Noel Andrew Warfel
- Associate Professor, Cellular and Molecular Medicine
- Member of the Graduate Faculty
- Associate Professor, Cancer Biology - GIDP
- Vice Chair, Cancer Biology - GIDP
2004 James Madison University
- BSc., Department of Integrated Science and Technology
2004 Johns Hopkins University
- MSc., Zanvyl Kreiger School of Arts and Science
2011 University of California, San Diego
- PhD., Biomedical Sciences graduate program
2015 – present Research Assistant Professor, University of Arizona
- Department of Cellular and Molecular Medicine
2013 – 2015 Research Assistant Professor, Medical University of South Carolina
- Joint appointment in the Hollings Cancer Center and Department of Biochemistry and Molecular Biology
2011 – 2013 Postdoctoral Fellow, Penn State University, HMC Cancer Institute
- Mentor: Dr. Wafik El-Deiry
2006 – 2011 Graduate Student, University of California, San Diego
- Mentor: Dr. Alexandra C. Newton
2004 – 2006 Graduate Research Fellow, National Cancer Insititute
- Mentor: Dr. Phillip Dennis
2017 American Lung Association Lung Cancer Discovery Award
2016 American Cancer Society Research Scholar Grant
2012 - 2014 Ruth L. Kirschstein National Research Service Award
- National Cancer Institute (1F32CA174138-01)
2009 – 2011 Predoctoral Traineeship Award
- DoD Breast Cancer Research Program (BC093021)
2009 Dissertation Award, CBCRP (Declined in favor of DoD award)
2006 – 2008 Pharmacology training grant, UCSD
- NIH (5 T32 GM07752-28)
2004 – 2006 Molecular Targets and Drug Discovery Fellowship
- Joint program - NIH/Johns Hopkins University
2013 AACR-Aflac Scholar in Training Award, AACR Annual Meeting
2012 Roland K. Robins Pharmacology Dissertation Award for best thesis, UCSD
2011 AACR Scholar in Training Award, Targeting PI3K/mTOR Signalling in Cancer
2004 Honors senior project in the department of ISAT, James Madison University
2015 – present University of Arizona Cancer Biology GIDP
2008 - present American Association for Cancer Research
2002 – 2006 Golden Key International Honor Society
SUPERVISION OF STUDENTS (2015-2016)
Supervision of 3 undergraduate students: Alva Sainz (entered Yale graduate program in biology fall of 2016), Isabella Brody-Calixito, and Ian Burton.
Supervision of 1 MSc student: Ashley Suiter
Supervision of 1 PhD student: Andrea Casillas
2016 - present UA COM MMI interviewer
2012 - present Peer reviewer for Cancer Biology and Therapy and Oncotarget
2009 Reviewer for Journal of Experimental & Clinical Cancer Research
2017 Poster presentation at AACR Prostate Cancer Meeting
2017 Invited speaker at Purdue University MCMP seminar series
2016 Poster presentation at 2016 AACR Annual Meeting
2016 Utilizing PIM Kinase inhibitors to target Nrf2-driven cancers. Invited Speaker, Pharmacology seminar series, Univ. of Arizona
2015 Targeting PIM Kinases to Overcome hypoxia-mediated therapeutic resistance. Invited Speaker, Cancer Biology seminar series, Univ. of Arizona Cancer Center
2014 Poster presentation at the 2014 Hollings Cancer Center Prostate Cancer Research Retreat
2013 Poster presentation at the 2013 AACR Annual Meeting
2013 Poster presentation at the 11th PSU postdoctoral society annual event
2012 Poster presentation at the 2012 AACR Annual Meeting
2011 Poster presentation at AACR PI3K/mTOR Signaling in Cancer meeting
2010 Invited Speaker at UCSD Biomedical Sciences annual graduate retreat
2010 Poster presentation at Salk Institute meeting on Protein Phosphorylation and Cell Signaling
2010 Poster presentation at CSHL PTEN and Pathways Meeting
2009 Poster presentation at 2009 AACR Annual Meeting
2008 Poster presentation at Salk meeting on protein phosphorylation
2008 Poster presentation at Experimental Biology Annual Meeting
2007 Invited Speaker at UCSD Biomedical Sciences annual graduate retreat
2006 Poster presentation at AACR Annual Meeting
PEER REVIEWED PUBLICATIONS
- N. A. Warfel, E. R. Lepper, C. Zhang, W. D. Figg, P. A. Dennis, Importance of the stress kinase p38alpha in mediating the direct cytotoxic effects of the thalidomide analogue, CPS49, in cancer cells and endothelial cells. Clin Cancer Res 12, 3502 (2006).
- C. A. Granville, N. Warfel, J. Tsurutani, M. C. Hollander, M. Robertson, S. D. Fox, T. D. Veenstra, H. J. Issaq, R. I. Linnoila, P. A. Dennis, Identification of a highly effective rapamycin schedule that markedly reduces the size, multiplicity, and phenotypic progression of tobacco carcinogen-induced murine lung tumors. Clin Cancer Res 13, 2281 (2007).
- J. J. Gills, S. S. Castillo, C. Zhang, P. A. Petukhov, R. M. Memmott, M. Hollingshead, N. Warfel, J. Han, A. P. Kozikowski, P. A. Dennis, Phosphatidylinositol ether lipid analogues that inhibit AKT also independently activate the stress kinase, p38alpha, through MKK3/6-independent and -dependent mechanisms. J Biol Chem 282, 27020 (2007).
- J. J. Gills, J. Lopiccolo, J. Tsurutani, R. H. Shoemaker, C. J. Best, M. S. Abu-Asab, J. Borojerdi, N. A. Warfel, E. R. Gardner, M. Danish, M. C. Hollander, S. Kawabata, M. Tsokos, W. D. Figg, P. S. Steeg, P. A. Dennis, Nelfinavir, A lead HIV protease inhibitor, is a broad-spectrum, anticancer agent that induces endoplasmic reticulum stress, autophagy, and apoptosis in vitro and in vivo. Clin Cancer Res 13, 5183 (2007).
- J. Brognard, M. Niederst, G. Reyes, N. Warfel, A. C. Newton, Common polymorphism in the phosphatase PHLPP2 results in reduced regulation of Akt and protein kinase C. J Biol Chem 284, 15215 (2009).
- N. A. Warfel, M. Niederst, M. W. Stevens, P. M. Brennan, M. C. Frame, A. C. Newton, Mislocalization of the E3 ligase, beta-transducin repeat-containing protein 1 (beta-TrCP1), in the pleckstrin homology domain leucine-rich repeat protein phosphatase 1 (PHLPP1) and Akt. J Biol Chem 286, 19777 (2011).
- N. A. Warfel, M. Niederst, A. C. Newton, Disruption of the interface between the PH and kinase domains of Akt is sufficient for hydrophobic motif site phosphorylation in the absence of mTORC2. J Biol Chem, (2011).
- N. A. Warfel, A. C. Newton, PH domain Leucine-rich Repeat Protein Phosphatase, PHLPP: a New Player in Cell Signaling. J Biol Chem, (2011).
- V.V. Prabhu, N.A. Warfel, W.S. El-Deiry, CTGF-mediated autophagy-senescence transition in tumor stroma promotes anabolic tumor growth and metastasis. Cell Cycle, (2012).
- N. A. Warfel, W. S. El-Deiry, p21WAF1 and tumourigenesis: 20 years after. Curr Opin Oncol 25, 52 (Jan, 2013).
- N.A. Warfel, N. Dolloff, Dicker D.T., J. Malysz, W. S. El-Diery, CDK1-mediated phosphorylation of Ser668 stabalizes HIF-1a to promote tumor growth. Cell Cycle (2013).
- N.A. Warfel and W.S. El-Deiry, HIF-1 Signaling in Drug Resistance to Chemotherapy. Curr Med Chem, (2014).
- N.A. Warfel and A.S. Kraft, PIM Kinase (and Akt) Biology and Signaling in Tumors. Pharmacology and Therapeutics, (2015).
- Zhang S., Zhou L., Hong B., van den Heuvel A.P., Prabhu V.V., Warfel N.A., Kline C.L., Dicker D.T., Kopelovich L., El-Deiry W.S. Small-Molecule NSC59984 Restores p53 Pathway Signaling and Antitumor Effects against Colorectal Cancer via p73 Activation and Degradation of Mutant p53. Cancer research, (2015)
- Song J.H., Padi S.K., Luevano L.A., Minden M.D., DeAngelo D.J., Hardiman G., Ball L.E., Warfel N.A., Kraft A.S. Insulin receptor substrate 1 is a substrate of the Pim protein kinases. Oncotarget. (2016).
- Warfel N.A*., Sainz A.G., Song J.H., Kraft A.S*. PIM Kinase Inhibitors Kill Hypoxic Tumor Cells by Reducing Nrf2 Signaling and Increasing Reactive Oxygen Species. Molecular Cancer Therapeutics. (2016). *co-corresponding authors.
- Warfel N.A. Targeting CDK4/6 to Oppose Hypoxia-Mediated Therapeutic Resistance. Cell Cycle, (2017).
- Casillas, A.L., Sainz, A.G., Toth, R.K., Singh, N., Desai, A., Kraft, A.S., and Warfel, N.A. Hypoxia-inducible PIM Kinase expression promotes resistance to anti-angiogenic agents. Clinical Cancer Research, (2018).
- Ph.D. Biomedical Sciences
- University of California, San Diego, La Jolla, California, United States
- Regulation of the PHLPP Phosphatases and Akt Signaling
- M.S. Biotechnology
- Johns Hopkins University, Baltimore, Maryland, United States
- Medical University of South Carolina (2013 - 2015)
- Penn State Hershey Cancer Institute (2011 - 2013)
- Certificate in Translational Medicine
- Eureka Institute for Translational Medicine, Spring 2019
In addition to my passion for research, I have taken advantage of the opportunity to serve as a mentor to clinical fellows, undergraduate, and graduate students. Since arriving at Univ. of Arizona, I have served as a mentor to Alva Sainz, a talented undergraduate in the UROC program. Her work will be included in two manuscripts, and she has recently been accepted into several graduate programs to study cancer biology. In addition, I had my first ABBS student, Andrea Casillas, rotate in my lab this winter. Her rotation project was to generate and characterize a somatic mutation in HIF-1 that is widely observed in clinical samples. In addition to my training, I have learned from mentoring students in my own lab. As a result, I have confidence in my ability to effectively train students in both the conceptual and technical aspects of how to study cancer biology through hypothesis driven science. I am extremely interested in serving as a mentor to train the next generation of scientist in cancer biology. In the coming year I hope to recruit a graduate student into my lab and train another undergraduate.
My laboratory concentrates on understanding the complex biological mechanisms that allow cancer cells to thrive in the hypoxic tumor microenvironment, with a focus on understanding how we can exploit such pathways to oppose the oncogenic properties of tumor hypoxia. In particular, we are currently focused on understanding how the Proviral Integration site for Moloney murine leukemia virus (PIM) kinases regulation of tumor angiogenesis and response to therapy. This research will be the first to describe a novel signaling pathway discovered in our lab in which PIM kinases control the amplitude of hypoxia-inducible factor-1 (HIF-1) signaling. Our working model places PIM kinases upstream of HIF-1 in the cellular response to hypoxia, making it a promising target for hypoxia-targeted therapy. We have developed innovative biochemical and imaging techniques to understand how aberrant PIM expression/activity impacts tumor angiogenesis and resistance to therapy in vitro and in vivo. Of particular significance, our preliminary data provide the rationale for a novel therapeutic strategy combining small molecule PIM kinase inhibitors and anti-angiogenic drugs that has the potential to change how we treat hypoxic tumors. My training is in two areas: cancer biology, focused on signal transduction, and drug development, focused on identifying and exploiting hypoxia in solid tumors; these skills remain pillars of my current research program studying prostate cancer biology and barriers to its effective treatment. My background greatly influences the approach I am currently taking to investigate the the cellular response to hypoxia, and this work will distinguish my laboratory as we pursue biological avenues that may have been overlooked by others.
Cancer BiologyCBIO 552 (Fall 2023)
DissertationCBIO 920 (Fall 2023)
Honors ThesisBIOC 498H (Fall 2023)
Prin of Cell BiologyCMM 577 (Fall 2023)
Prin of Cell BiologyMCB 577 (Fall 2023)
ResearchCBIO 900 (Fall 2023)
Research ConferenceCBIO 695A (Fall 2023)
Cell Biology of DiseaseCMM 404 (Summer I 2023)
Cell Biology of DiseaseCMM 504 (Summer I 2023)
CBIO GIDP Seminar SeriesCBIO 596H (Spring 2023)
Directed ResearchMCB 792 (Spring 2023)
DissertationCBIO 920 (Spring 2023)
ResearchCBIO 900 (Spring 2023)
Research ConferenceCBIO 695A (Spring 2023)
ThesisCMM 910 (Spring 2023)
Cancer BiologyCBIO 552 (Fall 2022)
DissertationCBIO 920 (Fall 2022)
Prin of Cell BiologyCMM 577 (Fall 2022)
Prin of Cell BiologyMCB 577 (Fall 2022)
ResearchCBIO 900 (Fall 2022)
Research ConferenceCBIO 695A (Fall 2022)
ThesisCMM 910 (Fall 2022)
Cell Biology of DiseaseCMM 504 (Summer I 2022)
CBIO GIDP Seminar SeriesCBIO 596H (Spring 2022)
Directed ResearchMCB 792 (Spring 2022)
DissertationCBIO 920 (Spring 2022)
Research ConferenceCBIO 695A (Spring 2022)
ThesisCMM 910 (Spring 2022)
Cancer BiologyCBIO 552 (Fall 2021)
Directed ResearchMCB 792 (Fall 2021)
DissertationCBIO 920 (Fall 2021)
Honors Independent StudyPSIO 499H (Fall 2021)
Prin of Cell BiologyCMM 577 (Fall 2021)
Prin of Cell BiologyMCB 577 (Fall 2021)
Research ConferenceCBIO 695A (Fall 2021)
ThesisCMM 910 (Fall 2021)
Cell Biology of DiseaseCMM 404 (Summer I 2021)
Cell Biology of DiseaseCMM 504 (Summer I 2021)
CBIO GIDP Seminar SeriesCBIO 596H (Spring 2021)
Directed ResearchMCB 792 (Spring 2021)
DissertationCBIO 920 (Spring 2021)
ResearchCBIO 900 (Spring 2021)
Research ConferenceCBIO 695A (Spring 2021)
ThesisCMM 910 (Spring 2021)
Cancer BiologyCBIO 552 (Fall 2020)
DissertationCBIO 920 (Fall 2020)
Prin of Cell BiologyCMM 577 (Fall 2020)
Prin of Cell BiologyMCB 577 (Fall 2020)
ResearchCBIO 900 (Fall 2020)
Research ConferenceCBIO 695A (Fall 2020)
ThesisCMM 910 (Fall 2020)
Cell Biology of DiseaseCMM 504 (Summer I 2020)
Adv Topics in Cancer BiologyCBIO 553 (Spring 2020)
CBIO GIDP Seminar SeriesCBIO 596H (Spring 2020)
DissertationCBIO 920 (Spring 2020)
Research ConferenceCBIO 695A (Spring 2020)
ThesisCMM 910 (Spring 2020)
Cancer BiologyCBIO 552 (Fall 2019)
DissertationCBIO 920 (Fall 2019)
Introduction to ResearchMCB 795A (Fall 2019)
Prin of Cell BiologyCMM 577 (Fall 2019)
Prin of Cell BiologyMCB 577 (Fall 2019)
Research ConferenceCBIO 695A (Fall 2019)
ThesisCMM 910 (Fall 2019)
Cell Biology of DiseaseCMM 404 (Summer I 2019)
Cell Biology of DiseaseCMM 504 (Summer I 2019)
ThesisCMM 910 (Summer I 2019)
CBIO GIDP Seminar SeriesCBIO 596H (Spring 2019)
ResearchCBIO 900 (Spring 2019)
Research ConferenceCBIO 695A (Spring 2019)
ThesisCMM 910 (Spring 2019)
Cancer BiologyCBIO 552 (Fall 2018)
DissertationCBIO 920 (Fall 2018)
Prin of Cell BiologyCMM 577 (Fall 2018)
Prin of Cell BiologyMCB 577 (Fall 2018)
ResearchCBIO 900 (Fall 2018)
Research ConferenceCBIO 695A (Fall 2018)
Adv Topics in Cancer BiologyCBIO 553 (Spring 2018)
ResearchCBIO 900 (Spring 2018)
Research ConferenceCBIO 695A (Spring 2018)
Cancer BiologyCBIO 552 (Fall 2017)
Introduction to ResearchMCB 795A (Fall 2017)
ResearchCBIO 900 (Fall 2017)
Research ConferenceCBIO 695A (Fall 2017)
ResearchCBIO 900 (Spring 2017)
Research ConferenceCBIO 695A (Spring 2017)
ResearchCBIO 900 (Fall 2016)
Research ConferenceCBIO 695A (Fall 2016)
ThesisCMM 910 (Fall 2016)
CBIO GIDP Seminar SeriesCBIO 596H (Spring 2016)
Directed RsrchMCB 492 (Spring 2016)
- Hill, S. M., Padilla-Rodriguez, M., Clements, A., Sweetland, J. A., Parker, S. S., Warfel, N. A., & Mouneimne, G. (2022). Optimized in vitro three-dimensional invasion assay for quantifying a wide range of cancer cell invasive behavior. STAR protocols, 3(3), 101516.More infoWe describe a three-dimensional (3D) in vitro assay for quantifying cancer cell invasion into a 3D microenvironment with defined biochemical and biophysical properties. Researchers can quantify invasion dynamics (e.g., cell motility and directionality) and examine morphological changes during invasion, using live-cell and confocal imaging techniques. Together, these advantages over existing in vitro invasion assays, such as transwell-based assays, provide researchers with a valuable tool to gain insight into the mechanisms regulating cancer cell invasion. For complete details on the use and execution of this protocol, please refer to Padilla-Rodriguez et al. (2018) and Watson et al. (2021).
- Warfel, N. A. (2022). Defining the mechanisms underlying cyclin dependent kinase control of HIF-1α. Oncotarget, 13(1), 454-455. doi:10.18632/oncotarget.28208
- Warfel, N. A., & Chauhan, S. S. (2022). Targeting mitochondrial dynamics to overcome therapeutic resistance. The Applied Biology & Chemistry Journal, 1-3. doi:10.52679/tabcj.2022.0001
- Warfel, N. A., Hernandez-Cortes, D., Gard, J. M., Knudsen, B. S., & Cress, A. E. (2022). Abstract 3835: Kindlin-2 complexes containing α6β1 integrin are responsive to hypoxia. Cancer Research, 82(12_Supplement), 3835-3835. doi:10.1158/1538-7445.am2022-3835
- 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.
- Cress, A. E., Warfel, N. A., Hernandez-Cortes, D., & Knudsen, B. S. (2021). Abstract LB256: Dynamic kindlin-2 complexes containing a laminin-binding integrin are responsive to hypoxia. Cancer Research, 81(13_Supplement), LB256-LB256. doi:10.1158/1538-7445.am2021-lb256
- Singh, N., Ramnarine, V. R., Song, J. H., Pandey, R., Padi, S. K., Nouri, M., Olive, V., Kobelev, M., Okumura, K., McCarthy, D., Hanna, M. M., Mukherjee, P., Sun, B., Lee, B. R., Parker, J. B., Chakravarti, D., Warfel, N. A., Zhou, M., Bearss, J. J., , Gibb, E. A., et al. (2021). The long noncoding RNA H19 regulates tumor plasticity in neuroendocrine prostate cancer. Nature communications, 12(1), 7349.More infoNeuroendocrine (NE) prostate cancer (NEPC) is a lethal subtype of castration-resistant prostate cancer (PCa) arising either de novo or from transdifferentiated prostate adenocarcinoma following androgen deprivation therapy (ADT). Extensive computational analysis has identified a high degree of association between the long noncoding RNA (lncRNA) H19 and NEPC, with the longest isoform highly expressed in NEPC. H19 regulates PCa lineage plasticity by driving a bidirectional cell identity of NE phenotype (H19 overexpression) or luminal phenotype (H19 knockdown). It contributes to treatment resistance, with the knockdown of H19 re-sensitizing PCa to ADT. It is also essential for the proliferation and invasion of NEPC. H19 levels are negatively regulated by androgen signaling via androgen receptor (AR). When androgen is absent SOX2 levels increase, driving H19 transcription and facilitating transdifferentiation. H19 facilitates the PRC2 complex in regulating methylation changes at H3K27me3/H3K4me3 histone sites of AR-driven and NEPC-related genes. Additionally, this lncRNA induces alterations in genome-wide DNA methylation on CpG sites, further regulating genes associated with the NEPC phenotype. Our clinical data identify H19 as a candidate diagnostic marker and predictive marker of NEPC with elevated H19 levels associated with an increased probability of biochemical recurrence and metastatic disease in patients receiving ADT. Here we report H19 as an early upstream regulator of cell fate, plasticity, and treatment resistance in NEPC that can reverse/transform cells to a treatable form of PCa once therapeutically deactivated.
- Toth, R. K., & Warfel, N. A. (2021). Targeting PIM Kinases to Overcome Therapeutic Resistance in Cancer. Molecular cancer therapeutics, 20(1), 3-10.More infoCancer progression and the onset of therapeutic resistance are often the results of uncontrolled activation of survival kinases. The proviral integration for the Moloney murine leukemia virus (PIM) kinases are oncogenic serine/threonine kinases that regulate tumorigenesis by phosphorylating a wide range of substrates that control cellular metabolism, proliferation, and survival. Because of their broad impact on cellular processes that facilitate progression and metastasis in many cancer types, it has become clear that the activation of PIM kinases is a significant driver of resistance to various types of anticancer therapies. As a result, efforts to target PIM kinases for anticancer therapy have intensified in recent years. Clinical and preclinical studies indicate that pharmacologic inhibition of PIM has the potential to significantly improve the efficacy of standard and targeted therapies. This review focuses on the signaling pathways through which PIM kinases promote cancer progression and resistance to therapy, as well as highlights biological contexts and promising strategies to exploit PIM as a therapeutic target in cancer.
- Warfel, N. A., & Toth, R. K. (2021). Targeting PIM Kinases to Overcome Therapeutic Resistance in Cancer. Molecular Cancer Therapeutics, 20(1), 3-10. doi:10.1158/1535-7163.mct-20-0535
- Casillas, A. L., Jensen, C. C., Warfel, N. A., Warfel, N. A., Toth, R. K., Chauhan, S. S., Casillas, A. L., Jensen, C. C., Warfel, N. A., Toth, R. K., & Chauhan, S. S. (2020). Abstract 1482: PIM1 promotes angiogenesis via phosphorylation and stabilization of HIF-1α. Cancer Research, 80(16_Supplement), 1482-1482. doi:10.1158/1538-7445.am2020-1482More infoAn essential process for the growth and dissemination of solid tumors is angiogenesis, or the formation of new blood vessels. Overexpression of Proviral Integration site for Moloney murine leukemia virus (PIM)-1, a serine-threonine kinase, has been implicated as a driver of aggressive prostate and colon cancer. PIM1 is known to promote tumor growth and survival, and we recently uncovered a role for PIM1 in promoting resistance to anti-angiogenic agents. Here, we identify a novel signaling pathway that directly links PIM to tumor angiogenesis. Immunohistochemical staining of prostate cancer tissue revealed a significant positive correlation between PIM1 and CD31 expression, a marker of endothelial cells. Using prostate cancer as a model, in vitro models of endothelial tube formation and DCE-MRI confirmed that PIM1 overexpression increases tumor angiogenesis. Gene expression analysis of PIM1 overexpressing cells showed that PIM kinases regulates the expression levels of many hypoxia inducible factor-1α (HIF-1α) target genes in normoxic conditions. In vitro, in vivo and kinase assays discovered a novel phosphorylation site within the oxygen-dependent degradation domain (ODDD) of HIF-1α that is a direct target of PIM1. Biochemical analysis demonstrates that phosphorylation of this site increases HIF-1α stability by decreasing PHD binding, hydroxylation, and subsequent proteasomal degradation of HIF-1α. Two CRISPR-generated cell lines with point mutations that mimic phosphorylation of this site were used in xenograft models of colon cancer to confirm the significance of this site to the identified PIM-HIF signaling and tumor angiogenesis. Importantly, we show that the anti-angiogenic and cytotoxic effects of PIM inhibitors are largely dependent on their ability to downregulate HIF-1α signaling. In summary, we uncovered a novel role for PIM1 in promoting tumor angiogenesis and identified a novel phosphorylation site that controls the stability of HIF-1α in normoxic and hypoxic conditions.This research yields important insight into the role of PIM signaling in solid tumors and provides preclinical evidence that to improve the translation of PIM kinase inhibitors in solid tumors. Citation Format: Andrea L. Casillas, Shailender S. Chauhan, Rachel K. Toth, Corbin C. Jensen, Noel A. Warfel. PIM1 promotes angiogenesis via phosphorylation and stabilization of HIF-1α [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1482.
- Chauhan, S. S., Toth, R. K., Jensen, C. C., Casillas, A. L., Kashatus, D. F., & Warfel, N. A. (2020). PIM kinases alter mitochondrial dynamics and chemosensitivity in lung cancer. Oncogene, 39(12), 2597-2611.More infoResistance to chemotherapy represents a major obstacle to the successful treatment of non-small-cell lung cancer (NSCLC). The goal of this study was to determine how PIM kinases impact mitochondrial dynamics, ROS production, and response to chemotherapy in lung cancer. Live-cell imaging and microscopy were used to determine the effect of PIM loss or inhibition on mitochondrial phenotype and ROS. Inhibition of PIM kinases caused excessive mitochondrial fission and significant upregulation of mitochondrial superoxide, increasing intracellular ROS. Mechanistically, we define a signaling axis linking PIM1 to Drp1 and mitochondrial fission in lung cancer. PIM inhibition significantly increased the protein levels and mitochondrial localization of Drp1, causing marked fragmentation of mitochondria. An inverse correlation between PIM1 and Drp1 was confirmed in NSCLC patient samples. Inhibition of PIM sensitized NSCLC cells to chemotherapy and produced a synergistic antitumor response in vitro and in vivo. Immunohistochemistry and transmission electron microscopy verified that PIM inhibitors promote mitochondrial fission and apoptosis in vivo. These data improve our knowledge about how PIM1 regulates mitochondria and provide justification for combining PIM inhibition with chemotherapy in NSCLC.
- Jensen, C. C., Warfel, N., Toth, R. K., Sainz, A. G., Miranti, C. K., Langlais, P. R., Cress, A. E., Clements, A. N., Chauhan, S. S., & Casillas, A. L. (2020). Direct Phosphorylation and Stabilization of HIF-1α By PIM1 Kinase Drives Angiogenesis in Solid Tumors. SSRN Electronic Journal. doi:10.2139/ssrn.3710750
- Warfel, N. A., Cress, A. E., Batai, K., Lee, B. R., Wong, A. C., Warfel, N. A., Pollock, G. R., Marr*, K., Marr, K. D., Lee, B. R., Ignatenko, N., Cress, A. E., & Batai, K. (2020). MP09-07 USE OF 3D VIDEO MICROSCOPY OF HUMAN-DERIVED PROSTATE ORGANOIDS TO ASSESS FEATURES OF EARLY INVASIVE PROSTATE CANCER. The Journal of Urology, 203, e118. doi:10.1097/ju.0000000000000829.07More infoINTRODUCTION AND OBJECTIVE:Patient-derived organoids provide a method for creating human prostate cancer cell lines in culture in three dimensions. We established living organoids using normal and ...
- Harryman, W. L., Warfel, N. A., Nagle, R. B., & Cress, A. E. (2019). The Tumor Microenvironments of Lethal Prostate Cancer. Advances in experimental medicine and biology, 1210, 149-170.More infoLocalized prostate cancer (confined to the gland) generally is considered curable, with nearly a 100% 5-year-survival rate. When the tumor escapes the prostate capsule, leading to metastasis, there is a poorer prognosis and higher mortality rate, with 5-year survival dropping to less than 30%. A major research question has been to understand the transition from indolent (low risk) disease to aggressive (high risk) disease. In this chapter, we provide details of the changing tumor microenvironments during prostate cancer invasion and their role in the progression and metastasis of lethal prostate cancer. Four microenvironments covered here include the muscle stroma, perineural invasion, hypoxia, and the role of microvesicles in altering the extracellular matrix environment. The adaptability of prostate cancer to these varied microenvironments and the cues for phenotypic changes are currently understudied areas. Model systems for understanding smooth muscle invasion both in vitro and in vivo are highlighted. Invasive human needle biopsy tissue and mouse xenograft tumors both contain smooth muscle invasion. In combination, the models can be used in an iterative process to validate molecular events for smooth muscle invasion in human tissue. Understanding the complex and interacting microenvironments in the prostate holds the key to early detection of high-risk disease and preventing tumor invasion through escape from the prostate capsule.
- 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.
- 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.
- Warfel, N. A., Nagle, R. B., Marr, K. D., Hinton, J. P., McGrath, J. E., Wang, M., Rubenstein, C. S., Gard, J. M., Ingabire, N., Simpson, S. J., Miranti, C. K., 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. doi:10.1158/0008-5472.can-19-0868
- Casillas, A. L., Toth, R. K., Sainz, A. G., Singh, N., Desai, A. A., Kraft, A. S., & Warfel, N. A. (2018). Hypoxia-Inducible PIM Kinase Expression Promotes Resistance to Antiangiogenic Agents. Clinical cancer research : an official journal of the American Association for Cancer Research, 24(1), 169-180.More infoPatients develop resistance to antiangiogenic drugs, secondary to changes in the tumor microenvironment, including hypoxia. PIM kinases are prosurvival kinases and their expression increases in hypoxia. The goal of this study was to determine whether targeting hypoxia-induced PIM kinase expression is effective in combination with VEGF-targeting agents. The rationale for this therapeutic approach is based on the fact that antiangiogenic drugs can make tumors hypoxic, and thus more sensitive to PIM inhibitors. Xenograft and orthotopic models of prostate and colon cancer were used to assess the effect of PIM activation on the efficacy of VEGF-targeting agents. IHC and imaging were used to analyze angiogenesis, apoptosis, proliferation, and metastasis. Biochemical studies were performed to characterize the novel signaling pathway linking PIM and HIF1. PIM was upregulated following treatment with anti-VEGF therapies, and PIM1 overexpression reduced the ability of these drugs to disrupt vasculature and block tumor growth. PIM inhibitors reduced HIF1 activity, opposing the shift to a pro-angiogenic gene signature associated with hypoxia. Combined inhibition of PIM and VEGF produced a synergistic antitumor response characterized by decreased proliferation, reduced tumor vasculature, and decreased metastasis. This study describes PIM kinase expression as a novel mechanism of resistance to antiangiogenic agents. Our data provide justification for combining PIM and VEGF inhibitors to treat solid tumors. The unique ability of PIM inhibitors to concomitantly target HIF1 and selectively kill hypoxic tumor cells addresses two major components of tumor progression and therapeutic resistance. .
- Chauhan, S. S., & Warfel, N. A. (2018). Targeting PIM kinases to oppose hypoxia-mediated therapeutic resistance. Oncoscience, 5(9-10), 254-255.
- Song, J. H., Singh, N., Luevano, L. A., Padi, S. K., Okumura, K., Olive, V., Black, S. M., Warfel, N. A., Goodrich, D. W., & Kraft, A. S. (2018). Mechanisms Behind Resistance to PI3K Inhibitor Treatment Induced by the PIM Kinase. Molecular cancer therapeutics, 17(12), 2710-2721.More infoCancer resistance to PI3K inhibitor therapy can be in part mediated by increases in the PIM1 kinase. However, the exact mechanism by which PIM kinase promotes tumor cell resistance is unknown. Our study unveils the pivotal control of redox signaling by PIM kinases as a driver of this resistance mechanism. PIM1 kinase functions to decrease cellular ROS levels by enhancing nuclear factor erythroid 2-related factor 2 (NRF2)/antioxidant response element activity. PIM prevents cell death induced by PI3K-AKT-inhibitory drugs through a noncanonical mechanism of NRF2 ubiquitination and degradation and translational control of NRF2 protein levels through modulation of eIF4B and mTORC1 activity. Importantly, PIM also controls NAD(P)H production by increasing glucose flux through the pentose phosphate shunt decreasing ROS production, and thereby diminishing the cytotoxicity of PI3K-AKT inhibitors. Treatment with PIM kinase inhibitors reverses this resistance phenotype, making tumors increasingly susceptible to small-molecule therapeutics, which block the PI3K-AKT pathway.
- Warfel, N. A., Black, S. M., Song, J. H., Singh, N., Luevano, L. A., Padi, S. K., Okumura, K., Olive, V., Goodrich, D. W., & Kraft, A. S. (2018). Mechanisms Behind Resistance to PI3K Inhibitor Treatment Induced by the PIM Kinase. Molecular Cancer Therapeutics, 17(12), 2710-2721. doi:10.1158/1535-7163.mct-18-0374
- Warfel, N. A., Desai, A. A., Casillas, A. L., Toth, R. K., Sainz, A. G., Singh, N., & Kraft, A. S. (2018). Hypoxia-Inducible PIM Kinase Expression Promotes Resistance to Antiangiogenic Agents. Clinical Cancer Research, 24(1), 169-180. doi:10.1158/1078-0432.ccr-17-1318
- Toth, R. K., & Warfel, N. A. (2017). Strange Bedfellows: Nuclear Factor, Erythroid 2-Like 2 (Nrf2) and Hypoxia-Inducible Factor 1 (HIF-1) in Tumor Hypoxia. Antioxidants (Basel, Switzerland), 6(2).More infoThe importance of the tumor microenvironment for cancer progression and therapeutic resistance is an emerging focus of cancer biology. Hypoxia, or low oxygen, is a hallmark of solid tumors that promotes metastasis and represents a significant obstacle to successful cancer therapy. In response to hypoxia, cancer cells activate a transcriptional program that allows them to survive and thrive in this harsh microenvironment. Hypoxia-inducible factor 1 (HIF-1) is considered the main effector of the cellular response to hypoxia, stimulating the transcription of genes involved in promoting angiogenesis and altering cellular metabolism. However, growing evidence suggests that the cellular response to hypoxia is much more complex, involving coordinated signaling through stress response pathways. One key signaling molecule that is activated in response to hypoxia is nuclear factor, erythroid 2 like-2 (Nrf2). Nrf2 is a transcription factor that controls the expression of antioxidant-response genes, allowing the cell to regulate reactive oxygen species. Nrf2 is also activated in various cancer types due to genetic and epigenetic alterations, and is associated with poor survival and resistance to therapy. Emerging evidence suggests that coordinated signaling through Nrf2 and HIF-1 is critical for tumor survival and progression. In this review, we discuss the distinct and overlapping roles of HIF-1 and Nrf2 in the cellular response to hypoxia, with a focus on how targeting Nrf2 could provide novel chemotherapeutic modalities for treating solid tumors.
- Warfel, N. A. (2017). Targeting CDK4/6 to oppose hypoxia-mediated therapeutic resistance. Cell Cycle, 16(13), 1241-1242. doi:10.1080/15384101.2017.1337975
- Warfel, N. A. (2017). Targeting CDK4/6 to oppose hypoxia-mediated therapeutic resistance. Cell cycle (Georgetown, Tex.), 16(13), 1241-1242.
- Kraft, A. S., Song, J. H., Warfel, N. A., & Sainz, A. G. (2016). PIM Kinase Inhibitors Kill Hypoxic Tumor Cells by Reducing Nrf2 Signaling and Increasing Reactive Oxygen Species. Molecular Cancer Therapeutics, 15(7), 1637-1647. doi:10.1158/1535-7163.mct-15-1018
- Song, J. H., Kraft, A. S., Warfel, N. A., Warfel, N. A., Warfel, N. A., Song, J. H., Sainz, A. G., & Kraft, A. S. (2016). Abstract LB-024: PIM kinase inhibitors selectively kill hypoxic cancer cells by reducing Nrf2 activity and increasing reactive oxygen species. Cancer Research, 76(14_Supplement), LB-024-LB-024. doi:10.1158/1538-7445.am2016-lb-024More infoProstate cancer (PCa) is the leading cause of cancer death in men worldwide. Patients diagnosed with metastatic prostate cancer initially respond to androgen deprivation therapy, but this response is not durable, and all patients develop hormone-refractory disease that ultimately leads to death. Current treatment options for metastatic PCa patients are limited (e.g., cytotoxic chemotherapy or radiation), and the response to these agents is invariably temporary and does not improve survival. A significant obstacle to the successful treatment of advanced PCa is tumor hypoxia. While hypoxia is toxic to normal cells, cancer cells take particular advantage of their ability to adapt to these harsh conditions by increasing the expression of proteins that promote angiogenesis and survival. Therefore, elucidating the molecular mechanisms controlling survival in hypoxia is important for the development of effective therapeutic strategies to selectively kill hypoxic tumor cells. The Proviral Integration site for Moloney murine leukemia virus (PIM) kinases represent a pro-survival pathawy that is upregulated in response to hypoxia in a HIF-1 independent manner. We demonstrate that pharmacological or genetic inhibition of PIM kinases is significantly more toxic toward cancer cells in hypoxia versus normoxia. Xenograft studies confirm that PIM kinase inhibitors impede tumor growth and selectively kill hypoxic tumor cells in vivo. Experiments show that PIM kinases enhance the ability of tumor cells to adapt to hypoxia-induced oxidative stress by increasing the nuclear localization and activity of Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2), which functions to increase the expression of antioxidant genes. Small molecule PIM kinase inhibitors reduce the ability of Nrf2 to enter the nucleus and block the transcrption of cytoprotective genes, leading to the accumulation of intracellular reactive oxygen species (ROS) to toxic levels in hypoxic tumor cells. This toxic effect of PIM inhibitors can be succesfully blocked by ROS scavengers, including N-acetyl cystine and superoxide dismutase. Thus, inhibition of PIM kinases has the potential to oppose hypoxia-mediate therapeutic resistance and induce cell death in the hypoxic tumor microenvironment. Citation Format: Alva G. Sainz, Jin H. Song, Andrew S. Kraft, Noel A. Warfel. PIM kinase inhibitors selectively kill hypoxic cancer cells by reducing Nrf2 activity and increasing reactive oxygen species. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-024.
- Song, J. H., Padi, S. K., Luevano, L. A., Minden, M. D., DeAngelo, D. J., Hardiman, G., Ball, L. E., Warfel, N. A., & Kraft, A. S. (2016). Insulin receptor substrate 1 is a substrate of the Pim protein kinases. Oncotarget, 7(15), 20152-65.More infoThe Pim family of serine/threonine protein kinases (Pim 1, 2, and 3) contribute to cellular transformation by regulating glucose metabolism, protein synthesis, and mitochondrial oxidative phosphorylation. Drugs targeting the Pim protein kinases are being tested in phase I/II clinical trials for the treatment of hematopoietic malignancies. The goal of these studies was to identify Pim substrate(s) that could help define the pathway regulated by these enzymes and potentially serve as a biomarker of Pim activity. To identify novel substrates, bioinformatics analysis was carried out to identify proteins containing a consensus Pim phosphorylation site. This analysis identified the insulin receptor substrate 1 and 2 (IRS1/2) as potential Pim substrates. Experiments were carried out in tissue culture, animals, and human samples from phase I trials to validate this observation and define the biologic readout of this phosphorylation. Our study demonstrates in both malignant and normal cells using either genetic or pharmacological inhibition of the Pim kinases or overexpression of this family of enzymes that human IRS1S1101 and IRS2S1149 are Pim substrates. In xenograft tumor experiments and in a human phase I clinical trial, a pan-Pim inhibitor administered in vivo to animals or humans decreased IRS1S1101 phosphorylation in tumor tissues. This phosphorylation was shown to have effects on the half-life of the IRS family of proteins, suggesting a role in insulin or IGF signaling. These results demonstrate that IRS1S1101 is a novel substrate for the Pim kinases and provide a novel marker for evaluation of Pim inhibitor therapy.
- Warfel, N. A., Sainz, A. G., Song, J. H., & Kraft, A. S. (2016). PIM Kinase Inhibitors Kill Hypoxic Tumor Cells by Reducing Nrf2 Signaling and Increasing Reactive Oxygen Species. Molecular cancer therapeutics, 15(7), 1637-47.More infoIntratumoral hypoxia is a significant obstacle to the successful treatment of solid tumors, and it is highly correlated with metastasis, therapeutic resistance, and disease recurrence in cancer patients. As a result, there is an urgent need to develop effective therapies that target hypoxic cells within the tumor microenvironment. The Proviral Integration site for Moloney murine leukemia virus (PIM) kinases represent a prosurvival pathway that is upregulated in response to hypoxia, in a HIF-1-independent manner. We demonstrate that pharmacologic or genetic inhibition of PIM kinases is significantly more toxic toward cancer cells in hypoxia as compared with normoxia. Xenograft studies confirm that PIM kinase inhibitors impede tumor growth and selectively kill hypoxic tumor cells in vivo Experiments show that PIM kinases enhance the ability of tumor cells to adapt to hypoxia-induced oxidative stress by increasing the nuclear localization and activity of nuclear factor-erythroid 2 p45-related factor 2 (Nrf2), which functions to increase the expression of antioxidant genes. Small molecule PIM kinase inhibitors prevent Nrf2 from accumulating in the nucleus, reducing the transcription of cytoprotective genes and leading to the build-up of intracellular reactive oxygen species (ROS) to toxic levels in hypoxic tumor cells. This toxic effect of PIM inhibitors can be successfully blocked by ROS scavengers, including N-acetyl cystine and superoxide dismutase. Thus, inhibition of PIM kinases has the potential to oppose hypoxia-mediated therapeutic resistance and induce cell death in the hypoxic tumor microenvironment. Mol Cancer Ther; 15(7); 1637-47. ©2016 AACR.
- Warfel, N. A., Song, J. H., Padi, S. K., Luevano, L. A., Minden, M. D., DeAngelo, D. J., Hardiman, G., Ball, L. E., & Kraft, A. S. (2016). Insulin receptor substrate 1 is a substrate of the Pim protein kinases. Oncotarget, 7(15), 20152-20165. doi:10.18632/oncotarget.7918
- Warfel, N. A., & Kraft, A. S. (2015). PIM kinase (and Akt) biology and signaling in tumors. Pharmacology & therapeutics, 151, 41-9.More infoThe initiation and progression of human cancer is frequently linked to the uncontrolled activation of survival kinases. Two such pro-survival kinases that are commonly amplified in cancer are PIM and Akt. These oncogenic proteins are serine/threonine kinases that regulate tumorigenesis by phosphorylating substrates that control the cell cycle, cellular metabolism, proliferation, and survival. Growing evidence suggests that cross-talk exists between the PIM and Akt kinases, indicating that they control partially overlapping survival signaling pathways that are critical to the initiation, progression, and metastatic spread of many types of cancer. The PI3K/Akt signaling pathway is activated in many human tumors, and it is well established as a promising anticancer target. Likewise, based on the role of PIM kinases in normal and tumor tissues, it is clear that this family of kinases represents an interesting target for anticancer therapy. Pharmacological inhibition of PIM has the potential to significantly influence the efficacy of standard and targeted therapies. This review focuses on the regulation of PIM kinases, their role in tumorigenesis, and the biological impact of their interaction with the Akt signaling pathway on the efficacy of cancer therapy.
- Zhang, S., Zhou, L., Hong, B., van den Heuvel, A. P., Prabhu, V. V., Warfel, N. A., Kline, C. L., Dicker, D. T., Kopelovich, L., & El-Deiry, W. S. (2015). Small-Molecule NSC59984 Restores p53 Pathway Signaling and Antitumor Effects against Colorectal Cancer via p73 Activation and Degradation of Mutant p53. Cancer research, 75(18), 3842-52.More infoThe tumor-suppressor p53 prevents cancer development via initiating cell-cycle arrest, cell death, repair, or antiangiogenesis processes. Over 50% of human cancers harbor cancer-causing mutant p53. p53 mutations not only abrogate its tumor-suppressor function, but also endow mutant p53 with a gain of function (GOF), creating a proto-oncogene that contributes to tumorigenesis, tumor progression, and chemo- or radiotherapy resistance. Thus, targeting mutant p53 to restore a wild-type p53 signaling pathway provides an attractive strategy for cancer therapy. We demonstrate that small-molecule NSC59984 not only restores wild-type p53 signaling, but also depletes mutant p53 GOF. NSC59984 induces mutant p53 protein degradation via MDM2 and the ubiquitin-proteasome pathway. NSC59984 restores wild-type p53 signaling via p73 activation, specifically in mutant p53-expressing colorectal cancer cells. At therapeutic doses, NSC59984 induces p73-dependent cell death in cancer cells with minimal genotoxicity and without evident toxicity toward normal cells. NSC59984 synergizes with CPT11 to induce cell death in mutant p53-expressing colorectal cancer cells and inhibits mutant p53-associated colon tumor xenograft growth in a p73-dependent manner in vivo. We hypothesize that specific targeting of mutant p53 may be essential for anticancer strategies that involve the stimulation of p73 in order to efficiently restore tumor suppression. Taken together, our data identify NSC59984 as a promising lead compound for anticancer therapy that acts by targeting GOF-mutant p53 and stimulates p73 to restore the p53 pathway signaling.
- Warfel, N. A., Warfel, N. A., & El-deiry, W. S. (2014). HIF-1 signaling in drug resistance to chemotherapy.. Current medicinal chemistry, 21(26), 3021-8. doi:10.2174/0929867321666140414101056More infoActivation of hypoxia-inducible factor 1 (HIF-1) signaling is observed in a broad range of human cancers due to tumor hypoxia and epigenetic mechanisms. HIF-1 activation leads to the transcription of a plethora of target genes that promote physiological changes associated with therapeutic resistance, including the inhibition of apoptosis and senescence and the activation of drug efflux and cellular metabolism. As a result, targeting HIF-1 represents an attractive strategy to enhance the efficacy of current therapies as well as reduce resistance to chemotherapy in tumors. Approaches to inhibit HIF-1 signaling have primarily focused on reducing HIF-1α protein levels, by inducing its degradation or inhibiting its transcription, inhibiting HIF-1-mediated transcription, or disrupting the formation of the HIF-1 transcription factor complex. To date, multiple preclinical and clinical agents have been identified that effectively inhibit HIF-1 activity through various mechanisms, likely accounting for a portion of their anti-tumor efficacy. This review aims to provide an overview of our current understanding of the role of HIF-1 in therapeutic resistance and discuss the ongoing effort to develop HIF-1 inhibitors as an anti-cancer strategy.
- Staveley-o'carroll, K. F., Kaifi, J. T., Warfel, N. A., Zheng, S., Warfel, N. A., Staveley-o'carroll, K. F., Kaifi, J. T., Harouaka, R., El-deiry, W. S., Dicker, D. T., & Das, A. (2013). Abstract 1452: Perioperative detection of circulating tumor cells in patients undergoing colorectal cancer liver and lung metastasectomy : Comparing CellSearch and FMSA technologies and investigating the role of EMT .. Cancer Research, 73, 1452-1452. doi:10.1158/1538-7445.am2013-1452More infoProceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Circulating tumor cells (CTCs) reflect disease burden in metastatic colorectal cancer (mCRC) and patients with resectable liver and lung metastases are potentially curable by surgery with a 5-year survival of 30% . Here we report a study where we are detecting CTCs from blood of such patients collected perioperatively, using simultaneously the FDA-approved Veridex CellSearch and the Flexible Micro Spring Array (FMSA) device developed by Penn State Bioengineering and have accrued 12 patients so far. 7.5 ml of blood is collected from the central venous catheter placed during surgery at incision, at the point of starting surgical resection of the mass, 30 minutes after complete removal of the mass and on the day after surgery. Variation in CTC counts between samples from the same patient is expected to provide valuable answers about the kinetics of tumor cell shedding and dissemination during surgery. Tissue from resected liver or lung is appropriately stored for benchtop studies to correlate molecular signatures with CTC counts. Preliminary results suggest a mean count of 36 cells per blood sample detected using FMSA technology, with counts varying between zero to 101 cells / 7.5 ml of blood, in contrast to only zero to 2 cells in similar samples detected using the CellSearch method. FMSA relies on size-based isolation of CTCs from blood which are identified as CK+, DAPI +, CD45- cells on further immunostaining .The trial provides an opportunity to compare the two technologies in an important clinical setting and understand the underlying molecular mechanisms for the difference in counts . Aggressive tumors have been hypothesized to shed cells that have undergone EMT and thus evade detection by the EpCAM-based CellSearch detection method . We have noted expression of the mesenchymal marker vimentin in the CTCs captured by FMSA device thus documenting EMT . We plan to do additional stem cell and prognostic marker analysis on FMSA-captured CTCs . Both liver and lung metastases are being studied to compare two organs with different vascular anatomies and divergent surgical techniques. Correlating cell counts with disease-free survival as well as relapse will be helpful in validating intra-operative CTC isolation technique as means of prognosticating post-surgical recovery and gain insights into link between possible vascular tumor cell shedding and the surgical and vascular ligation technique that is followed. Future directions include comparing results with a patient population undergoing hepatic or pulmonary surgical procedures for non-malignant disease as well as extend the trial to include patients who are candidates for adjuvant chemotherapy and explore the role of intraoperative CTC counts and marker expression in choice of regime and predicting response. Citation Format: Avisnata Das, David T. Dicker, Ramdane Harouaka, Siyang Zheng, Noel Warfel, Kevin F. Staveley-O'Carroll, Wafik S. El-Deiry, Jussuf T. Kaifi. Perioperative detection of circulating tumor cells in patients undergoing colorectal cancer liver and lung metastasectomy : Comparing CellSearch and FMSA technologies and investigating the role of EMT . [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 1452. doi:10.1158/1538-7445.AM2013-1452
- Warfel, N. A., Warfel, N. A., & El-deiry, W. S. (2013). p21WAF1 and tumourigenesis: 20 years after.. Current opinion in oncology, 25(1), 52-8. doi:10.1097/cco.0b013e32835b639eMore infoThis review provides an overview of the structure, regulation and physiological functions of p21, the product of the cyclin-dependent kinase inhibitor 1A (CDKN1A) gene, with a focus on its dual role in promoting and repressing biological processes that are hallmarks of tumourigenesis..Recent work has provided a better understanding of the molecular mechanisms of how oncogenic signalling pathways influence p21 expression. In response to cellular stimuli, p21 expression is tightly regulated at transcriptional and post-translational levels through mechanisms involving RNA stabilization, phosphorylation and ubiquitination. As a result, growing evidence reveals that several important tumour suppressor and oncogenic signalling pathways alter p21 expression to elicit their effects on cell cycle progression and survival. Thus, p21 expression can both promote and inhibit tumourigenic processes, depending on the cellular context..Since its discovery, it has become increasingly clear that p21 can function as both a classical tumour suppressor and an oncogene. In order to effectively utilize p21 as a therapeutic target, it will be necessary to design therapeutic strategies that preferentially block the ability of p21 to promote senescence, stem cell renewal and cyclin/CDK activation, while leaving its tumour suppressive functions intact.
- Warfel, N. A., Warfel, N. A., Malysz, J., El-deiry, W. S., Dolloff, N. G., & Dicker, D. T. (2013). CDK1 stabilizes HIF-1α via direct phosphorylation of Ser668 to promote tumor growth.. Cell cycle (Georgetown, Tex.), 12(23), 3689-701. doi:10.4161/cc.26930More infoHypoxia-inducible factor 1 (HIF-1) is a major mediator of tumor physiology, and its activation is correlated with tumor progression, metastasis, and therapeutic resistance. HIF-1 is activated in a broad range of solid tumors due to intratumoral hypoxia or genetic alterations that enhance its expression or inhibit its degradation. As a result, decreasing HIF-1α expression represents an attractive strategy to sensitize hypoxic tumors to anticancer therapies. Here, we show that cyclin-dependent kinase 1 (CDK1) regulates the expression of HIF-1α, independent of its known regulators. Overexpression of CDK1 and/or cyclin B1 is sufficient to stabilize HIF-1α under normoxic conditions, whereas inhibition of CDK1 enhances the proteasomal degradation of HIF-1α, reducing its half-life and steady-state levels. In vitro kinase assays reveal that CDK1 directly phosphorylates HIF-1α at a previously unidentified regulatory site, Ser668. HIF-1α is stabilized under normoxic conditions during G 2/M phase via CDK1-mediated phosphorylation of Ser668. A phospho-mimetic construct of HIF-1α at Ser668 (S668E) is significantly more stable under both normoxic and hypoxic conditions, resulting in enhanced transcription of HIF-1 target genes and increased tumor cell invasion and migration. Importantly, HIF-1α (S668E) displays increased tumor angiogenesis, proliferation, and tumor growth in vivo compared with wild-type HIF-1α. Thus, we have identified a novel link between CDK1 and HIF-1α that provides a potential molecular explanation for the elevated HIF-1 activity observed in primary and metastatic tumors, independent of hypoxia, and offers a molecular rationale for the clinical translation of CDK inhibitors for use in tumors with constitutively active HIF-1.
- Warfel, N. A., Warfel, N. A., Zhou, L., Zhou, L., Zhang, S., Zhang, S., Warfel, N. A., Warfel, N. A., Kopelovich, L., Kopelovich, L., Hong, B., Hong, B., Heuvel, A. V., Heuvel, A. V., El-deiry, W. S., El-deiry, W. S., Dicker, D. T., & Dicker, D. T. (2013). Abstract 2171: A new small molecule compound restoring p53 pathway induces cell death via active p73 and degradation of mutant p53 in colorectal cancer cells.. Cancer Research, 73, 2171-2171. doi:10.1158/1538-7445.am2013-2171More infoProceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Tumor suppressor p53 is critical for protection against cancer. Over 50% of human cancers harbor mutant p53 which is hyper-stabilized and blocks p53 family member p73/p63 activity. Mutations not only abrogate the p53 tumor suppressor function, but also endow mutant p53 with gain-of-function (GOF) as pro-oncogene which contributes to tumor progression and chemotherapy resistance. Targeting mutant p53 to restore the p53 pathway is a promising strategy for cancer therapy. Small molecule restoring p53 pathway is mostly based on conformational change in mutant p53 or upregulation of wild-type p53. It is barely reported about small molecule compound with ability to both restore p53 pathway and deplete mutant p53 GOF. In this study, we describe a new small molecular compound, NCI-8 that specifically depletes mutant p53 protein and activates p73 to induce p53 responsive transcriptional activity and cell death in mutant p53 colorectal cancer cells. NCI-8 treatment increased p53 responsive bioluminescence and p53 target gene expression such as P21, Puma and DR5 specifically in mutant p53 colorectal cancer cells. Accompanied with p53 pathway restoration, NCI-8 down-regulated mutant p53 in cancer cells with no evident wild-type conformational change in mutant p53. NCI-8- mediated down-regulation of mutant p53 was rescued by MG132, a proteasome inhibitor and nutlin-3, an MDM2 inhibitor. NCI-8 induced ubiquitination of mutant p53. Taken together these results indicate that NCI-8 induces mutant p53 protein degradation via an MDM2-mediated ubiquitin-proteasome pathway. Further, we examined the role of p73 in NCI-8 action to p53 pathway restoration. NCI-8 enhanced p53-responsive bioluminescence with10-fold increase in cells overexpressing p73, but only 2-fold increase in p53-overexpressing cells. NCI-8- induced p53-responsive bioluminescence was significantly reduced by knock-down of p73, but not by knock-down of mutant p53. Knock-down of p73 significantly reduced NCI-8 induction of p53 target genes in mutant p53 cancer cells. These results indicate that p73 is required for NCI-8 to restore the p53 pathway in mutant p53-expressing cancer cells. Importantly, NCI-8 induced cell death in cancer cells, but not in normal cells. No genotoxicity of NCI-8 was detected in cancer and normal cells. Knock-down of p73 reduced cell apoptosis in mutant p53-expressing cancer cells treated with NCI-8, while, overexpression of p73 sensitized these cancer cells to NCI-8 induced cell death. Combination treatments demonstrate that NCI-8 synergizes with CPT11chemotherapy to induce cell death in mutant p53 colorectal cancer cells. These results suggest that p53 pathway restoration and cell death induced by NCI-8 occur via activation of p73 and depletion of mutant p53 GOF. NCI-8 is a promising lead compound specifically targeting mutant p53 for the development of new anti-cancer drugs. Citation Format: Shengliang Zhang, Lanlan Zhou, Bo Hong, Noel Warfel, Antonius Van den heuvel, David Dicker, Levy Kopelovich, Wafik El-Deiry. A new small molecule compound restoring p53 pathway induces cell death via active p73 and degradation of mutant p53 in colorectal cancer cells. [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 2171. doi:10.1158/1538-7445.AM2013-2171
- Warfel, N. A., Newton, A. C., Warfel, N. A., & Newton, A. C. (2012). Pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP): a new player in cell signaling.. The Journal of biological chemistry, 287(6), 3610-6. doi:10.1074/jbc.r111.318675More infoPrecise balance between phosphorylation, catalyzed by protein kinases, and dephosphorylation, catalyzed by protein phosphatases, is essential for cellular homeostasis. Deregulation of this balance leads to pathophysiological states that drive diseases such as cancer, heart disease, and diabetes. The recent discovery of the PHLPP (pleckstrin homology domain leucine-rich repeat protein phosphatase) family of Ser/Thr phosphatases adds a new player to the cast of phosphate-controlling enzymes in cell signaling. PHLPP isozymes catalyze the dephosphorylation of a conserved regulatory motif, the hydrophobic motif, on the AGC kinases Akt, PKC, and S6 kinase, as well as an inhibitory site on the kinase Mst1, to inhibit cellular proliferation and induce apoptosis. The frequent deletion of PHLPP in cancer, coupled with the development of prostate tumors in mice lacking PHLPP1, identifies PHLPP as a novel tumor suppressor. This minireview discusses the structure, function, and regulation of PHLPP, with particular focus on its role in disease.
- Warfel, N. A., Warfel, N. A., Prabhu, V. V., & El-deiry, W. S. (2012). CTGF-mediated autophagy-senescence transition in tumor stroma promotes anabolic tumor growth and metastasis.. Cell cycle (Georgetown, Tex.), 11(14), 2592-3. doi:10.4161/cc.21240More infoComment on: Capparelli C, et al. Cell Cycle 2012; 11:2272-84 and Capparelli C, et al. Cell Cycle 2012; 11:2285-302.
- Warfel, N. A., Newton, A. C., Warfel, N. A., Niederst, M., & Newton, A. C. (2011). Disruption of the interface between the pleckstrin homology (PH) and kinase domains of Akt protein is sufficient for hydrophobic motif site phosphorylation in the absence of mTORC2.. The Journal of biological chemistry, 286(45), 39122-9. doi:10.1074/jbc.m111.278747More infoThe pro-survival kinase Akt requires phosphorylation at two conserved residues, the activation loop site (Thr-308) and the hydrophobic motif site (Ser-473), for maximal activation. Previous reports indicate that mTORC2 is necessary for phosphorylation of the hydrophobic motif and that this site is not phosphorylated in cells lacking components of the mTORC2 complex, such as Sin1. Here we show that Akt can be phosphorylated at the hydrophobic motif site (Ser-473) in the absence of mTORC2. First, increasing the levels of PIP(3) in Sin1(-/-) MEFs by (i) expression of a constitutively active PI3K or (ii) relief of a negative feedback loop on PI3K by prolonged inhibition of mTORC1 or S6K is sufficient to rescue hydrophobic motif phosphorylation of Akt. The resulting accumulation of PIP(3) at the plasma membrane results in Ser-473 phosphorylation. Second, constructs of Akt in which the PH domain is constitutively disengaged from the kinase domain are phosphorylated at the hydrophobic motif site in Sin1(-/-) MEFs; both myristoylated-Akt and Akt lacking the PH domain are phosphorylated at Ser-473. Thus, disruption of the interface between the PH and kinase domains of Akt bypasses the requirement for mTORC2. In summary, these data support a model in which Akt can be phosphorylated at Ser-473 and activated in the absence of mTORC2 by mechanisms that depend on removal of the PH domain from the kinase domain.
- Warfel, N. A., Newton, A. C., Warfel, N. A., Stevens, M. W., Niederst, M. J., Newton, A. C., Frame, M. C., & Brennan, P. M. (2011). Mislocalization of the E3 ligase, β-transducin repeat-containing protein 1 (β-TrCP1), in glioblastoma uncouples negative feedback between the pleckstrin homology domain leucine-rich repeat protein phosphatase 1 (PHLPP1) and Akt.. The Journal of biological chemistry, 286(22), 19777-88. doi:10.1074/jbc.m111.237081More infoThe PH domain leucine-rich repeat protein phosphatase, PHLPP, plays a central role in controlling the amplitude of growth factor signaling by directly dephosphorylating and thereby inactivating Akt. The cellular levels of PHLPP1 have recently been shown to be enhanced by its substrate, activated Akt, via modulation of a phosphodegron recognized by the E3 ligase β-TrCP1, thus providing a negative feedback loop to tightly control cellular Akt output. Here we show that this feedback loop is lost in aggressive glioblastoma but not less aggressive astrocytoma. Overexpression and pharmacological studies reveal that loss of the feedback loop does not result from a defect in PHLPP1 protein or in the upstream kinases that control its phosphodegron. Rather, the defect arises from altered localization of β-TrCP1; in astrocytoma cell lines and in normal brain tissue the E3 ligase is predominantly cytoplasmic, whereas in glioblastoma cell lines and patient-derived tumor neurospheres, the E3 ligase is confined to the nucleus and thus spatially separated from PHLPP1, which is cytoplasmic. Restoring the localization of β-TrCP1 to the cytosol of glioblastoma cells rescues the ability of Akt to regulate PHLPP1 stability. Additionally, we show that the degradation of another β-TrCP1 substrate, β-catenin, is impaired and accumulates in the cytosol of glioblastoma cell lines. Our findings reveal that the cellular localization of β-TrCP1 is altered in glioblastoma, resulting in dysregulation of PHLPP1 and other substrates such as β-catenin.
- Newton, A. C., Warfel, N. A., Warfel, N. A., Reyes, G., Niederst, M. J., Newton, A. C., & Brognard, J. (2009). Common polymorphism in the phosphatase PHLPP2 results in reduced regulation of Akt and protein kinase C.. The Journal of biological chemistry, 284(22), 15215-23. doi:10.1074/jbc.m901468200More infoPHLPP2 (PH domain leucine-rich repeat protein phosphatase 2) terminates Akt and protein kinase C (PKC) activity by specifically dephosphorylating these kinases at a key regulatory site, the hydrophobic motif (Ser-473 in Akt1). Here we identify a polymorphism that results in an amino acid change from a Leu to Ser at codon 1016 in the phosphatase domain of PHLPP2, which reduces phosphatase activity toward Akt both in vitro and in cells, in turn resulting in reduced apoptosis. Depletion of endogenous PHLPP2 variants in breast cancer cells revealed the Ser-1016 variant is less functional toward both Akt and PKC. In pair-matched high grade breast cancer samples we observed retention of only the Ser allele from heterozygous patients (identical results were observed in a pair-matched normal and tumor cell line). Thus, we have identified a functional polymorphism that impairs the activity of PHLPP2 and correlates with elevated Akt phosphorylation and increased PKC levels.
- Dennis, P. A., Warfel, N. A., Warfel, N. A., Veenstra, T. D., Tsurutani, J., Robertson, M., Linnoila, R. I., Issaq, H. J., Hollander, M. C., Granville, C. A., & Fox, S. D. (2007). Identification of a highly effective rapamycin schedule that markedly reduces the size, multiplicity, and phenotypic progression of tobacco carcinogen-induced murine lung tumors.. Clinical cancer research : an official journal of the American Association for Cancer Research, 13(7), 2281-9. doi:10.1158/1078-0432.ccr-06-2570More infoHuman and murine preneoplastic lung lesions induced by tobacco exposure are characterized by increased activation of the Akt/mammalian target of rapamycin (mTOR) pathway, suggesting a role for this pathway in lung cancer development. To test this, we did studies with rapamycin, an inhibitor of mTOR, in A/J mice that had been exposed to the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)..Tumorigenesis was induced by i.p. injection of NNK, and rapamycin was administered 1 or 26 weeks after NNK administration. Biomarkers associated with mTOR inhibition were assessed in lung and/or surrogate tissues using immunohistochemistry and immunoblotting. Rapamycin levels were measured using mass spectroscopy..Rapamycin was administered on a daily (5 of 7 days) regimen beginning 26 weeks after NNK decreased tumor size, proliferative rate, and mTOR activity. Multiplicity was not affected. Comparing this regimen with an every-other-day (qod) regimen revealed that rapamycin levels were better maintained with qod administration, reaching a nadir of 16.4 ng/mL, a level relevant in humans. When begun 1 week after NNK, this regimen was well tolerated and decreased tumor multiplicity by 90%. Tumors that did develop showed decreased phenotypic progression and a 74% decrease in size that correlated with decreased proliferation and inhibition of mTOR..Tobacco carcinogen-induced lung tumors in A/J mice are dependent upon mTOR activity because rapamycin markedly reduced the development and growth of tumors. Combined with the Food and Drug Administration approval of rapamycin and broad clinical experience, these studies provide a rationale to assess rapamycin in trials with smokers at high risk to develop lung cancer.
- Dennis, P. A., Warfel, N. A., Zhang, C., Warfel, N. A., Petukhov, P. A., Memmott, R. M., Kozikowski, A. P., Hollingshead, M. G., Han, J., Gills, J. J., & Castillo, S. S. (2007). Phosphatidylinositol ether lipid analogues that inhibit AKT also independently activate the stress kinase, p38alpha, through MKK3/6-independent and -dependent mechanisms.. The Journal of biological chemistry, 282(37), 27020-27029. doi:10.1074/jbc.m701108200More infoPreviously, we identified five active phosphatidylinositol ether lipid analogues (PIAs) that target the pleckstrin homology domain of Akt and selectively induce apoptosis in cancer cells with high levels of Akt activity. To examine specificity, PIAs were screened against a panel of 29 purified kinases. No kinase was inhibited, but one isoform of p38, p38alpha, was uniformly activated 2-fold. Molecular modeling of p38alpha revealed the presence of two regions that could interact with PIAs, one in the activation loop and a heretofore unappreciated region in the upper lobe that resembles a pleckstrin homology domain. In cells, two phases of activation were observed, an early phase that was independent of the upstream kinase MKK3/6 and inhibited by the p38 inhibitor SB203580 and a latter phase that was coincident with MKK3/6 activation. In short term xenograft experiments that employed immunohistochemistry and immunoblotting, PIA administration increased phosphorylation of p38 but not MKK3/6 in tumors in a statistically significant manner. Although PIAs rapidly activated p38 with similar time and dose dependence as Akt inhibition, p38 activation and Akt inhibition were independent events induced by PIAs. Using SB203580 or p38alpha(-/-) cells, we showed that p38alpha is not required for PIA-induced apoptosis but is required for H(2)O(2)- and anisomycin-induced apoptosis. Nonetheless, activation of p38a contributes to PIA-induced apoptosis, because reconstitution of p38a into p38alpha(-/-) cells increased apoptosis. These studies indicate that p38alpha is activated by PIAs through a novel mechanism and show that p38alpha activation contributes to PIA-induced cell death. Independent modulation of Akt and p38alpha could account for the profound cytotoxicity of PIAs.
- Warfel, N. A., Dennis, P. A., Warfel, N. A., Tsurutani, J., Tsokos, M., Steeg, P. S., Shoemaker, R. H., Lopiccolo, J., Kawabata, S., Hollander, M. C., Gills, J. J., Gardner, E. R., Figg, W. D., Danish, M., Borojerdi, J. P., Best, C. J., & Abu-asab, M. S. (2007). Nelfinavir, A lead HIV protease inhibitor, is a broad-spectrum, anticancer agent that induces endoplasmic reticulum stress, autophagy, and apoptosis in vitro and in vivo.. Clinical cancer research : an official journal of the American Association for Cancer Research, 13(17), 5183-94. doi:10.1158/1078-0432.ccr-07-0161More infoThe development of new cancer drugs is slow and costly. HIV protease inhibitors are Food and Drug Administration approved for HIV patients. Because these drugs cause toxicities that can be associated with inhibition of Akt, an emerging target in cancer, we assessed the potential of HIV protease inhibitors as anticancer agents..HIV protease inhibitors were screened in vitro using assays that measure cellular proliferation, apoptotic and nonapoptotic cell death, endoplasmic reticulum (ER) stress, autophagy, and activation of Akt. Nelfinavir was tested in non-small cell lung carcinoma (NSCLC) xenografts with biomarker assessment..Three of six HIV protease inhibitors, nelfinavir, ritonavir, and saquinavir, inhibited proliferation of NSCLC cells, as well as every cell line in the NCI60 cell line panel. Nelfinavir was most potent with a mean 50% growth inhibition of 5.2 micromol/L, a concentration achievable in HIV patients. Nelfinavir caused two types of cell death, caspase-dependent apoptosis and caspase-independent death that was characterized by induction of ER stress and autophagy. Autophagy was protective because an inhibitor of autophagy increased nelfinavir-induced death. Akt was variably inhibited by HIV protease inhibitors, but nelfinavir caused the greatest inhibition of endogenous and growth factor-induced Akt activation. Nelfinavir decreased the viability of a panel of drug-resistant breast cancer cell lines and inhibited the growth of NSCLC xenografts that was associated with induction of ER stress, autophagy, and apoptosis..Nelfinavir is a lead HIV protease inhibitor with pleiotropic effects in cancer cells. Given its wide spectrum of activity, oral availability, and familiarity of administration, nelfinavir is a Food and Drug Administration-approved drug that could be repositioned as a cancer therapeutic.
- Warfel, N. A., Dennis, P. A., Warfel, N. A., Veenstra, T. D., Tsurutani, J., Linnoila, I., Isaaq, H. J., Granville, C. A., & Fox, S. D. (2006). Identification of a highly effective rapamycin schedule that markedly reduces the size, multiplicity and phenotypic progression of tobacco carcinogen-induced murine lung tumors.. Cancer Epidemiology and Prevention Biomarkers, 15.More infoFifth AACR International Conference on Frontiers in Cancer Prevention Research, Nov 12-15, 2006 A77 Human and murine pre-neoplastic lung lesions induced by tobacco exposure are characterized by increased activation of the kinases Akt and mTOR, suggesting that activation of this pathway is important for the development of lung cancer. To test this hypothesis, we administered rapamycin, an inhibitor of mTOR, to A/J mice that had been exposed to the tobacco carcinogen, NNK. When rapamycin was given on a daily 5 of 7 day regimen, mTOR activity was inhibited. Tumor size but not multiplicity was reduced. To optimize rapamycin delivery, the pharmacokinetics and pharmacodynamics of a daily 5/7 regimen were compared to an every-other-day regimen. Immunohistochemical analysis of S6 phosphorylation in lung tissue from this study revealed that although mTOR was inhibited equally immediately after rapamycin delivery, recovery of mTOR activity was different with the two regimens. With the daily 5/7 regimen, mTOR activity completely recovered after the 72 hour rest-period, which corresponded to blood levels of rapamycin that were undetectable. With every-other-day rapamycin, recovery of S6 phosphorylation was incomplete after the washout period, and rapamycin levels in this group nadired at 16.4 ng/ml, which is comparable to levels reached in humans. These studies suggested that an every-other-day regimen might be more effective than a daily 5/7 regimen. To test this, every-other-day rapamycin (1.5 mg/kg) was administered one week after NNK exposure, prior to tumor development. Rapamycin was continued for 12 wk and was well tolerated. At 16 wk, rapamycin reduced the multiplicity of tobacco-carcinogen induced tumors by 90%. Tumors that did develop showed decreased phenotypic progression and size that was associated with a decreased rate of proliferation. Inhibition of tumorigenesis by rapamycin was associated with mTOR inhibition in lung tumors, lung tissues, and surrogate tissues. These studies identify a highly effective treatment schedule of rapamycin that markedly reduced the burden of tobacco carcinogen-induced lung tumors. Given the FDA-approval of rapamycin to prevent rejection of kidney transplants and the broad clinical experience with rapamycin, these studies provide a strong rationale to pursue prevention trials with rapamycin in smokers at high risk to develop lung cancer.
- Warfel, N. A., Dennis, P. A., Zhang, C., Warfel, N. A., Lepper, E. R., & Figg, W. D. (2006). Importance of the stress kinase p38alpha in mediating the direct cytotoxic effects of the thalidomide analogue, CPS49, in cancer cells and endothelial cells.. Clinical cancer research : an official journal of the American Association for Cancer Research, 12(11 Pt 1), 3502-9. doi:10.1158/1078-0432.ccr-05-1837More infoThalidomide has gained renewed interest as a cancer therapeutic due to its potential antiangiogenic effects. The thalidomide analogues CPS11 and CPS49 are active in preclinical angiogenesis assays and xenograft model systems, but the biochemical basis for these observations is unclear..To address this question, we assessed the toxicity of these thalidomide analogues in cancer cells, endothelial cells, and genetically modified cells using assays that measure apoptotic and nonapoptotic cell death. Phosphospecific and native antibodies were used in immunoblotting and immunohistochemical experiments to assess the activation states of kinases that control cellular survival in vitro and in vivo..CPS49 predominantly induced nonapoptotic cell death in lung cancer cells, prostate cancer cells, and endothelial cells in a dose-dependent manner, whereas CPS11 was not cytotoxic. CPS49 did not inhibit kinases that promote survival, such as Akt or extracellular signal-regulated kinase, but rather rapidly activated the stress kinase p38 pathway in both cancer cells and endothelial cells. CPS49 activated p38 in tumor xenografts. Using p38alpha-/- cells or an inhibitor of p38, we show that the presence and activation of p38alpha is important for cytotoxicity in all cell types examined..Our studies identify a unifying mechanism of action for cytotoxicity of the tetraflourinated thalidomide analogue, CPS49, and suggest that activation of p38 could serve as a biomarker in clinical trials with CPS49.
- Jensen, C. C., Warfel, N. A., Warfel, N. A., Toth, R. K., Kashatus, D. F., Chauhan, S. S., & Casillas, A. L. (2020). Abstract 6332: Pim kinase inhibition promotes mitochondrial fission to combat chemoresistance in non-small cell lung cancer. In Experimental and Molecular Therapeutics, 80, 6332-6332.
- Sainz, A. G., Song, J., Kraft, A., & Warfel, N. A. (2016, 07-15-2016). PIM kinase inhibitors selectively kill hypoxic cancer cells by reducing Nrf2 activity and increasing reactive oxygen species. In AACR Annual Meeting, Cancer Research, Volume 76, Issue 14.