance. We and others have shown that ovarian cancer cells exploit the physiologic function(s) of these structures for omental metastatic colonization. The purpose of this study was to identify cellular and molecular mechanisms responsible for ovarian cancer homing to and growth within milky spot structures. Experimental Procedures: Quantitative in vivo and ex vivo assays were used to evaluate human (SKOV3ip.1, HEYA8, and CaOV3) and murine (ID8) ovarian cancer cell localization to milky spots on the murine omental fat band. Assays were conducted using C57/Bl6 mice or those lacking B cells (Igh6 -/- ); T cells (Nude); B and T cells (Rag1 -/- ); or B, T, and NK cells (BN XID). In vitro assays were used to assess the migration-promoting activity of omentum- and macrophage-conditioned media. Standard approaches were used to assess protein expression, cell growth and viability, etc. Rationale: Milky spots provide resident tissue macrophages and lymphocytes needed for peritoneal homeostasis. Macrophages and stromal cells secrete chemokines promoting peritoneal lymphocyte homing to the omentum. In response to peritoneal irritants, activated CD11b + milky spot macrophages organize coordinated expansion of vascular and stromal compartments. The increase in both the number and size of milky spots is needed to process particulates, resolve infections, and encapsulate foreign bodies. Hypothesis: CD11b+ cells secrete homeostatic chemokines promoting G-protein-dependent migration of ovarian cancer cells to milky spots. Ovarian cancer cell binding to adhesion molecules on the milky spot surface activates CD11b+ cell-dependent tissue remodeling, creating a microenvironment promoting ovarian cancer growth. New Findings: Consistent with our hypothesis, in vivo assays showed that macrophage depletion prior to injection of ID8 and SKOV3ip.1 cells prevented microscopic metastasis formation. In vitro assays found that macrophages are required for ovarian cancer cell localization to milky spots. These data prompt the hypothesis that CD11b + cells produce a factor(s) responsible for the migration-promoting ability of omentum-conditioned media. To test this, media was conditioned by omental adipose isolated from mice after macrophage depletion. In support of our hypothesis, the migration-promoting activity of macrophage-depleted omentum-conditioned media was on a par with that of media conditioned by milky spot-deficient adipose. Further, media conditioned by CD11b + cells recapitulates the migration-promoting activity exhibited by omentum-conditioned media. In vitro and ex vivo assays were used to test whether ovarian cancer cells utilize mechanisms analogous to lymphocyte trafficking for milky spot homing. Specifically, cells were pretreated with pertussis toxin or vehicle alone, and then evaluated for migration in response to omentum-conditioned media and milky spot localization ex vivo. Consistent with data reported for lymphocyte homing, pertussis toxin pre-treatment caused a 40% to 50% reduction in ovarian cancer cell homing. Conclusions and Current Efforts: Our data support a model in which CD11b+ macrophages secrete one or more chemokines promoting G-protein receptor-dependent ovarian cancer cell migration, and potentially integrin activation, which mediate milky spot homing. Current experiments focus on identification of integrin-ligand interactions, CD11b+ cell activation, and defining the link between ovarian cancer cell growth and increase in milky spot size and number. Citation Format: Venkatesh Krishnan, Kelly Mitchell, Jason Miska, Sophia George, Patricia Shaw, Victoria Seewaldt, Maciej Lesniak, Marina Chekmareva, Lev Becker, Vinita Parkash, Cindy Miranti, Carrie Rinker-Schaeffer. Ovarian cancer cells hijack immune functions of omental milky spots for metastatic colonization. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A82.
- Meng, X., Vander Ark, A., Lee, P., Hostetter, G., Bhowmick, N. A., Matrisian, L. M., Williams, B. O., Miranti, C. K., & Li, X. (2016). Myeloid-specific TGF-β signaling in bone promotes basic-FGF and breast cancer bone metastasis. Oncogene, 35(18), 2370-8.More infoBreast cancer (BCa) bone metastases cause osteolytic bone lesions, which result from the interactions of metastatic BCa cells with osteoclasts and osteoblasts. Osteoclasts differentiate from myeloid lineage cells. To understand the cell-specific role of transforming growth factor beta (TGF-β) in the myeloid lineage, in BCa bone metastases, MDA-MB-231 BCa cells were intra-tibially or intra-cardially injected into LysM(Cre)/Tgfbr2(floxE2/floxE2) knockout (LysM(Cre)/Tgfbr2 KO) or Tgfbr2(floxE2/floxE2) mice. Metastatic bone lesion development was compared by analysis of both lesion number and area. We found that LysM(Cre)/Tgfbr2 knockout significantly decreased MDA-MB-231 bone lesion development in both the cardiac and tibial injection models. LysM(Cre)/Tgfbr2 knockout inhibited the tumor cell proliferation, angiogenesis and osteoclastogenesis of the metastatic bones. Cytokine array analysis showed that basic fibroblast growth factor (bFGF) was downregulated in MDA-MB-231-injected tibiae from the LysM(Cre)/Tgfbr2 KO group, and intravenous injection of the recombinant bFGF to LysM(Cre)/Tgfbr2 KO mice rescued the inhibited metastatic bone lesion development. The mechanism by which bFGF rescued the bone lesion development was by promotion of tumor cell proliferation through the downstream mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase (ERK)-cFos pathway after binding to the FGF receptor 1 (FGFR1). Consistent with animal studies, we found that in human BCa bone metastatic tissues, TGF-β type II receptor (TβRII) and p-Smad2 were expressed in osteoclasts and tumor cells, and were correlated with the expression of FGFR1. Our studies suggest that myeloid-specific TGF-β signaling-mediated bFGF in the bone promotes BCa bone metastasis.
- Tesfay, L., Schulz, V. V., Frank, S. B., Lamb, L. E., & Miranti, C. K. (2016). Receptor tyrosine kinase Met promotes cell survival via kinase-independent maintenance of integrin α3β1. Molecular biology of the cell, 27(15), 2493-504.More infoMatrix adhesion via integrins is required for cell survival. Adhesion of epithelial cells to laminin via integrin α3β1 was previously shown to activate at least two independent survival pathways. First, integrin α3β1 is required for autophagy-induced cell survival after growth factor deprivation. Second, integrin α3β1 independently activates two receptor tyrosine kinases, EGFR and Met, in the absence of ligands. EGFR signaling to Erk promotes survival independently of autophagy. To determine how Met promotes cell survival, we inhibited Met kinase activity or blocked its expression with RNA interference. Loss of Met expression, but not inhibition of Met kinase activity, induced apoptosis by reducing integrin α3β1 levels, activating anoikis, and blocking autophagy. Met was specifically required for the assembly of autophagosomes downstream of LC3II processing. Reexpression of wild-type Met, kinase-dead Met, or integrin α3 was sufficient to rescue death upon removal of endogenous Met. Integrin α3β1 coprecipitated and colocalized with Met in cells. The extracellular and transmembrane domain of Met was required to fully rescue cell death and restore integrin α3 expression. Thus Met promotes survival of laminin-adherent cells by maintaining integrin α3β1 via a kinase-independent mechanism.
- Watson, M., Miranti, C. K., Winn, M. E., & Berger, P. L. (2016). Abstract B20: Key intermediate progenitor in luminal prostate epithelial differentiation dictates susceptibility to Myc overexpression and Pten loss in prostate cancer cell of origin. Molecular Cancer Research, 14. doi:10.1158/1557-3125.devbiolca15-b20More infoOverexpression of Myc and loss of Pten are very common oncogenic events in prostate cancer. However, why these specific oncogenic events, as opposed to others, are involved in prostate cancer develop remains unclear. While the impact of Myc and Pten on cell proliferation is well-characterized, their role in differentiation and the resulting impact on tumorigenesis is poorly understood. We recently developed an in vitro differentiation model in which basal prostate epithelial cells (PrECs) can be differentiated into secretory luminal cells. Our model offers several advantages, in that it is human-specific and readily manipulated genetically and biochemically; providing a rare opportunity to dissect the specific genetic and biochemical events associated with human PrEC differentiation and determining how it is impacted by specific oncogenic events. Using this model, we tested the hypothesis that overexpression of Myc and loss of Pten induce prostate cancer because their dysregulation impairs differentiation of an intermediate progenitor cell population that marks the tumor cell of origin. Transient induction of both Pten and Myc are required for normal PrEC differentiation. Constitutive Myc overexpression initially accelerates differentiation, but the differentiated cells ultimately die via a p53-independent mechanism. Overexpression of Myc in combination with loss of Pten (Myc+shPten), is required for tumorigenesis because it rescues cell survival and blocks terminal differentiation. We identified the chromatin binding protein, ING4, as a Myc target required for PrEC terminal differentiation. ING4 is lost in over 60% of human prostate tumors and in the Myc+shPten cells, ING4 expression is blocked. Loss of ING4 in Myc overexpressing cells is sufficient to replace loss of Pten; ING4 loss, like Pten loss, is required for tumorigenesis to block terminal differentiation and keep cells alive. We further identified Miz1, a component of the Myc repressor complex, as a direct target of ING4 required for terminal PrEC differentiation. Myc/Miz1 is required to suppress integrin α6 and β1 expression during terminal PrEC differentiation; in Myc+shPten cells, Miz1 fails to be induced and integrin α6β1 is aberrantly co-expressed with AR in an intermediate progenitor and tumorigenic cell population. Conclusions: Prostate cancer oncogenesis requires dysregulation of Myc and Pten, because they are required for the normal PrEC terminal differentiation pathway. Pten loss is required to remove the ING4/Miz1-mediated terminal differentiation program initiated by Myc overexpression, to maintain the intermediate progenitor cells in a partially differentiated, yet proliferative state, and to evade cell death. Citation Format: Penny Berger, McLane Watson, Mary Winn, Cindy K. Miranti. Key intermediate progenitor in luminal prostate epithelial differentiation dictates susceptibility to Myc overexpression and Pten loss in prostate cancer cell of origin. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr B20.
- Zarif, J. C., & Miranti, C. K. (2016). The importance of non-nuclear AR signaling in prostate cancer progression and therapeutic resistance. Cellular signalling, 28(5), 348-56.More infoThe androgen receptor (AR) remains the major oncogenic driver of prostate cancer, as evidenced by the efficacy of androgen deprivation therapy (ADT) in naïve patients, and the continued effectiveness of second generation ADTs in castration resistant disease. However, current ADTs are limited to interfering with AR ligand binding, either through suppression of androgen production or the use of competitive antagonists. Recent studies demonstrate 1) the expression of constitutively active AR splice variants that no longer depend on androgen, and 2) the ability of AR to signal in the cytoplasm independently of its transcriptional activity (non-genomic); thus highlighting the need to consider other ways to target AR. Herein, we review canonical AR signaling, but focus on AR non-genomic signaling, some of its downstream targets and how these effectors contribute to prostate cancer cell behavior. The goals of this review are to 1) re-highlight the continued importance of AR in prostate cancer as the primary driver, 2) discuss the limitations in continuing to use ligand binding as the sole targeting mechanism, 3) discuss the implications of AR non-genomic signaling in cancer progression and therapeutic resistance, and 4) address the need to consider non-genomic AR signaling mechanisms and pathways as a viable targeting strategy in combination with current therapies.
- Berger, P. L., Winn, M. E., & Miranti, C. K. (2017). Miz1, a Novel Target of ING4, Can Drive Prostate Luminal Epithelial Cell Differentiation. The Prostate, 77(1), 49-59.More infoHow prostate epithelial cells differentiate and how dysregulation of this process contributes to prostate tumorigenesis remain unclear. We recently identified a Myc target and chromatin reader protein, ING4, as a necessary component of human prostate luminal epithelial cell differentiation, which is often lost in primary prostate tumors. Furthermore, loss of ING4 in the context of oncogenic mutations is required for prostate tumorigenesis. Identifying the gene targets of ING4 can provide insight into how its loss disrupts differentiation and leads to prostate cancer.
- Frank, S. B., Berger, P. L., & Miranti, C. K. (2015). Abstract B18: Myc governs a prostate epithelial differentiation program involving chromatin remodeling protein ING4 and Notch3: Disruption of which is necessary for human prostate cancer development. Molecular Cancer Research, 13. doi:10.1158/1557-3125.myc15-b18More infoMyc is overexpressed in the majority of human prostate cancers and is a known determinant of cell fate, yet the cell of origin from which prostate cancers arise is controversial. Furthermore, the mechanisms by which oncogenes such as Myc disrupt prostate epithelial cell fate are poorly understood. Using a novel human in vitro differentiation model in which prostate basal epithelial cells are induced to differentiate into lumenal cells, we previously demonstrated that Myc-driven prostate cancer develops in an intermediate progenitor cell population whose full differentiation is derailed upon oncogenic transformation (Berger et al, Cancer Res 74:3357-68, 2014). In basal prostate epithelial cells, Myc is required for transient expression of the chromatin-binding protein ING4, which is required for lumenal cell differentiation. In human tissues, ING4 expression is lost in >60% of primary prostate tumors. Loss of ING4 prevented differentiation and was necessary for Myc-dependent tumorigenesis in vivo. ING4 loss generated Myc-dependent tumor cells co-expressing basal and lumenal markers, indicating Myc-dependent oncogenesis disrupted an intermediate step in the prostate epithelial differentiation program. Our objective for this study was to further elucidate the mechanisms by which Myc controls prostate epithelial cell fate. Myc is a known downstream target of Notch1, and several studies suggest Notch signaling is aberrant in prostate cancer; although the mechanistic details are vague. We found that Notch3 is required for lumenal cell differentiation and hypothesized that Notch3 expression is directly controlled by Myc. Inhibition of total Notch signaling with a γ-secretase inhibitor (RO4929097) prevented differentiation. Total Notch1 mRNA and protein levels change very little during differentiation; whereas both Notch3 mRNA and protein increase dramatically. Knock-down of Notch3 by shRNA blocked differentiation, while over expression of active Notch3 (NCID3) induced spontaneous differentiation. Less than 15% of the increase in Notch3 mRNA was attributable to increased mRNA stability, and required new protein synthesis. Temporally, Myc mRNA and protein levels increase prior to Notch3. Blocking Myc expression prevented Notch3 induction. The 2kb proximal promoter region of Notch3 lacked the elements that promote Notch3 induction. We identified a Notch3 enhancer element with Myc binding motifs that support differentiation-induced luciferase reporter activity. We further determined that p38α-MAPK is required for Myc and Notch3 induction. We are currently determining how Myc-dependent regulation of Notch3 influences Myc-dependent regulation of ING4. Thus, our studies demonstrate that at least 2 targets of Myc, ING4 and Notch3, control prostate epithelial cell fate, and that disruption of at least one of them is required for Myc-driven human prostate cancer development. In depth understanding of Myc-driven differentiation pathways will provide new insights into how oncogenic transformation by Myc in intermediate progenitor prostate epithelial cells gives rise to prostate cancer. Funding was provided by the Association for International Cancer Research, NIH/NCI CA154835, Department of Defense W81XWH-14-10479, and the Van Andel Research Institute. Citation Format: Sander B. Frank, Penny L. Berger, Cindy K. Miranti. Myc governs a prostate epithelial differentiation program involving chromatin remodeling protein ING4 and Notch3: Disruption of which is necessary for human prostate cancer development. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr B18.
- Tesfay, L., Clausen, K. A., Kim, J. W., Hegde, P., Wang, X., Miller, L. D., Deng, Z., Blanchette, N., Arvedson, T., Miranti, C. K., Babitt, J. L., Lin, H. Y., Peehl, D. M., Torti, F. M., & Torti, S. V. (2015). Hepcidin regulation in prostate and its disruption in prostate cancer. Cancer research, 75(11), 2254-63.More infoHepcidin is a circulating peptide hormone made by the liver that is a central regulator of systemic iron uptake and recycling. Here, we report that prostate epithelial cells also synthesize hepcidin, and that synthesis and secretion of hepcidin are markedly increased in prostate cancer cells and tissue. Prostatic hepcidin functions as an autocrine hormone, decreasing cell surface ferroportin, an iron exporter, increasing intracellular iron retention, and promoting prostate cancer cell survival. Synthesis of hepcidin in prostate cancer is controlled by a unique intersection of pathways that involves BMP4/7, IL6, Wnt, and the dual BMP and Wnt antagonist, SOSTDC1. Epigenetic silencing of SOSTDC1 through methylation is increased in prostate cancer and is associated with accelerated disease progression in patients with prostate cancer. These results establish a new connection between iron metabolism and prostate cancer, and suggest that prostatic dysregulation of hepcidin contributes to prostate cancer growth and progression.
- Uchtmann, K., Park, E. R., Bergsma, A., Segula, J., Edick, M. J., & Miranti, C. K. (2015). Homozygous loss of mouse tetraspanin CD82 enhances integrin αIIbβ3 expression and clot retraction in platelets. Experimental cell research, 339(2), 261-9.More infoIntegrin αIIbβ3 is critical for platelet-mediated blood clotting. Tetraspanins are well-established regulators of integrins and genetic loss of tetraspanin CD151 or TSSC6 in mice leads to increased bleeding due to inadequate integrin αIIbβ3 outside-in signaling. Conversely, mild but enhanced integrin αIIbβ3 activation and hyperaggregation is observed in CD9 and CD63 null mice respectively. CD82 is reportedly expressed in platelets; however its function is unknown. Using genetically engineered CD82 null mice, we investigated the role of the tetraspanin CD82 in platelet activation. Loss of CD82 resulted in reduced bleed times in vivo. CD82 was present on the surface of both human and mouse platelets, and its levels did not change upon platelet activation or degranulation. No differences in platelet activation, degranulation, or aggregation in response to ADP or collagen were detected in CD82 null mice. However, the kinetics of clot retraction was enhanced, which was intrinsic to the CD82-null platelets. Integrin αIIbβ3 surface expression was elevated on the platelets from CD82 null mice and they displayed enhanced adhesion and tyrosine kinase signaling on fibrinogen. This is the first report on CD82 function in platelets; which we found intrinsically modulates clot retraction, integrin αIIbβ3 expression, cell adhesion, and tyrosine signaling.
- Zarif, J. C., Lamb, L. E., Schulz, V. V., Nollet, E. A., & Miranti, C. K. (2015). Androgen receptor non-nuclear regulation of prostate cancer cell invasion mediated by Src and matriptase. Oncotarget, 6(9), 6862-76.More infoCastration-resistant prostate cancers still depend on nuclear androgen receptor (AR) function despite their lack of dependence on exogenous androgen. Second generation anti-androgen therapies are more efficient at blocking nuclear AR; however resistant tumors still develop. Recent studies indicate Src is highly active in these resistant tumors. By manipulating AR activity in several different prostate cancer cell lines through RNAi, drug treatment, and the use of a nuclear-deficient AR mutant, we demonstrate that androgen acting on cytoplasmic AR rapidly stimulates Src tyrosine kinase via a non-genomic mechanism. Cytoplasmic AR, acting through Src enhances laminin integrin-dependent invasion. Active Matriptase, which cleaves laminin, is elevated within minutes after androgen stimulation, and is subsequently shed into the medium. Matriptase activation and shedding induced by cytoplasmic AR is dependent on Src. Concomitantly, CDCP1/gp140, a Matriptase and Src substrate that controls integrin-based migration, is activated. However, only inhibition of Matriptase, but not CDCP1, suppresses the AR/Src-dependent increase in invasion. Matriptase, present in conditioned medium from AR-stimulated cells, is sufficient to enhance invasion in the absence of androgen. Thus, invasion is stimulated by a rapid but sustained increase in Src activity, mediated non-genomically by cytoplasmic AR, leading to rapid activation and shedding of the laminin protease Matriptase.
- Berger, P. L., Frank, S. B., Schulz, V. V., Nollet, E. A., Edick, M. J., Holly, B., Chang, T. T., Hostetter, G., Kim, S., & Miranti, C. K. (2014). Transient induction of ING4 by Myc drives prostate epithelial cell differentiation and its disruption drives prostate tumorigenesis. Cancer research, 74(12), 3357-68.More infoThe mechanisms by which Myc overexpression or Pten loss promotes prostate cancer development are poorly understood. We identified the chromatin remodeling protein, ING4, as a crucial switch downstream of Myc and Pten that is required for human prostate epithelial differentiation. Myc-induced transient expression of ING4 is required for the differentiation of basal epithelial cells into luminal cells, while sustained ING4 expression induces apoptosis. ING4 expression is lost in >60% of human primary prostate tumors. ING4 or Pten loss prevents epithelial cell differentiation, which was necessary for tumorigenesis. Pten loss prevents differentiation by blocking ING4 expression, which is rescued by ING4 re-expression. Pten or ING4 loss generates tumor cells that co-express basal and luminal markers, indicating prostate oncogenesis occurs through disruption of an intermediate step in the prostate epithelial differentiation program. Thus, we identified a new epithelial cell differentiation switch involving Myc, Pten, and ING4, which when disrupted leads to prostate tumorigenesis. Myc overexpression and Pten loss are common genetic abnormalities in prostate cancer, whereas loss of the tumor suppressor ING4 has not been reported. This is the first demonstration that transient ING4 expression is absolutely required for epithelial differentiation, its expression is dependent on Myc and Pten, and it is lost in the majority of human prostate cancers. This is the first demonstration that loss of ING4, either directly or indirectly through loss of Pten, promotes Myc-driven oncogenesis by deregulating differentiation. The clinical implication is that Pten/ING4 negative and ING4-only negative tumors may reflect two distinct subtypes of prostate cancer.
- Ganguly, S. S., Li, X., & Miranti, C. K. (2014). The host microenvironment influences prostate cancer invasion, systemic spread, bone colonization, and osteoblastic metastasis. Frontiers in oncology, 4, 364.More infoProstate cancer (PCa) is the second leading cause of cancer death in men worldwide. Most PCa deaths are due to osteoblastic bone metastases. What triggers PCa metastasis to the bone and what causes osteoblastic lesions remain unanswered. A major contributor to PCa metastasis is the host microenvironment. Here, we address how the primary tumor microenvironment influences PCa metastasis via integrins, extracellular proteases, and transient epithelia-mesenchymal transition (EMT) to promote PCa progression, invasion, and metastasis. We discuss how the bone-microenvironment influences metastasis; where chemotactic cytokines favor bone homing, adhesion molecules promote colonization, and bone-derived signals induce osteoblastic lesions. Animal models that fully recapitulate human PCa progression from primary tumor to bone metastasis are needed to understand the PCa pathophysiology that leads to bone metastasis. Better delineation of the specific processes involved in PCa bone metastasize is needed to prevent or treat metastatic PCa. Therapeutic regimens that focus on the tumor microenvironment could add to the PCa pharmacopeia.
- Akfirat, C., Zhang, X., Ventura, A., Berel, D., Colangelo, M. E., Miranti, C. K., Krajewska, M., Reed, J. C., Higano, C. S., True, L. D., Vessella, R. L., Morrissey, C., & Knudsen, B. S. (2013). Tumour cell survival mechanisms in lethal metastatic prostate cancer differ between bone and soft tissue metastases. The Journal of pathology, 230(3), 291-7.More infoThe complexity of survival mechanisms in cancer cells from patients remains poorly understood. To obtain a comprehensive picture of tumour cell survival in lethal prostate cancer metastases, we examined five survival proteins that operate within three survival pathways in a cohort of 185 lethal metastatic prostate metastases obtained from 44 patients. The expression levels of BCL-2, BCL-XL, MCL-1, cytoplasmic survivin, nuclear survivin, and stathmin were measured by immunohistochemistry in a tissue microarray. Simultaneous expression of three or more proteins occurred in 81% of lethal prostate cancer metastases and BCL-2, cytoplasmic survivin and MCL-1 were co-expressed in 71% of metastatic sites. An unsupervised cluster analysis separated bone and soft tissue metastases according to patterns of survival protein expression. BCL-2, cytoplasmic survivin and MCL-1 had significantly higher expression in bone metastases (p
- Frank, S. B., & Miranti, C. K. (2013). Disruption of prostate epithelial differentiation pathways and prostate cancer development. Frontiers in oncology, 3, 273.More infoOne of the foremost problems in the prostate cancer (PCa) field is the inability to distinguish aggressive from indolent disease, which leads to difficult prognoses and thousands of unnecessary surgeries. This limitation stems from the fact that the mechanisms of tumorigenesis in the prostate are poorly understood. Some genetic alterations are commonly reported in prostate tumors, including upregulation of Myc, fusion of Ets genes to androgen-regulated promoters, and loss of Pten. However, the specific roles of these aberrations in tumor initiation and progression are poorly understood. Likewise, the cell of origin for PCa remains controversial and may be linked to the aggressive potential of the tumor. One important clue is that prostate tumors co-express basal and luminal protein markers that are restricted to their distinct cell types in normal tissue. Prostate epithelium contains layer-specific stem cells as well as rare bipotent cells, which can differentiate into basal or luminal cells. We hypothesize that the primary oncogenic cell of origin is a transient-differentiating bipotent cell. Such a cell must maintain tight temporal and spatial control of differentiation pathways, thus increasing its susceptibility for oncogenic disruption. In support of this hypothesis, many of the pathways known to be involved in prostate differentiation can be linked to genes commonly altered in PCa. In this article, we review what is known about important differentiation pathways (Myc, p38MAPK, Notch, PI3K/Pten) in the prostate and how their misregulation could lead to oncogenesis. Better understanding of normal differentiation will offer new insights into tumor initiation and may help explain the functional significance of common genetic alterations seen in PCa. Additionally, this understanding could lead to new methods for classifying prostate tumors based on their differentiation status and may aid in identifying more aggressive tumors.
- Zarif, J. C., Zarif, J. C., Lamb, L. E., Lamb, L. E., Miranti, C. K., & Miranti, C. K. (2013). Abstract 3801: Androgen Receptor non-genomic regulation of prostate tumor cell invasion and metastasis through Src signaling and Matriptase .. Cancer Research, 73, 3801-3801. doi:10.1158/1538-7445.am2013-3801More infoProceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Prostate cancer is the leading cause of cancer deaths in men here in the U.S. and over 240,000 new cases are predicted to be diagnosed this year. Initially, tumors arising in this gland rely on the androgen receptor (AR) and its ligand, dihydrotestosterone (DHT) for growth and survival. At this stage, androgen deprivation therapy (ADT) can lead to tumor regression. Unfortunately, patients typically relapse within two to three years and tumors no longer respond to ADT; this stage is termed castration-resistant prostate cancer (CRPC) for which there is no cure. At this stage, AR expression is either increased or mutationally activated and non-responsive to physiological levels of androgen. Several reports have shown that AR has both genomic signaling within nucleus and non-genomic signaling within the cytoplasm. Using prostate tumor cell line PC-3, expressing wild-type AR (PC3-AR), we reported that AR genomic signaling leads to an increase in integrin α6β1 transcription and expression, which in turn increases Bcl-xL expression, a mechanism that regulates prostate cancer cell survival. However, a role for AR and integrin α6β1 in prostate cancer metastasis has not been examined. In addition to promoting cell survival, integrins are critically important for cell movement and can activate Src. We observed that in the absence of androgen, PC3-AR cells have increased Src activity as well as enhanced cell migration and invasion relative to cells that do not expressing AR. Androgen responsive prostate cancer cell lines LNCaP, VCaP and C42 which express AR endogenously, have enhanced invasion and Src activity once stimulated with androgen. We found that knock-down of AR decreased Src activity in PC3-AR cells, as well as in LNCaP and C42 cells. We also found that inhibiting the expression of either AR or Src blocked the ability of cells to invade through matrix in vitro. However, neither AR nor Src possess the ability to degrade ECM proteins. Thus, we hypothesize that AR activates Src in a non-genomic fashion to promote a metastatic phenotype by the regulation of a downstream protease. To test our hypothesis, we set out to determine how AR/Src signaling controls the expression and/or activity of a protease that can degrade proteins in the ECM to promote metastasis. Our data shows that the protease downstream of AR/Src signaling is matriptase, a transmembrane serine protease that has been recently reported to cleave laminin. We observe matriptase cellular expression levels decrease in androgen responsive cell lines once stimulated with androgen as early as twenty minutes. This is suggestive that this is regulated through a non-genomic signaling mechanism. Also seen in these androgen stimulated cells within twenty minutes is that matriptase is shed and found within the media at higher levels relative to the media of non-stimulated cells. Citation Format: Jelani C. Zarif, Laura E. Lamb, Cindy K. Miranti. Androgen Receptor non-genomic regulation of prostate tumor cell invasion and metastasis through Src signaling and Matriptase . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3801. doi:10.1158/1538-7445.AM2013-3801
- Kim, S., Miranti, C. K., & Berger, P. L. (2012). Abstract C63: Understanding prostate cancer initiation and development through differentiation: Role of ING4 and Myc in prostate differentiation and cancer development. Cancer Research, 72(4 Supplement), C63-C63. doi:10.1158/1538-7445.prca2012-c63More infoAbstract Introduction: Genes involved in differentiation and cell fate determination are frequently altered in cancers. Understanding how normal differentiation is controlled, and how its deregulation leads to cancer development is paramount to developing better diagnostic and therapeutic strategies for prostate cancer. Our laboratory recently developed an in vitro differentiation model in which human AR-negative basal prostate epithelial cells can be differentiated in to AR-positive secretory cells. Loss of integrin expression and cell-matrix adhesion is crucial to generating stable AR-expressing cells. This is consistent with the observation that integrins are only expressed in basal cells and AR is only expressed in secretory cells. However, in prostate cancer basal cells are lost and AR and integrin α6β1 are co-expressed in the tumor cells. The mechanism by which this occurs is unknown. Our hypothesis is that oncogenic conversion disrupts an intermediate step in normal prostate epithelial differentiation, causing retention of integrin α6β1 and AR expression in the same cell. The earliest alterations in cell adhesion during differentiation are the loss of the extracellular matrix laminin 5 and its respective ligand-specific integrin subunits, α3 and β4. This results in the pairing of the α6 and β1 subunits to generate α6β1, which is not lost until several days later. Several lines of evidence suggest Myc is required to suppress α6β1 expression. Misregulated expression of Myc may contribute to prostate cancer development through up-regulation of integrin α6β1. ING4, a PHD finger containing nuclear protein, is a tumor suppressor that blocks Myc-induced hyperplasia. We hypothesize that ING4 controls Myc, is vital for the normal prostate epithelial differentiation, and loss of ING4 is required for tumorigenesis. Methods: We manipulated Myc and ING4 expression in differentiating human prostate epithelial cells and measured the effects on differentiation. We generated tumorigenic variants by over expressing Myc and Erg, and suppressing Pten. We measured ING4 expression in prostate cancer tissues. Results: Myc over expression resulted in faster differentiation, i.e. loss of integrin expression and induction of AR-responsive genes. However, the differentiated Myc over expressing cells eventually died while control cells did not. ING4 expression increased transiently during differentiation. Loss of ING4 expression did not affect early differentiation; however, cells could not complete differentiation. Myc over expression resulted in an increase in ING4 expression, and ING4 over expression, like Myc, initially augmented differentiation, but induced death of the differentiated cells. Knock-down of ING4 reversed the effect of Myc over expression. Epithelial cells over expressing Myc, Erg, and shPten were tumorigenic in mice, but failed to properly differentiate in vitro. They initiated the AR program, but failed to switch off integrin expression. Over-expression of ING4 in the tumorigenic cells restored normal differentiation. In a cohort of 50 patients, ING4 expression was lost in 67% of primary tumors. Conclusions: These data indicate Myc stimulates ING4 expression in mid differentiation and ING4 is required transiently for proper differentiation and loss of integrin expression. Sustained over expression of ING4, directly or indirectly by Myc, results in death of the differentiated cells. In tumorigenic cells, the differentiation program is altered such that Myc over expression no longer promotes full differentiation, ING4 is not turned on, and cells survive. We hypothesize that ING4 loss is required for prostate cancer development to overcome the death-inducing effects of Myc over expression. Funding: Association for International Cancer Research and VARI. Citation Format: Penny Berger, Suwon Kim, Cindy K. Miranti. Understanding prostate cancer initiation and development through differentiation: Role of ING4 and Myc in prostate differentiation and cancer development [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr C63.
- Klionsky, D. J., Abdalla, F. C., Abeliovich, H., Abraham, R. T., Acevedo-Arozena, A., Adeli, K., Agholme, L., Agnello, M., Agostinis, P., Aguirre-Ghiso, J. A., Ahn, H. J., Ait-Mohamed, O., Ait-Si-Ali, S., Akematsu, T., Akira, S., Al-Younes, H. M., Al-Zeer, M. A., Albert, M. L., Albin, R. L., , Alegre-Abarrategui, J., et al. (2012). Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy, 8(4), 445-544.More infoIn 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
- Lamb, L. E., Zarif, J. C., & Miranti, C. K. (2011). The androgen receptor induces integrin α6β1 to promote prostate tumor cell survival via NF-κB and Bcl-xL Independently of PI3K signaling. Cancer research, 71(7), 2739-49.More infoRecent studies indicate that androgen receptor (AR) signaling is critical for prostate cancer cell survival, even in castration-resistant disease wherein AR continues to function independently of exogenous androgens. Integrin-mediated adhesion to the extracellular matrix is also important for prostate cell survival. AR-positive prostate cancer cells express primarily integrin α6β1 and adhere to a laminin-rich matrix. In this study, we show that active nuclear-localized AR protects prostate cancer cells from death induced by phosphoinositide 3-kinase (PI3K) inhibition when cells adhere to laminin. Resistance to PI3K inhibition is mediated directly by an AR-dependent increase in integrin α6β1 mRNA transcription and protein expression. Subsequent signaling by integrin α6β1 in AR-expressing cells increased NF-κB activation and Bcl-xL expression. Blocking AR, integrin α6, NF-κB, or Bcl-xL concurrent with inhibition of PI3K was sufficient and necessary to trigger death of laminin-adherent AR-expressing cells. Taken together, these results define a novel integrin-dependent survival pathway in prostate cancer cells that is regulated by AR, independent of and parallel to the PI3K pathway. Our findings suggest that combined targeting of both the AR/α6β1 and PI3K pathways may effectively trigger prostate cancer cell death, enhancing the potential therapeutic value of PI3K inhibitors being evaluated in this setting.
- Lamb, L. E., Knudsen, B. S., & Miranti, C. K. (2010). E-cadherin-mediated survival of androgen-receptor-expressing secretory prostate epithelial cells derived from a stratified in vitro differentiation model. Journal of cell science, 123(Pt 2), 266-76.More infoThe androgen receptor (AR) is expressed in differentiated secretory prostate epithelial cells in vivo. However, in the human prostate, it is unclear whether androgens directly promote the survival of secretory cells, or whether secretory cells survive through androgen-dependent signals from the prostate stroma. Biochemical and mechanistic studies have been hampered by inadequate cell-culture models. In particular, large-scale differentiation of prostate epithelial cells in culture has been difficult to achieve. Here, we describe the development of a differentiation system that is amenable to functional and biochemical analysis and its application to deciphering the survival pathways in differentiated AR-expressing epithelial cells. Confluent prostate epithelial cell cultures were treated with keratinocyte growth factor (KGF) and dihydrotestosterone. After 2 weeks, a suprabasal cell layer was formed in which cells no longer expressed alpha2, alpha3, alpha6, alphav, beta1 or beta4 integrins or p63, K5, K14, EGFR, FGFR2IIIb or Bcl-2, but instead expressed AR and androgen-induced differentiation markers, including K18, K19, TMPRSS2, Nkx3.1, PMSA, KLK2 and secreted prostate-specific antigen (PSA). Differentiated prostate cell survival depended on E-cadherin and PI3K, but not KGF, androgen, AR or MAPK. Thus survival of differentiated prostate epithelial cells is mediated by cell-cell adhesion, and not through androgen activity or prostate stroma-derived KGF.
- Miranti, C. K. (2009). Controlling cell surface dynamics and signaling: how CD82/KAI1 suppresses metastasis. Cellular signalling, 21(2), 196-211.More infoThe recent identification of metastasis suppressor genes, uniquely responsible for negatively controlling cancer metastasis, are providing inroads into the molecular machinery involved in metastasis. While the normal function of a few of these genes is known; the molecular events associated with their loss that promotes tumor metastasis is largely not understood. KAI1/CD82, whose loss is associated with a wide variety of metastatic cancers, belongs to the tetraspanin family. Despite intense scrutiny, many aspects of how CD82 specifically functions as a metastasis suppressor and its role in normal biology remain to be determined. This review will focus on the molecular events associated with CD82 loss, the potential impact on signaling pathways that regulate cellular processes associated with metastasis, and its relationship with other metastasis suppressor genes.
- Putnam, A. J., Schulz, V. V., Freiter, E. M., Bill, H. M., & Miranti, C. K. (2009). Src, PKCalpha, and PKCdelta are required for alphavbeta3 integrin-mediated metastatic melanoma invasion. Cell communication and signaling : CCS, 7, 10.More infoIntegrins, cell-surface receptors that mediate adhesive interactions between cells and the extracellular matrix (ECM), play an important role in cancer progression. Expression of the vitronectin receptor alphavbeta3 integrin correlates with increased invasive and metastatic capacity of malignant melanomas, yet it remains unclear how expression of this integrin triggers melanoma invasion and metastasis.
- Edick, M. J., Tesfay, L., Lamb, L. E., Knudsen, B. S., & Miranti, C. K. (2007). Inhibition of integrin-mediated crosstalk with epidermal growth factor receptor/Erk or Src signaling pathways in autophagic prostate epithelial cells induces caspase-independent death. Molecular biology of the cell, 18(7), 2481-90.More infoIn vivo in the prostate gland, basal epithelial cells adhere to laminin 5 (LM5) via alpha3beta1 and alpha6beta4 integrins. When placed in culture primary prostate basal epithelial cells secrete and adhere to their own LM5-rich matrix. Adhesion to LM5 is required for cell survival that is dependent on integrin-mediated, ligand-independent activation of the epidermal growth factor receptor (EGFR) and the cytoplasmic tyrosine kinase Src, but not PI-3K. Integrin-mediated adhesion via alpha3beta1, but not alpha6beta4 integrin, supports cell survival through EGFR by signaling downstream to Erk. PC3 cells, which do not activate EGFR or Erk on LM5-rich matrices, are not dependent on this pathway for survival. PC3 cells are dependent on PI-3K for survival and undergo caspase-dependent death when PI-3K is inhibited. The death induced by inhibition of EGFR or Src in normal primary prostate cells is not mediated through or dependent on caspase activation, but depends on the induction of reactive oxygen species. In addition the presence of an autophagic pathway, maintained by adhesion to matrix through alpha3beta1 and alpha6beta4, prevents the induction of caspases when EGFR or Src is inhibited. Suppression of autophagy is sufficient to induce caspase activation and apoptosis in LM5-adherent primary prostate epithelial cells.
- Tolbert, W. D., Daugherty, J., Gao, C., Xie, Q., Miranti, C., Gherardi, E., Vande Woude, G., & Xu, H. E. (2007). A mechanistic basis for converting a receptor tyrosine kinase agonist to an antagonist. Proceedings of the National Academy of Sciences of the United States of America, 104(37), 14592-7.More infoHepatocyte growth factor (HGF) activates the Met receptor tyrosine kinase by binding and promoting receptor dimerization. Here we describe a mechanistic basis for designing Met antagonists based on NK1, a natural variant of HGF containing the N-terminal and the first kringle domain. Through detailed biochemical and structural analyses, we demonstrate that both mouse and human NK1 induce Met dimerization via a conserved NK1 dimer interface. Mutations designed to alter the NK1 dimer interface abolish its ability to promote Met dimerization but retain full Met-binding activity. Importantly, these NK1 mutants act as Met antagonists by inhibiting HGF-mediated cell scattering, proliferation, branching, and invasion. The ability to separate the Met-binding activity of NK1 from its Met dimerization activity thus provides a rational basis for designing Met antagonists. This strategy of antagonist design may be applicable for other growth factor receptors by selectively abolishing the receptor activation ability but not the receptor binding of the growth factors.
- Wang, X., Zhu, J., Zhao, P., Jiao, Y., Xu, N., Grabinski, T., Liu, C., Miranti, C. K., Fu, T., & Cao, B. B. (2007). In vitro efficacy of immuno-chemotherapy with anti-EGFR human Fab-Taxol conjugate on A431 epidermoid carcinoma cells. Cancer biology & therapy, 6(6), 980-7.More infoThe aims of this study were to generate a human Fab fragment against EGFR; conjugate it to paclitaxel (Taxol) as an immuno-chemotherapy agent; and investigate its in vitro anti-tumor efficacy on A431 epidermoid carcinoma cells. A431 cells (EGFR-positive), NIH 3T3 cells (EGFR-negative), and purified EGFR were used for subtractive panning on a human naïve Fab phage library to generate a human anti-EGFR Fab fragment that binds the EGFR extracellular domain in native conformation and subsequently internalizes it into the cytosol. The Fab was then conjugated with the chemotherapeutic Taxol, and cell proliferation inhibition and apoptosis (TUNEL) assays were conducted to determine the effect of this Fab-drug conjugate on A431 cells. The specificity and internalization property of this Fab were characterized by immunoprecipitation, fluorescence staining, flow cytometry, and Hum-Zap assay. The binding affinity to purified EGFR was 30 nM. The Fab-Taxol conjugate inhibited A431 cell proliferation at low concentrations and in a dose-responsive manner; more than 70% inhibition was observed at 52 pM. Furthermore, almost 100% of cells underwent apoptosis after treatment with Fab-Taxol at 26 pM for 48 hours. Our findings suggest that this Fab-Taxol conjugate could be a potential immuno-chemotherapeutic drug for clinical treatment of EGFR-overexpressing tumors.
- Knudsen, B. S., & Miranti, C. K. (2006). The impact of cell adhesion changes on proliferation and survival during prostate cancer development and progression. Journal of cellular biochemistry, 99(2), 345-61.More infoIn the normal prostate epithelium, androgen receptor (AR) negative basal epithelial cells adhere to the substratum, while AR expressing secretory cells lose substratum adhesion. In contrast, prostate cancer cells both express AR and adhere to a tumor basement membrane. In this review, we describe the differential expression of integrins, growth factor receptors (GFRs), and AR in normal and cancerous epithelium. In addition, we discuss how signals from integrins, GFRs, and AR are integrated to regulate the proliferation and survival of normal and malignant prostate epithelial cells. While cell adhesion is likely of great importance when considering therapeutic approaches for treatment of metastatic prostate cancer, no data on integrin expression are available from tissues of prostate cancer metastasis. However, several drug targets that are upregulated after androgen ablative therapy regulate cell adhesion and thus novel targeted therapies indirectly interfere with cell adhesion mechanisms in prostate cancer cells.
- Sridhar, S. C., & Miranti, C. K. (2006). Tetraspanin KAI1/CD82 suppresses invasion by inhibiting integrin-dependent crosstalk with c-Met receptor and Src kinases. Oncogene, 25(16), 2367-78.More infoKAI1/CD82, a tetraspanin protein, was first identified as a metastasis suppressor in prostate cancer. How loss of CD82 expression promotes cancer metastasis is unknown. Restoration of CD82 expression to physiological levels in the metastatic prostate cell line PC3 inhibits integrin-mediated cell migration and invasion, but does not affect integrin expression. Integrin-dependent activation of the receptor kinase c-Met is dramatically reduced in CD82-expressing cells, as is c-Met activation by its ligand HGF/SF. CD82 expression also reduced integrin-induced activation and phosphorylation of the cytoplasmic tyrosine kinase Src, and its downstream substrates p130Cas and FAK Y861. Inhibition of c-Met expression or Src kinase function reduced matrigel invasion of PC3 cells to the same extent as CD82 expression. These data indicate that CD82 functions to suppress integrin-induced invasion by regulating signaling to c-Met and Src kinases, and suggests that CD82 loss may promote metastasis by removing a negative regulator of c-Met and Src signaling.
- Bill, H. M., Knudsen, B., Moores, S. L., Muthuswamy, S. K., Rao, V. R., Brugge, J. S., & Miranti, C. K. (2004). Epidermal growth factor receptor-dependent regulation of integrin-mediated signaling and cell cycle entry in epithelial cells. Molecular and cellular biology, 24(19), 8586-99.More infoIntegrin-mediated adhesion of epithelial cells to extracellular matrix (ECM) proteins induces prolonged tyrosine phosphorylation and partial activation of epidermal growth factor receptor (EGFR) in an integrin-dependent and EGFR ligand-independent manner. Integrin-mediated activation of EGFR in epithelial cells is required for multiple signal transduction events previously shown to be induced by cell adhesion to matrix proteins, including tyrosine phosphorylation of Shc, Cbl, and phospholipase Cgamma, and activation of the Ras/Erk and phosphatidylinositol 3'-kinase/Akt signaling pathways. In contrast, activation of focal adhesion kinase, Src, and protein kinase C, adhesion to matrix proteins, cell spreading, migration, and actin cytoskeletal rearrangements are induced independently of EGFR kinase activity. The ability of integrins to induce the activation of EGFR and its subsequent regulation of Erk and Akt activation permitted adhesion-dependent induction of cyclin D1 and p21, Rb phosphorylation, and activation of cdk4 in epithelial cells in the absence of exogenous growth factors. Adhesion of epithelial cells to the ECM failed to efficiently induce degradation of p27, to induce cdk2 activity, or to induce Myc and cyclin A synthesis; subsequently, cells did not progress into S phase. Treatment of ECM-adherent cells with EGF, or overexpression of EGFR or Myc, resulted in restoration of late-G(1) cell cycle events and progression into S phase. These results indicate that partial activation of EGFR by integrin receptors plays an important role in mediating events triggered by epithelial cell attachment to ECM; EGFR is necessary for activation of multiple integrin-induced signaling enzymes and sufficient for early events in G(1) cell cycle progression. Furthermore, these findings suggest that EGFR or Myc overexpression may provoke ligand-independent proliferation in matrix-attached cells in vivo and could contribute to carcinoma development.
- Bromberg-White, J. L., Webb, C. P., Patacsil, V. S., Miranti, C. K., Williams, B. O., & Holmen, S. L. (2004). Delivery of short hairpin RNA sequences by using a replication-competent avian retroviral vector. Journal of virology, 78(9), 4914-6.More infoWhile recent studies have demonstrated that retroviral vectors can be used to stably express short hairpin RNA (shRNA) to inhibit gene expression, these studies have utilized replication-defective retroviruses. We describe the creation of a replication-competent, Gateway-compatible retroviral vector capable of expressing shRNA that inhibits the expression of specific genes.
- Lee, C. C., Putnam, A. J., Miranti, C. K., Gustafson, M., Wang, L. M., Vande Woude, G. F., & Gao, C. F. (2004). Overexpression of sprouty 2 inhibits HGF/SF-mediated cell growth, invasion, migration, and cytokinesis. Oncogene, 23(30), 5193-202.More infoA strict regulation of hepatocyte growth factor/scatter factor (HGF/SF)-Met signaling is essential for its appropriate function. Several negative regulators of Met signaling have been identified. Here we report that human Spry2 is induced by HGF/SF and negatively regulates HGF/SF-Met signaling. We show that overexpression of Spry2 inhibits cell proliferation, anchorage-independent cell growth, and migration in wound-healing and in vitro invasion assays. Measured in an electric cell-substrate impedance sensing biosensor, cell movement is restricted, because Spry2 dramatically facilitates cell attachment and spreading by enhancing focal adhesions and increasing stress fibers. An analysis of cell cycle distribution shows, unexpectedly, that Spry2-GFP cells are polyploid. Thus, as with FGF and EGF receptors, Spry2-GFP tempers downstream Met signaling in addition to its pronounced effect on cell adhesion, and it has properties suitable to be considered a tumor-suppressor protein.
- Miranti, C. K. (2002). Application of cell adhesion to study signaling networks. Methods in cell biology, 69, 359-83.
- Miranti, C. K., & Brugge, J. S. (2002). Sensing the environment: a historical perspective on integrin signal transduction. Nature cell biology, 4(4), E83-90.More infoCell adhesion mediated by integrin receptors has a critical function in organizing cells in tissues and in guiding haematopoietic cells to their sites of action. However, integrin adhesion receptors have broader functions in regulating cell behaviour through their ability to transduce bi-directional signals into and out of the cell and to engage in reciprocal interactions with other cellular receptors. This historical perspective traces the key findings that have led to our current understanding of these important functions of integrins.
- Woodside, D. G., Obergfell, A., Leng, L., Wilsbacher, J. L., Miranti, C. K., Brugge, J. S., Shattil, S. J., & Ginsberg, M. H. (2001). Activation of Syk protein tyrosine kinase through interaction with integrin beta cytoplasmic domains. Current biology : CB, 11(22), 1799-804.More infoSyk protein tyrosine kinase is essential for immune system development and function and for the maintenance of vascular integrity [2,3]. In leukocytes, Syk is activated by binding to diphosphorylated immune receptor tyrosine-based activation motifs (pITAMs). Syk can also be activated by integrin adhesion receptors [4,5], but the mechanism of its activation is unknown. Here we report a novel mechanism for Syk's recruitment and activation, which requires that Syk bind to the integrin beta3 cytoplasmic tail. We found that both Syk and the related kinase ZAP-70 bound the beta3 cytoplasmic tail through their tandem SH2 domains. However, unlike Syk binding to pITAMs, this interaction was independent of tyrosine phosphorylation and of the phosphotyrosine binding function of Syk's tandem SH2 domains. Deletion of the four C-terminal residues of the beta3 cytoplasmic tail [beta3(759X)] decreased Syk binding and disrupted its physical association with integrin alphaIIbbeta3. Furthermore, cells expressing alphaIIbbeta3(759X) failed to exhibit Syk activation or lamellipodia formation upon cell adhesion to the alphaIIbbeta3 ligand, fibrinogen. In contrast, FAK phosphorylation and focal adhesion formation were unimpaired by this mutation. Thus, the direct binding of Syk kinase to the integrin beta3 cytoplasmic tail is a novel and functionally significant mechanism for the regulation of this important non-receptor tyrosine kinase.
- Miranti, C. K., Ohno, S., & Brugge, J. S. (1999). Protein kinase C regulates integrin-induced activation of the extracellular regulated kinase pathway upstream of Shc. The Journal of biological chemistry, 274(15), 10571-81.More infoAdhesion of fibroblasts to extracellular matrices via integrin receptors is accompanied by extensive cytoskeletal rearrangements and intracellular signaling events. The protein kinase C (PKC) family of serine/threonine kinases has been implicated in several integrin-mediated events including focal adhesion formation, cell spreading, cell migration, and cytoskeletal rearrangements. However, the mechanism by which PKC regulates integrin function is not known. To characterize the role of PKC family kinases in mediating integrin-induced signaling, we monitored the effects of PKC inhibition on fibronectin-induced signaling events in Cos7 cells using pharmacological and genetic approaches. We found that inhibition of classical and novel isoforms of PKC by down-regulation with 12-0-tetradeconoyl-phorbol-13-acetate or overexpression of dominant-negative mutants of PKC significantly reduced extracellular regulated kinase 2 (Erk2) activation by fibronectin receptors in Cos7 cells. Furthermore, overexpression of constitutively active PKCalpha, PKCdelta, or PKCepsilon was sufficient to rescue 12-0-tetradeconoyl-phorbol-13-acetate-mediated down-regulation of Erk2 activation, and all three of these PKC isoforms were activated following adhesion. PKC was required for maximal activation of mitogen-activated kinase kinase 1, Raf-1, and Ras, tyrosine phosphorylation of Shc, and Shc association with Grb2. PKC inhibition does not appear to have a generalized effect on integrin signaling, because it does not block integrin-induced focal adhesion kinase or paxillin tyrosine phosphorylation. These results indicate that PKC activity enhances Erk2 activation in response to fibronectin by stimulating the Erk/mitogen-activated protein kinase pathway at an early step upstream of Shc.
- Miranti, C. K., Leng, L., Maschberger, P., Brugge, J. S., & Shattil, S. J. (1998). Identification of a novel integrin signaling pathway involving the kinase Syk and the guanine nucleotide exchange factor Vav1. Current biology : CB, 8(24), 1289-99.More info. Integrins induce the formation of large complexes of cytoskeletal and signaling proteins, which regulate many intracellular processes. The activation and assembly of signaling complexes involving focal adhesion kinase (FAK) occurs late in integrin signaling, downstream from actin polymerization. Our previous studies indicated that integrin-mediated activation of the non-receptor tyrosine kinase Syk in hematopoietic cells is independent of FAK and actin polymerization, and suggested the existence of a distinct signaling pathway regulated by Syk.
- Xia, Z., Dudek, H., Miranti, C. K., & Greenberg, M. E. (1996). Calcium influx via the NMDA receptor induces immediate early gene transcription by a MAP kinase/ERK-dependent mechanism. The Journal of neuroscience : the official journal of the Society for Neuroscience, 16(17), 5425-36.More infoThe regulation of gene expression by neurotransmitters is likely to play a key role in neuroplasticity both during development and in the adult animal. Therefore, it is important to determine the mechanisms of neuronal gene regulation to understand fully the mechanisms of learning, memory, and other long-term adaptive changes in neurons. The neurotransmitter glutamate stimulates rapid and transient induction of many genes, including the c-fos proto-oncogene. The c-fos promoter contains several critical regulatory elements, including the serum response element (SRE), that mediate glutamate-induced transcription in neurons; however, the mechanism by which the SRE functions in neurons has not been defined. In this study, we sought to identify transcription factors that mediate glutamate induction of transcription through the SRE in cortical neurons and to elucidate the mechanism(s) of transcriptional activation by these factors. To facilitate this analysis, we developed an improved calcium phosphate coprecipitation procedure to transiently introduce DNA into primary neurons, both efficiently and consistently. Using this protocol, we demonstrate that the transcription factors serum response factor (SRF) and Elk-1 can mediate glutamate induction of transcription through the SRE in cortical neurons. There are at least two distinct pathways by which glutamate signals through the SRE: an SRF-dependent pathway that can operate in the absence of Elk and an Elk-dependent pathway. Activation of the Elk-dependent pathway of transcription seems to require phosphorylation of Elk-1 by extracellular signal-regulated kinases (ERKs), providing evidence for a physiological function of ERKs in glutamate signaling in neurons. Taken together, these findings suggest that SRF, Elk, and ERKs may have important roles in neuroplasticity.
- Miranti, C. K., Ginty, D. D., Huang, G., Chatila, T., & Greenberg, M. E. (1995). Calcium activates serum response factor-dependent transcription by a Ras- and Elk-1-independent mechanism that involves a Ca2+/calmodulin-dependent kinase. Molecular and cellular biology, 15(7), 3672-84.More infoEnhanced levels of cytoplasmic Ca2+ due to membrane depolarization with elevated levels of KCl or exposure to the Ca2+ ionophore ionomycin stimulate serum response element (SRE)-dependent transcription in the pheochromocytoma cell line PC12. By using altered binding specificity mutants of transcription factors that bind to the SRE, it was demonstrated that in contrast to treatment with purified growth factors, such as nerve growth factor, the serum response factor (SRF), but not Elk-1, mediates Ca(2+)-regulated SRE-dependent transcription. Enhanced levels of cytoplasmic Ca2+ were found to trigger SRE-dependent transcription via a Ras-independent signaling pathway that appears to involve a Ca2+/calmodulin-dependent kinase (CaMK). Overexpression of a constitutively active form of CaMKIV stimulated SRF-dependent transcription. Taken together, these findings indicate that SRF is a versatile transcription factor that, when bound to the SRE, can function by distinct mechanisms and can mediate transcriptional responses to both CaMK- and Ras-dependent signaling pathways.
- Misra, R. P., Bonni, A., Miranti, C. K., Rivera, V. M., Sheng, M., & Greenberg, M. E. (1994). L-type voltage-sensitive calcium channel activation stimulates gene expression by a serum response factor-dependent pathway. The Journal of biological chemistry, 269(41), 25483-93.More infoA mechanism by which calcium-induced signals are transduced to the nucleus to activate transcription of the c-fos proto-oncogene has been characterized. The serum response element (SRE), a region of the c-fos gene which controls growth factor-induced transcription, is now shown to mediate c-fos transcription in response to activation of L-type voltage-sensitive calcium channels. Calcium-dependent transcriptional activation through the SRE is mediated by the serum response factor (SRF). Membrane depolarization induces phosphorylation of SRF at Ser-103, an event shown to enhance the ability of SRF to bind the SRE. Ca(2+)-induced SRF phosphorylation occurs via a pathway that may involve Ca2+/calmodulin-dependent kinases.
- Rivera, V. M., Miranti, C. K., Misra, R. P., Ginty, D. D., Chen, R. H., Blenis, J., & Greenberg, M. E. (1993). A growth factor-induced kinase phosphorylates the serum response factor at a site that regulates its DNA-binding activity. Molecular and cellular biology, 13(10), 6260-73.More infoA signaling pathway by which growth factors may induce transcription of the c-fos proto-oncogene has been characterized. Growth factor stimulation of quiescent fibroblasts activates a protein kinase cascade that leads to the rapid and transient phosphorylation of the serum response factor (SRF), a regulator of c-fos transcription. The in vivo kinetics of SRF phosphorylation and dephosphorylation parallel the activation and subsequent repression of c-fos transcription, suggesting that this phosphorylation event plays a critical role in the control of c-fos expression. The ribosomal S6 kinase pp90rsk, a growth factor-inducible kinase, phosphorylates SRF in vitro at serine 103, the site that becomes newly phosphorylated upon growth factor stimulation in vivo. Phosphorylation of serine 103 significantly enhances the affinity and rate with which SRF associates with its binding site, the serum response element, within the c-fos promoter. These results suggest a model in which the growth factor-induced phosphorylation of SRF at serine 103 contributes to the activation of c-fos transcription by facilitating the formation of an active transcription complex at the serum response element.
- Guitart, X., Thompson, M. A., Mirante, C. K., Greenberg, M. E., & Nestler, E. J. (1992). Regulation of cyclic AMP response element-binding protein (CREB) phosphorylation by acute and chronic morphine in the rat locus coeruleus. Journal of neurochemistry, 58(3), 1168-71.More infoPrevious studies have implicated adaptations in the cyclic AMP system in mechanisms of opiate tolerance, dependence, and withdrawal in the rat locus coeruleus. It has been speculated that such adaptations may occur at the level of gene expression. To understand better the mechanism by which opiates produce these intracellular adaptations, we studied morphine regulation of the state of phosphorylation of cyclic AMP response element-binding protein (CREB), a transcription factor that mediates some of the effects of the cyclic AMP system on gene expression. We show here, by use of a back phosphorylation and immunoprecipitation procedure, that acute morphine decreases the state of phosphorylation of CREB, an effect that becomes completely attenuated after chronic morphine administration. In contrast, acute precipitation of opiate withdrawal, via administration of an opiate receptor antagonist, increases the phosphorylation state of CREB. Such regulation of CREB phosphorylation could be part of the molecular pathway by which opiates produce changes in gene expression that lead to addiction.
- Miranti, C., & Puck, T. T. (1990). Gene regulation in reverse transformation: cyclic AMP-induced actin homolog in CHO cells. Somatic cell and molecular genetics, 16(1), 67-78.More infoReverse transformation (RT) presents a challenge in understanding of the role of protein-genome interaction in regulating gene expression in normal and transformed cells. Early during RT of CHO-K1 cells by cyclic AMP a new protein, mol wt = 43,000 and pI = 5.3 +/- 0.2, was rapidly and specifically induced. This cAMP-induced protein (CIP) is a phosphorylated actin homolog. Induction required new protein synthesis. Actinomycin D treatment failed to inhibit CIP induction, suggesting the existence of an untranslated or sequestered mRNA in untreated cells. Expression of CIP was not dependent upon cell shape or cytoskeletal integrity as are other steps in RT. CIP was detectable only in cAMP-treated cells, whether transformed or nontransformed, and cAMP treatment inhibited growth of both cell types. CIP was associated with soluble cell fractions and not with F-actin. We propose that CIP plays an early role in RT, that is necessary but not sufficient for the complete RT process, and that it participates in the cAMP signaling pathway of cells through changes in the cytoskeleton. This pathway inhibits cell growth as required in the differentiated phenotype. A molecular model is presented for the RT reaction in CHO-K1, which also explains cAMP effects on transformed cells such as the S49 lymphoma and other malignancies.
- Goren, M. B., Swendsen, C. L., Fiscus, J., & Miranti, C. (1984). Fluorescent markers for studying phagosome-lysosome fusion. Journal of leukocyte biology, 36(3), 273-92.More infoLysosomotropic fluorescent aminoacridines such as acridine orange and quinacrine have achieved prominence as markers for studying lysosome-phagosomes fusion, especially in macrophages. Experiments described demonstrate that because the aminoacridines traverse biological membranes with facility, they diffuse throughout the system, and ultimately accumulate intra- or extracellularly where they are most efficiently bound. Their presence or absence in phagosomes is therefore not unequivocally indicative of fusion or nonfusion. Alternative fluorescent lysosomal markers are described, and systems defined for which the aminoacridines may probably be used with confidence.
- Miranti, C. K., Zohar, Y., Kraft, A. S., Zohar, Y., Tran, M., Tahsin, S., Miranti, C. K., Kraft, A. S., Jiang, L., Ivich, F., & Frank, S. B. (2018). Application of a Microfluidic-Based Model of a Human Prostate Gland for Cancer Research. In 2018 IEEE 12th International Conference on Nano/Molecular Medicine and Engineering (NANOMED), 109-112.More infoA Microfluidic-based model of a human prostate gland has been developed featuring a 3D co-culture of epithelial and stromal cells. The model was used to investigate the effects of normal stroma on normal luminal epithelial cell differentiation, and to measure the ability of cancerous epithelium to convert normal stroma to cancer stroma. The ability of PIM1 kinase to induce cancer stroma was also tested in the model. The results demonstrate a promising potential of the model for cancer research applications.
- Watson, M., Winn, M. E., Miranti, C. K., Frank, S. B., & Berger, P. L. (2018). Abstract B031: CREB1 and ATF1 differentially regulate terminal prostate luminal differentiation by controlling the timing of ING4 expression, while CREB1 prevents ING4 expression upon PTEN loss in prostate cancer. In Poster Presentations - Proffered Abstracts.
- Miranti, C. (2019, Spring). Attacking the Tumor Microenvironment in Castration Resistant Prostate Cancer.. 3rd Prostate Cancer Workshop: Reducing the Burden of Bone Metastatic Prostate Cancer, San Diego, CA.
- Miranti, C. (2018, Fall). Attacking the Tumor Microenvironment in Castration Resistant Prostate Cancer.. Invited Speaker: 12th Prostate Cancer Symposium at Clark Atlanta University.
- Miranti, C. (2018, Fall). How and Why Integrin α6β1 Drives Prostate Cancer.. Seminar: Harvard Medical School, Dana Farber Cancer Institute.
- Miranti, C. (2018, Fall). Targeting the Tumor Microenvironment in Bone Metastatic Castration-Resistant Prostate Cancer.. Seminar: Purdue University, Cancer Center.
- Miranti, C. (2018, Spring). Attacking the Tumor Microenvironment in Drug-Resistant Prostate Cancer. Seminar: University of Washington, Spokane Medical School.
- Miranti, C. (2018, Spring). Targeting the Tumor Microenvironment in Bone Metastatic Castration-Resistant Prostate Cancer.. Seminar: University of West Virginia, Cancer Center.
- Miranti, C. (2018, Spring). Tetraspanin CD82 is Required for Normal Actin Dynamics and Efficient Bone Remodeling by Osteoclasts.. Invited Speaker: 9th International Tetraspanin Conference.
- Miranti, C. (2017, Apr 21). Pten Loss Promotes Prostate Cancer Tumorigenesis through Disruption of a CREB-Dependent Differentiation Pathway. University of Arizona Cancer Center Retreat. Tucson, AZ: UACC.
- Miranti, C. (2017, Mar 9-11). Notch3 Drives Osteoblastic PCa Bone Metastasis. 2nd Prostate Cancer Workshop: Reducing the Burden of Bone Metastatic Prostate Cancer. San Diego: UCSD.
- Miranti, C. (2017, Nov 13). Pten Loss Disrupts a CREB-Dependent Differentiation Pathway during Prostate Cancer Oncogenesis. Ventana Medical Systems Seminar Series. Oro Valley, AZ: Ventana Medical Systems.
- Miranti, C. (2017, Nov 9-12). Pten Loss Promotes Prostate Cancer Tumorigenesis through Disruption of a CREB-Dependent Differentiation Pathway.. SBUR Annual Meeting: Multiple Genetic and Epigenetic Mechanisms of Urologic Disease,. Tampa, FL: SBUR.
- Miranti, C. (2017, Sep 18). Attacking the Tumor Microenvironment in Drug-Resistant Prostate Cancer. CBIO Graduate Program Seminar. Tucson, AZ: CBIO Graduate Students.
- Miranti, C. (2016, Oct 8 - 9). Tetraspanin CD82 in Osteoclast and Osteoblast Differentiation in the Bone. Department of Cellular and Molecular Medicine Retreat. Oro Valley, AZ: CMM.
- Miranti, C. (2016, Sept 26-28). Pten and CREB Cooperativity during Prostate Luminal Epithelial Differentiation is Disrupted in Prostate Cancer. 11th Prostate Cancer Symposium at Clark Atlanta University. Altanta, GA: Clark University.
- Miranti, C. (2016, Sept 7-9). Defective Osteoclast Adhesion and Bone Degradation in CD82 null mice. 7th European Conference on Tetraspanins. Sheffield England.
- Miranti, C. (2019, Fall). Extranuclear Nucleolin induces ITGα6 expression in prostate cancer independent of androgen receptor.. SBUR Annual Meeting: Novel Discoveries in Urology, New Orleans, LA,.
- Miranti, C. (2019, Fall). GATA-2 and Twist-1 as targets of CREB-1 in prostate cancer development.. SBUR Annual Meeting: Novel Discoveries in Urology.
- Miranti, C. (2019, Fall). Loss of androgen receptor in prostate cancer stroma inhibits luminal epithelial cell differentiation.. SBUR Annual Meeting: Novel Discoveries in Urology, New Orleans, LA.
- Miranti, C. (2019, Fall). Notch3 promotes prostate cancer-induced bone lesion development by modulating the mone microenvironment via MMP-3.. SBUR Annual Meeting: Novel Discoveries in Urology.