Ghassan Mouneimne

Interests

Research

My laboratory concentrates on understanding how the organization of the actin cytoskeleton regulates cellular behavior and how aberrations in this organization lead to grave consequences, such as cancer cell invasion and ultimately tumor metastasis. We are investigating estrogen receptor (ER) regulation of invasion of ER positive breast cancer by focusing on hormonal regulation of the actin cytoskeletal architecture. These studies foreground the unexpected alterations in the actin architecture caused by suppressing ER transcriptional activity and how these alterations promote cellular changes, such as hyperactive protrusions, which promote invasion. My expertise is in two areas: analytical cell biology, focused on quantitative cellular microscopy, and cancer biology, focused on invasion and metastasis of breast cancer. My background greatly influences the approach I am currently taking to investigate the invasive behavior of breast tumors and distinguishes my laboratory by being adherent to a highly quantitative methodology that will allow us to identify even subtle changes in cellular behavior. Such analytical approaches are crucial when studying invasion of cancer cells since even subtle changes might have significant effects on disease progression – which is something that we found to be true in many cancer models in vivo and in vitro. In addition, my current position at the University of Arizona Cancer Center (UACC) allows me to develop a translational research program through the collaborative interactions with resident basic and clinical researchers.

Teaching

I believe that teaching is one of our basic responsibilities as scientists, which is to nurture young analytical minds and help them grow towards fulfilling their highest potential, academically and intellectually.As a student at the American University of Beirut (AUB), I began to appreciate the impact a strong teaching program has on developing students. The American University is one of the leading educational institutions in the Middle East. Based on the American liberal arts model of higher education, AUB is a teaching-centered research university. At AUB, I was a teaching assistant for an entry-level general biology course and an advanced cell biology course, offered to sophomore and senior students, respectively. My roles in these classes included writing exam questions, giving lectures, and running laboratory sections and discussion groups. These two courses exposed me to two different types of experiences in teaching: helping sophomores develop their interests horizontally by exposing them to different scientific areas; and enticing seniors to develop their learning vertically by getting a deeper understanding of a specific area of research, including conducting short experimental projects.As a graduate student and postdoctoral fellow at Albert Einstein College of Medicine and Harvard Medical School, respectively, I mentored a number of graduate and undergraduate students. Working with these students was a symbiotic experience. On the one hand, I contributed to the students’ research projects by helping them develop and execute their ideas. While on the other hand, I honed my skills as a mentor by developing the ability to build an individual relationship with each student based on his/her competence and general interests, learning how to guide students through the different phases of scientific discovery - especially the unrewarding ones - and acquiring a deep appreciation of being part of a dynamic research team.

Courses

Scholarly Contributions

Journals/Publications

• Charest, P. G., Mouneimne, G., Werner, A. N., Collins, S. E., Wiegand, M. E., Brown, I. N., Mundo, M. I., & Swango, D. J. (2023). Ras-mediated activation of mTORC2 promotes breast epithelial cell migration and invasion. Molecular Biology of the Cell, 34(2). doi:10.1091/mbc.e22-06-0236
• Collins, S. E., Werner, A. N., Mouneimne, G., Charest, P. G., Mundo, I. M., Brown, I. N., & Wiegand, M. E. (2022). Abstract 2416: Ras-mediated activation of mTORC2 drives breast cancer cell migration and invasion. Cancer Research, 82(12_Supplement), 2416-2416. doi:10.1158/1538-7445.am2022-2416
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  Abstract We previously identified the mechanistic Target of Rapamycin Complex 2 (mTORC2) as an effector of Ras for the control of directed cell migration in Dictyostelium. Recently, this relationship was found to be conserved in mammalian cells, and mTORC2 was shown to be an effector of oncogenic Ras. Deregulated mTORC2 signaling in cancer is mostly associated with increased survival and metabolic reprogramming, but mTORC2 has also been linked to cancer cell migration, particularly in breast cancer. Interestingly, although Ras is rarely mutated in breast cancer, it is often upregulated due to amplification and overexpression of growth factor receptors. Here, we investigated the role of Ras in promoting the migration and invasion of breast cancer cells through mTORC2. We observed that Ras and mTORC2 promote the migration of breast cancer cells, independently of the breast cancer molecular subtype. Using breast epithelial MCF10A cells transformed with HER2 or mutant Ras, we found that Ras promotes mTORC2 activation, and mTORC2-dependent migration and invasion. We further observed that while mutant Ras-transformed MCF10A cells display uncontrolled cell proliferation and an invasive phenotype, silencing of the mTORC2-dependent component Rictor mostly leads to loss of invasiveness. Together, our findings suggest that, whereas Ras activation of mTORC2 is likely to play a minor role in breast tumor formation, the Ras-mTORC2 pathway plays a key and general role in promoting the migration and invasion of breast cancer cells. Citation Format: Mollie E. Wiegand, Shannon E. Collins, Isabella N. Brown, Alyssa N. Werner, Isabelle M. Mundo, Ghassan Mouneimne, Pascale G. Charest. Ras-mediated activation of mTORC2 drives breast cancer cell migration and invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2416.
• Hwang, T., Parker, S. S., Hill, S. M., Grant, R. A., Ilunga, M. W., Sivaraman, V., Mouneimne, G., & Keating, A. E. (2022). Native proline-rich motifs exploit sequence context to target actin-remodeling Ena/VASP protein ENAH. eLife, 11.
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  The human proteome is replete with short linear motifs (SLiMs) of four to six residues that are critical for protein-protein interactions, yet the importance of the sequence surrounding such motifs is underexplored. We devised a proteomic screen to examine the influence of SLiM sequence context on protein-protein interactions. Focusing on the EVH1 domain of human ENAH, an actin regulator that is highly expressed in invasive cancers, we screened 36-residue proteome-derived peptides and discovered new interaction partners of ENAH and diverse mechanisms by which context influences binding. A pocket on the ENAH EVH1 domain that has diverged from other Ena/VASP paralogs recognizes extended SLiMs and favors motif-flanking proline residues. Many high-affinity ENAH binders that contain two proline-rich SLiMs use a noncanonical site on the EVH1 domain for binding and display a thermodynamic signature consistent with the two-motif chain engaging a single domain. We also found that photoreceptor cilium actin regulator (PCARE) uses an extended 23-residue region to obtain a higher affinity than any known ENAH EVH1-binding motif. Our screen provides a way to uncover the effects of proteomic context on motif-mediated binding, revealing diverse mechanisms of control over EVH1 interactions and establishing that SLiMs can't be fully understood outside of their native context.
• Vanderah, T. W., Ramonett, A., Pan, C. C., Ortiz, H. R., Mythreye, K., Mouneimne, G., Mohler, P. J., Lochhead, J. J., Lee, Y. S., Lee, N. Y., Largent-milnes, T., Langlais, P. R., Kwak, E., Kumar, S., Hund, T. J., Georgieva, T. G., Ellis, N. A., Cruz-flores, P., & Ahmed, T. (2022). βIV-spectrin as a stalk cell-intrinsic regulator of VEGF signaling.. Nature communications, 13(1), 1326. doi:10.1038/s41467-022-28933-1
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  Defective angiogenesis underlies over 50 malignant, ischemic and inflammatory disorders yet long-term therapeutic applications inevitably fail, thus highlighting the need for greater understanding of the vast crosstalk and compensatory mechanisms. Based on proteomic profiling of angiogenic endothelial components, here we report βIV-spectrin, a non-erythrocytic cytoskeletal protein, as a critical regulator of sprouting angiogenesis. Early loss of endothelial-specific βIV-spectrin promotes embryonic lethality in mice due to hypervascularization and hemorrhagic defects whereas neonatal depletion yields higher vascular density and tip cell populations in developing retina. During sprouting, βIV-spectrin expresses in stalk cells to inhibit their tip cell potential by enhancing VEGFR2 turnover in a manner independent of most cell-fate determining mechanisms. Rather, βIV-spectrin recruits CaMKII to the plasma membrane to directly phosphorylate VEGFR2 at Ser984, a previously undefined phosphoregulatory site that strongly induces VEGFR2 internalization and degradation. These findings support a distinct spectrin-based mechanism of tip-stalk cell specification during vascular development.
• Hwang, T., Parker, S. S., Hill, S. M., Ilunga, M. W., Grant, R. A., Mouneimne, G., & Keating, A. E. (2021). A distributed residue network permits conformational binding specificity in a conserved family of actin remodelers. eLife, 10.
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  Metazoan proteomes contain many paralogous proteins that have evolved distinct functions. The Ena/VASP family of actin regulators consists of three members that share an EVH1 interaction domain with a 100 % conserved binding site. A proteome-wide screen revealed photoreceptor cilium actin regulator (PCARE) as a high-affinity ligand for ENAH EVH1. Here, we report the surprising observation that PCARE is ~100-fold specific for ENAH over paralogs VASP and EVL and can selectively bind ENAH and inhibit ENAH-dependent adhesion in cells. Specificity arises from a mechanism whereby PCARE stabilizes a conformation of the ENAH EVH1 domain that is inaccessible to family members VASP and EVL. Structure-based modeling rapidly identified seven residues distributed throughout EVL that are sufficient to differentiate binding by ENAH vs. EVL. By exploiting the ENAH-specific conformation, we rationally designed the tightest and most selective ENAH binder to date. Our work uncovers a conformational mechanism of interaction specificity that distinguishes highly similar paralogs and establishes tools for dissecting specific Ena/VASP functions in processes including cancer cell invasion.
• Kraft, A. S., Fernandes, N., Mouneimne, G., Song, J. H., Bearss, J. J., Padi, S. K., Singh, N., Cardo‐Vila, M., Li, Y., Harter, M. R., Gard, J. M., Cress, A. E., Peti, W., Nelson, A. D., Buchan, J. R., & Okumura, K. (2021). EDC3 phosphorylation regulates growth and invasion through controlling P‐body formation and dynamics. EMBO reports, 22(4). doi:10.15252/embr.202050835
• Padi, M., Mouneimne, G., Romanoski, C. E., & Cusanovich, D. A. (2021). Breast tumor stiffness instructs bone metastasis via maintenance of mechanical conditioning. Cell Reports.
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  The mechanical microenvironment of primary breast tumors plays a substantial role in promoting tumor progression. While the transitory response of cancer cells to pathological stiffness in their native microenvironment has been well described, it is still unclear how mechanical stimuli in the primary tumor influence distant, late-stage metastatic phenotypes across time and space in absentia. Here, we show that primary tumor stiffness promotes stable, non-genetically heritable phenotypes in breast cancer cells. This mechanical memory instructs cancer cells to adopt and maintain increased cytoskeletal dynamics, traction force, and 3D invasion in vitro, in addition to promoting osteolytic bone metastasis in vivo. Furthermore, we established a mechanical conditioning (MeCo) score comprised of mechanically-regulated genes as a global gene expression measurement of tumor stiffness response. Clinically, we show that a high MeCo score is strongly associated with bone metastasis in patients. Using a discovery approach, we mechanistically traced mechanical memory in part to ERK-mediated mechanotransductive activation of RUNX2, an osteogenic gene bookmarker and bone metastasis driver. The combination of these RUNX2 traits permits the stable transactivation of osteolytic target genes that remain upregulated after cancer cells disseminate from their activating microenvironment in order to modify a distant microenvironment. Using genetic, epigenetic, and functional approaches, we were able to simulate, repress, select and extend RUNX2-mediated mechanical memory and alter cancer cell behavior accordingly. In concert with previous studies detailing the influence of biochemical properties of the primary tumor stroma on distinct metastatic phenotypes, our findings detailing the influence of biomechanical properties support a generalized model of cancer progression in which the integrated properties of the primary tumor microenvironment govern the secondary tumor microenvironment, i.e., soil instructs soil.
• Watson, A. W., Grant, A. D., Parker, S. S., Hill, S., Whalen, M. B., Chakrabarti, J., Harman, M. W., Roman, M. R., Forte, B. L., Gowan, C. C., Castro-Portuguez, R., Stolze, L. K., Franck, C., Cusanovich, D. A., Zavros, Y., Padi, M., Romanoski, C. E., & Mouneimne, G. (2021). Breast tumor stiffness instructs bone metastasis via maintenance of mechanical conditioning. Cell reports, 35(13), 109293.
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  While the immediate and transitory response of breast cancer cells to pathological stiffness in their native microenvironment has been well explored, it remains unclear how stiffness-induced phenotypes are maintained over time after cancer cell dissemination in vivo. Here, we show that fibrotic-like matrix stiffness promotes distinct metastatic phenotypes in cancer cells, which are preserved after transition to softer microenvironments, such as bone marrow. Using differential gene expression analysis of stiffness-responsive breast cancer cells, we establish a multigenic score of mechanical conditioning (MeCo) and find that it is associated with bone metastasis in patients with breast cancer. The maintenance of mechanical conditioning is regulated by RUNX2, an osteogenic transcription factor, established driver of bone metastasis, and mitotic bookmarker that preserves chromatin accessibility at target gene loci. Using genetic and functional approaches, we demonstrate that mechanical conditioning maintenance can be simulated, repressed, or extended, with corresponding changes in bone metastatic potential.
• Merad, M., Posey, A. D., Olivero, O., Singh, P. K., Mouneimne, G., Li, L., Wallace, L. M., & Hayes, T. K. (2020). Diversity Is a Strength of Cancer Research in the U.S. Cancer cell, 38(3), 297-300.
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  We stand against racism and discrimination in cancer research in the U.S. By sharing the stories of scientists from different ethnicities, identities, and national origins, we want to promote change through mentoring, active participation, and policy changes and to inspire the next generation of cancer researchers: we make better science together.
• Saini, H., Rahmani Eliato, K., Veldhuizen, J., Zare, A., Allam, M., Silva, C., Kratz, A., Truong, D., Mouneimne, G., LaBaer, J., Ros, R., & Nikkhah, M. (2020). The role of tumor-stroma interactions on desmoplasia and tumorigenicity within a microengineered 3D platform. Biomaterials, 247, 119975.
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  The tumor microenvironment has been demonstrated to play a crucial role in modulating cancer progression. Amongst various cell types within the tumor microenvironment, cancer associated fibroblasts (CAFs) are in abundance, serving to modulate the biophysical properties of the stromal matrix, through excessive deposition of extracellular matrix (ECM) proteins that leads to enhanced tumor progression. There is still a critical need to develop a fundamental framework on the role of tumor-stromal cell interactions on desmoplasia and tumorigenicity. Herein, we developed a 3D microengineered organotypic tumor-stroma model incorporated with breast cancer cells surrounded by CAF-embedded collagen matrix. We further integrated our platform with atomic force microscopy (AFM) to study the dynamic changes in stromal stiffness during active tumor invasion. Our findings primarily demonstrated enhanced tumor progression in the presence of CAFs. Furthermore, we highlighted the crucial role of crosstalk between tumor cells and CAFs on stromal desmoplasia, where we identified the role of tumor-secreted PDGF-AA/-BB on elevated matrix stiffness. Inhibition of the activity of PDGFRs in CAFs led to attenuation of stromal stiffness. Overall, our work presents a well-controlled tumor microenvironment model capable of dissecting specific biophysical and biochemical signaling cues which lead to stromal desmoplasia and tumor progression.
• Krantz, J., Parker, S. S., Barker, N. K., Deer, C. G., Mouneimne, G., & Langlais, P. R. (2019). Insulin Induces Microtubule Stabilization and Regulates the Microtubule Plus-end Tracking Protein Network in Adipocytes.. Molecular & cellular proteomics : MCP.
• Mouneimne, G., Gertler, F. B., Ross, R., Roe, D., Saboda, K., Peng, L., Eliato, K. R., Watson, A. W., Parker, S. S., Roman, M. R., & Puleo, J. I. (2019). Mechanosensing during directed cell migration requires dynamic actin polymerization at focal adhesions. Journal of Cell Biology.
• Mouneimne, G., Roe, D. J., Peng, L., Parker, S. S., Puleo, J. I., Roman, M. R., Watson, A. W., Eliato, K. R., Saboda, K., Ros, R., & Gertler, F. B. (2019). Mechanosensing during directed cell migration requires dynamic actin polymerization at focal adhesions. Journal of Cell Biology, 218(12), 4215-4235. doi:10.1083/jcb.201902101
• Mouneimne, G., Nagaraju, S., Truong, D., & Nikkhah, M. (2018). Microfluidic Tumor–Vascular Model to Study Breast Cancer Cell Invasion and Intravasation. Advanced Healthcare Materials, 7(9), 1701257. doi:10.1002/adhm.201701257
• Nagaraju, S., Truong, D., Mouneimne, G., & Nikkhah, M. (2018). Microfluidic Tumor-Vascular Model to Study Breast Cancer Cell Invasion and Intravasation. Advanced healthcare materials.
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  Cancer is a major leading cause of disease-related death in the world. The severe impact of cancer can be attributed to poor understanding of the mechanisms involved in earliest steps of the metastatic cascade, specifically invasion into the surrounding stroma and intravasation into the blood capillaries. However, conducting integrated biological studies of invasion and intravasation have been challenging, within in vivo models and traditional in vitro assay, due to difficulties in establishing a precise tumor microenvironment. To that end, in this work, a novel 3D microfluidic platform comprised of concentric three-layer cell-laden hydrogels for simultaneous investigation of breast cancer cell invasion and intravasation as well as vasculature maturation influenced by tumor-vascular crosstalk is developed. It was demonstrated that the presence of spontaneously formed vasculature enhance MDA-MB-231 invasion into the 3D stroma. Following invasion, cancer cells are visualized intravasating into the outer vasculature. Additionally, invading cancer cells significantly reduce vessel diameter while increasing permeability, consistent with previous in vivo studies. Major signaling cytokines involved in tumor-vascular crosstalk that govern cancer cell invasion and intravasation are further identified. Taken together, this platform will enable unique insights of critical biological events within the metastatic cascade, with significant potential for developing efficient cancer therapeutics.
• Padilla-Rodriguez, M., Parker, S. S., Adams, D. G., Westerling, T., Puleo, J. I., Watson, A. W., Hill, S. M., Noon, M., Gaudin, R., Aaron, J., Tong, D., Roe, D. J., Knudsen, B., & Mouneimne, G. (2018). The actin cytoskeletal architecture of estrogen receptor positive breast cancer cells suppresses invasion. Nature communications, 9(1), 2980.
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  Estrogen promotes growth of estrogen receptor-positive (ER+) breast tumors. However, epidemiological studies examining the prognostic characteristics of breast cancer in postmenopausal women receiving hormone replacement therapy reveal a significant decrease in tumor dissemination, suggesting that estrogen has potential protective effects against cancer cell invasion. Here, we show that estrogen suppresses invasion of ER+ breast cancer cells by increasing transcription of the Ena/VASP protein, EVL, which promotes the generation of suppressive cortical actin bundles that inhibit motility dynamics, and is crucial for the ER-mediated suppression of invasion in vitro and in vivo. Interestingly, despite its benefits in suppressing tumor growth, anti-estrogenic endocrine therapy decreases EVL expression and increases local invasion in patients. Our results highlight the dichotomous effects of estrogen on tumor progression and suggest that, in contrast to its established role in promoting growth of ER+ tumors, estrogen has a significant role in suppressing invasion through actin cytoskeletal remodeling.
• Parker, S. S., Moutal, A., Cai, S., Chandrasekaran, S., Roman, M. R., Koshy, A. A., Khanna, R., Zinsmaier, K. E., & Mouneimne, G. (2018). High Fidelity Cryopreservation and Recovery of Primary Rodent Cortical Neurons. eNeuro, 5(5).
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  Cell cryopreservation improves reproducibility and enables flexibility in experimental design. Although conventional freezing methodologies have been used to preserve primary neurons, poor cell viability and reduced survival severely limited their utility. We screened several high-performance freezing media and found that CryoStor10 (CS10) provided superior cryoprotection to primary mouse embryonic cortical neurons compared to other commercially-available or traditional reagents, permitting the recovery of 68.8% of cells relative to a fresh dissection. We characterized developmental, morphometric, and functional indicators of neuron maturation and found that, without exception, neurons recovered from cryostorage in CS10 media faithfully recapitulate neurodevelopment in-step with neurons obtained by fresh dissection. Our method establishes cryopreserved neurons as a reliable, efficient, and equivalent model to fresh neuron cultures.
• Mouneimne, G., Peela, N., Barrientos, E. S., Truong, D., & Nikkhah, M. (2017). Effect of suberoylanilide hydroxamic acid (SAHA) on breast cancer cells within a tumor–stroma microfluidic model. Integrative Biology, 9(12), 988-999. doi:10.1039/c7ib00180k
• Peela, N., Barrientos, E. S., Truong, D., Mouneimne, G., & Nikkhah, M. (2017). Effect of suberoylanilide hydroxamic acid (SAHA) on breast cancer cells within a tumor-stroma microfluidic model. Integrative biology : quantitative biosciences from nano to macro, 9(12), 988-999.
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  Metastatic cancer is regarded as one of the largest contributors to disease-related deaths worldwide. Poor patient prognosis and treatment outcome is tied to the lack of efficacious anti-cancer therapies, which is due in part to the lack of physiologically relevant in vitro screening systems that can mimic the native tumor microenvironment. Conventional drug-screening platforms, which are often used in the pharmaceutical industry, are either two-dimensional (2D) assays or three-dimensional (3D) hydrogel-based matrices that lack precise control over cell distribution, matrix architecture, and organization. Despite the significance of in vivo models, they have limitations as it is difficult to control and analyze the influence of specific variables within their tumor microenvironment. Thus, there is still a crucial need to develop tumor models that enable precise control of microenvironmental cues (e.g. matrix composition, soluble factors, cellular organization) to assess the efficacy of anti-cancer drugs. Herein, we report the development and validation of a 3D microfluidic invasion platform for anti-cancer drug studies. Our platform allowed for compartmentalization of tumor and stromal fibroblasts in a defined architecture, thereby enabling pharmacokinetic drug transport to a cell-dense tumor region. We analyzed the effect of suberoylanilide hydroxamic acid (SAHA), a histone deacetylase (HDAC) inhibitor, on the behavior of SUM159 breast cancer cells. Many HDAC inhibitors, including SAHA, have been a subject of controversy with highly conflicting results for the treatment of solid tumors in vitro as well as in clinical trials. We found that SAHA significantly inhibited cellular migration/proliferation, and decreased microtubule polarization.
• Carmona, G., Perera, U., Gillett, C., Naba, A., Law, A. L., Sharma, V. P., Wang, J., Wyckoff, J., Balsamo, M., Mosis, F., De Piano, M., Monypenny, J., Woodman, N., McConnell, R. E., Mouneimne, G., Van Hemelrijck, M., Cao, Y., Condeelis, J., Hynes, R. O., , Gertler, F. B., et al. (2016). Lamellipodin promotes invasive 3D cancer cell migration via regulated interactions with Ena/VASP and SCAR/WAVE. Oncogene, 35(39), 5155-69.
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  Cancer invasion is a hallmark of metastasis. The mesenchymal mode of cancer cell invasion is mediated by elongated membrane protrusions driven by the assembly of branched F-actin networks. How deregulation of actin regulators promotes cancer cell invasion is still enigmatic. We report that increased expression and membrane localization of the actin regulator Lamellipodin correlate with reduced metastasis-free survival and poor prognosis in breast cancer patients. In agreement, we find that Lamellipodin depletion reduced lung metastasis in an orthotopic mouse breast cancer model. Invasive 3D cancer cell migration as well as invadopodia formation and matrix degradation was impaired upon Lamellipodin depletion. Mechanistically, we show that Lamellipodin promotes invasive 3D cancer cell migration via both actin-elongating Ena/VASP proteins and the Scar/WAVE complex, which stimulates actin branching. In contrast, Lamellipodin interaction with Scar/WAVE but not with Ena/VASP is required for random 2D cell migration. We identified a phosphorylation-dependent mechanism that regulates selective recruitment of these effectors to Lamellipodin: Abl-mediated Lamellipodin phosphorylation promotes its association with both Scar/WAVE and Ena/VASP, whereas Src-dependent phosphorylation enhances binding to Scar/WAVE but not to Ena/VASP. Through these selective, regulated interactions Lamellipodin mediates directional sensing of epidermal growth factor (EGF) gradients and invasive 3D migration of breast cancer cells. Our findings imply that increased Lamellipodin levels enhance Ena/VASP and Scar/WAVE activities at the plasma membrane to promote 3D invasion and metastasis.
• Peela, N., Sam, F. S., Christenson, W., Truong, D., Watson, A. W., Mouneimne, G., Ros, R., & Nikkhah, M. (2016). A three dimensional micropatterned tumor model for breast cancer cell migration studies. Biomaterials, 81, 72-83.
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  Breast cancer cell invasion is a highly orchestrated process driven by a myriad of complex microenvironmental stimuli, making it difficult to isolate and assess the effects of biochemical or biophysical cues (i.e. tumor architecture, matrix stiffness) on disease progression. In this regard, physiologically relevant tumor models are becoming instrumental to perform studies of cancer cell invasion within well-controlled conditions. Herein, we explored the use of photocrosslinkable hydrogels and a novel, two-step photolithography technique to microengineer a 3D breast tumor model. The microfabrication process enabled precise localization of cell-encapsulated circular constructs adjacent to a low stiffness matrix. To validate the model, breast cancer cell lines (MDA-MB-231, MCF7) and non-tumorigenic mammary epithelial cells (MCF10A) were embedded separately within the tumor model, all of which maintained high viability throughout the experiments. MDA-MB-231 cells exhibited extensive migratory behavior and invaded the surrounding matrix, whereas MCF7 or MCF10A cells formed clusters that stayed confined within the circular tumor regions. Additionally, real-time cell tracking indicated that the speed and persistence of MDA-MB-231 cells were substantially higher within the surrounding matrix compared to the circular constructs. Z-stack imaging of F-actin/α-tubulin cytoskeletal organization revealed unique 3D protrusions in MDA-MB-231 cells and an abundance of 3D clusters formed by MCF7 and MCF10A cells. Our results indicate that gelatin methacrylate (GelMA) hydrogel, integrated with the two-step photolithography technique, has great promise in the development of 3D tumor models with well-defined architecture and tunable stiffness.
• Truong, D., Puleo, J., Llave, A., Mouneimne, G., Kamm, R. D., & Nikkhah, M. (2016). Breast Cancer Cell Invasion into a Three Dimensional Tumor-Stroma Microenvironment. Scientific reports, 6, 34094.
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  In this study, to model 3D chemotactic tumor-stroma invasion in vitro, we developed an innovative microfluidic chip allowing side-by-side positioning of 3D hydrogel-based matrices. We were able to (1) create a dual matrix architecture that extended in a continuous manner, thus allowing invasion from one 3D matrix to another, and (2) establish distinct regions of tumor and stroma cell/ECM compositions, with a clearly demarcated tumor invasion front, thus allowing us to quantitatively analyze progression of cancer cells into the stroma at a tissue or single-cell level. We showed significantly enhanced cancer cell invasion in response to a transient gradient of epidermal growth factor (EGF). 3D tracking at the single-cell level displayed increased migration speed and persistence. Subsequently, we analyzed changes in expression of EGF receptors, cell aspect ratio, and protrusive activity. These findings show the unique ability of our model to quantitatively analyze 3D chemotactic invasion, both globally by tracking the progression of the invasion front, and at the single-cell level by examining changes in cellular behavior and morphology using high-resolution imaging. Taken together, we have shown a novel model recapitulating 3D tumor-stroma interactions for studies of real-time cell invasion and morphological changes within a single platform.
• Hager, M. H., Morley, S., Bielenberg, D. R., Gao, S., Morello, M., Holcomb, I. N., Liu, W., Mouneimne, G., Demichelis, F., Kim, J., Solomon, K. R., Adam, R. M., Isaacs, W. B., Higgs, H. N., Vessella, R. L., Di Vizio, D., & Freeman, M. R. (2012). DIAPH3 governs the cellular transition to the amoeboid tumour phenotype. EMBO molecular medicine, 4(8), 743-60.
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  Therapies for most malignancies are generally ineffective once metastasis occurs. While tumour cells migrate through tissues using diverse strategies, the signalling networks controlling such behaviours in human tumours are poorly understood. Here we define a role for the Diaphanous-related formin-3 (DIAPH3) as a non-canonical regulator of metastasis that restrains conversion to amoeboid cell behaviour in multiple cancer types. The DIAPH3 locus is close to RB1, within a narrow consensus region of deletion on chromosome 13q in prostate, breast and hepatocellular carcinomas. DIAPH3 silencing in human carcinoma cells destabilized microtubules and induced defective endocytic trafficking, endosomal accumulation of EGFR, and hyperactivation of EGFR/MEK/ERK signalling. Silencing also evoked amoeboid properties, increased invasion and promoted metastasis in mice. In human tumours, DIAPH3 down-regulation was associated with aggressive or metastatic disease. DIAPH3-silenced cells were sensitive to MEK inhibition, but showed reduced sensitivity to EGFR inhibition. These findings have implications for understanding mechanisms of metastasis, and suggest that identifying patients with chromosomal deletions at DIAPH3 may have prognostic value.
• Mouneimne, G., Hansen, S. D., Selfors, L. M., Petrak, L., Hickey, M. M., Gallegos, L. L., Simpson, K. J., Lim, J., Gertler, F. B., Hartwig, J. H., Mullins, R. D., & Brugge, J. S. (2012). Differential remodeling of actin cytoskeleton architecture by profilin isoforms leads to distinct effects on cell migration and invasion. Cancer cell, 22(5), 615-30.
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  Dynamic actin cytoskeletal reorganization is integral to cell motility. Profilins are well-characterized regulators of actin polymerization; however, functional differences among coexpressed profilin isoforms are not well defined. Here, we demonstrate that profilin-1 and profilin-2 differentially regulate membrane protrusion, motility, and invasion; these processes are promoted by profilin-1 and suppressed by profilin-2. Compared to profilin-1, profilin-2 preferentially drives actin polymerization by the Ena/VASP protein, EVL. Profilin-2 and EVL suppress protrusive activity and cell motility by an actomyosin contractility-dependent mechanism. Importantly, EVL or profilin-2 downregulation enhances invasion in vitro and in vivo. In human breast cancer, lower EVL expression correlates with high invasiveness and poor patient outcome. We propose that profilin-2/EVL-mediated actin polymerization enhances actin bundling and suppresses breast cancer cell invasion.
• Kim, J. H., Cho, A., Yin, H., Schafer, D. A., Mouneimne, G., Simpson, K. J., Nguyen, K., Brugge, J. S., & Montell, D. J. (2011). Psidin, a conserved protein that regulates protrusion dynamics and cell migration. Genes & development, 25(7), 730-41.
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  Dynamic assembly and disassembly of actin filaments is a major driving force for cell movements. Border cells in the Drosophila ovary provide a simple and genetically tractable model to study the mechanisms regulating cell migration. To identify new genes that regulate cell movement in vivo, we screened lethal mutations on chromosome 3R for defects in border cell migration and identified two alleles of the gene psidin (psid). In vitro, purified Psid protein bound F-actin and inhibited the interaction of tropomyosin with F-actin. In vivo, psid mutations exhibited genetic interactions with the genes encoding tropomyosin and cofilin. Border cells overexpressing Psid together with GFP-actin exhibited altered protrusion/retraction dynamics. Psid knockdown in cultured S2 cells reduced, and Psid overexpression enhanced, lamellipodial dynamics. Knockdown of the human homolog of Psid reduced the speed and directionality of migration in wounded MCF10A breast epithelial monolayers, whereas overexpression of the protein increased migration speed and altered protrusion dynamics in EGF-stimulated cells. These results indicate that Psid is an actin regulatory protein that plays a conserved role in protrusion dynamics and cell migration.
• Desmarais, V., Yamaguchi, H., Oser, M., Soon, L., Mouneimne, G., Sarmiento, C., Eddy, R., & Condeelis, J. (2009). N-WASP and cortactin are involved in invadopodium-dependent chemotaxis to EGF in breast tumor cells. Cell motility and the cytoskeleton, 66(6), 303-16.
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  Metastatic mammary carcinoma cells, which have previously been observed to form mature, matrix degrading invadopodia on a thick ECM matrix, are able to form invadopodia with similar characteristics on glass without previously applied matrix. They form in response to epidermal growth factor (EGF), and contain the usual invadopodium core proteins N-WASP, Arp2/3, cortactin, cofilin, and F-actin. The study of invadopodia on glass allows for higher resolution analysis including the use of total internal reflection microscopy and analysis of their relationship to other cell motility events, in particular, lamellipodium extension and chemotaxis toward an EGF gradient. Invadopodium formation on glass requires N-WASP and cortactin but not microtubules. In a gradient of EGF more invadopodia form on the side of the cells facing the source of EGF. In addition, depletion of N-WASP or cortactin, which blocks invadopodium fromation, inhibits chemotaxis of cells towards EGF. This appears to be a localized defect in chemotaxis since depletion of N-WASP or cortactin via siRNA had no effect on lamellipodium protrusion or barbed end generation at the lamellipodium's leading edge. Since chemotaxis to EGF by breast tumor cells is involved in metastasis, inhibiting N-WASP activity in breast tumor cells might prevent metastasis of tumor cells while not affecting chemotaxis-dependent innate immunity which depends on WASp function in macrophages.
• Mouneimne, G., & Brugge, J. S. (2009). YB-1 translational control of epithelial-mesenchyme transition. Cancer cell, 15(5), 357-9.
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  Transitions between epithelial and mesenchmal phenotypes play critical roles in normal development and cancer progression. In this issue of Cancer Cell, Evdokimova et al. demonstrate that YB-1 regulates epithelial-mesenchyme transition (EMT) by inducing cap-independent translation of mRNAs encoding EMT-promoting factors and suppressing cap-dependent translation of mRNAs encoding growth-promoting factors.
• Lapidus, K., Wyckoff, J., Mouneimne, G., Lorenz, M., Soon, L., Condeelis, J. S., & Singer, R. H. (2007). ZBP1 enhances cell polarity and reduces chemotaxis. Journal of cell science, 120(Pt 18), 3173-8.
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  The interaction of beta-actin mRNA with zipcode-binding protein 1 (ZBP1) is necessary for its localization to the lamellipod of fibroblasts and plays a crucial role in cell polarity and motility. Recently, we have shown that low ZBP1 levels correlate with tumor-cell invasion and metastasis. In order to establish a cause and effect relationship, we expressed ZBP1 in a metastatic rat mammary adenocarcinoma cell line (MTLn3) that has low endogenous ZBP1 levels and delocalized beta-actin mRNA. This leads to localization of beta-actin mRNA, and eventually reduces the chemotactic potential of the cells as well as their ability to move and orient towards vessels in tumors. To determine how ZBP1 leads to these two apparently contradictory aspects of cell behavior--increased cell motility but decreased chemotaxis--we examined cell motility in detail, both in cell culture and in vivo in tumors. We found that ZBP1 expression resulted in tumor cells with a stable polarized phenotype, and reduced their ability to move in response to a gradient in culture. To connect these results on cultured cells to the reduced metastatic ability of these cells, we used multiphoton imaging in vivo to examine tumor cell behavior in primary tumors. We found that ZBP1 expression actually reduced tumor cell motility and chemotaxis, presumably mediating their decreased metastatic potential by reducing their ability to respond to signals necessary for invasion.
• Mouneimne, G., & Brugge, J. S. (2007). Tensins: a new switch in cell migration. Developmental cell, 13(3), 317-9.
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  Epidermal Growth Factor (EGF) is an important regulator of normal epithelial and carcinoma cell migration. The mechanism by which EGF induces cell migration is not fully understood. A recent report in Nature Cell Biology (Katz et al., 2007) demonstrates that EGF regulates migration through a switch in the expression of two tensin isoforms, weakening the association of beta1 integrin with the actin cytoskeleton in focal adhesions.
• Overholtzer, M., Mailleux, A. A., Mouneimne, G., Normand, G., Schnitt, S. J., King, R. W., Cibas, E. S., & Brugge, J. S. (2007). A nonapoptotic cell death process, entosis, that occurs by cell-in-cell invasion. Cell, 131(5), 966-79.
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  Epithelial cells require attachment to extracellular matrix (ECM) to suppress an apoptotic cell death program termed anoikis. Here we describe a nonapoptotic cell death program in matrix-detached cells that is initiated by a previously unrecognized and unusual process involving the invasion of one cell into another, leading to a transient state in which a live cell is contained within a neighboring host cell. Live internalized cells are either degraded by lysosomal enzymes or released. We term this cell internalization process entosis and present evidence for entosis as a mechanism underlying the commonly observed "cell-in-cell" cytological feature in human cancers. Further we propose that entosis is driven by compaction force associated with adherens junction formation in the absence of integrin engagement and may represent an intrinsic tumor suppression mechanism for cells that are detached from ECM.
• Sidani, M., Wessels, D., Mouneimne, G., Ghosh, M., Goswami, S., Sarmiento, C., Wang, W., Kuhl, S., El-Sibai, M., Backer, J. M., Eddy, R., Soll, D., & Condeelis, J. (2007). Cofilin determines the migration behavior and turning frequency of metastatic cancer cells. The Journal of cell biology, 179(4), 777-91.
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  We have investigated the effects of inhibiting the expression of cofilin to understand its role in protrusion dynamics in metastatic tumor cells, in particular. We show that the suppression of cofilin expression in MTLn3 cells (an apolar randomly moving amoeboid metastatic tumor cell) caused them to extend protrusions from only one pole, elongate, and move rectilinearly. This remarkable transformation was correlated with slower extension of fewer, more stable lamellipodia leading to a reduced turning frequency. Hence, the loss of cofilin caused an amoeboid tumor cell to assume a mesenchymal-type mode of movement. These phenotypes were correlated with the loss of uniform chemotactic sensitivity of the cell surface to EGF stimulation, demonstrating that to chemotax efficiently, a cell must be able to respond to chemotactic stimulation at any region on its surface. The changes in cell shape, directional migration, and turning frequency were related to the re-localization of Arp2/3 complex to one pole of the cell upon suppression of cofilin expression.
• Yip, S., El-Sibai, M., Coniglio, S. J., Mouneimne, G., Eddy, R. J., Drees, B. E., Neilsen, P. O., Goswami, S., Symons, M., Condeelis, J. S., & Backer, J. M. (2007). The distinct roles of Ras and Rac in PI 3-kinase-dependent protrusion during EGF-stimulated cell migration. Journal of cell science, 120(Pt 17), 3138-46.
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  Cell migration involves the localized extension of actin-rich protrusions, a process that requires Class I phosphoinositide 3-kinases (PI 3-kinases). Both Rac and Ras have been shown to regulate actin polymerization and activate PI 3-kinase. However, the coordination of Rac, Ras and PI 3-kinase activation during epidermal growth factor (EGF)-stimulated protrusion has not been analyzed. We examined PI 3-kinase-dependent protrusion in MTLn3 rat adenocarcinoma cells. EGF-stimulated phosphatidyl-inositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P(3)] levels showed a rapid and persistent response, as PI 3-kinase activity remained elevated up to 3 minutes. The activation kinetics of Ras, but not Rac, coincided with those of leading-edge PtdIns(3,4,5)P(3) production. Small interfering RNA (siRNA) knockdown of K-Ras but not Rac1 abolished PtdIns(3,4,5)P(3) production at the leading edge and inhibited EGF-stimulated protrusion. However, Rac1 knockdown did inhibit cell migration, because of the inhibition of focal adhesion formation in Rac1 siRNA-treated cells. Our data show that in EGF-stimulated MTLn3 carcinoma cells, Ras is required for both PtdIns(3,4,5)P(3) production and lamellipod extension, whereas Rac1 is required for formation of adhesive structures. These data suggest an unappreciated role for Ras during protrusion, and a crucial role for Rac in the stabilization of protrusions required for cell motility.
• Mouneimne, G., DesMarais, V., Sidani, M., Scemes, E., Wang, W., Song, X., Eddy, R., & Condeelis, J. (2006). Spatial and temporal control of cofilin activity is required for directional sensing during chemotaxis. Current biology : CB, 16(22), 2193-205.
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  Previous work has led to the hypothesis that cofilin severing, as regulated by PLC, is involved in chemotactic sensing. We have tested this hypothesis by investigating whether activation of endogenous cofilin is spatially and temporally linked to sensing an EGF point source in carcinoma cells.
• Song, X., Chen, X., Yamaguchi, H., Mouneimne, G., Condeelis, J. S., & Eddy, R. J. (2006). Initiation of cofilin activity in response to EGF is uncoupled from cofilin phosphorylation and dephosphorylation in carcinoma cells. Journal of cell science, 119(Pt 14), 2871-81.
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  It has been demonstrated that the actin-severing activity of cofilin can be downregulated by LIM kinase (LIMK)-dependent phosphorylation at residue Ser3. Chemotactic stimulation in various cell types induces cofilin dephosphorylation, suggesting that cofilin activation in these cells occurs by a dephosphorylation mechanism. However, resting metastatic carcinoma cells have the majority of their cofilin in a dephosphorylated but largely inactive state. Stimulation with epidermal growth factor (EGF) induces an increase in cofilin activity after 60 seconds together with an increase in phosphorylated cofilin (p-cofilin), indicating that cofilin dephosphorylation is not coupled to cofilin activation in these cells. Suppression of LIMK function by inhibiting Rho-associated protein kinase (ROCK) or LIMK siRNA inhibited the EGF-induced cofilin phosphorylation but had no effect on cofilin activity or cofilin-dependent lamellipod protrusion induced by EGF. Correlation analysis revealed that cofilin, p-cofilin and LIMK are not colocalized, and changes in the location of these proteins upon stimulation with EGF indicate that they are not functionally coupled. Phospholipase C, which has been implicated in cofilin activation following stimulation with EGF, does not regulate p-cofilin levels following stimulation with EGF. Therefore, our results do not support a model for the initial activation of cofilin by dephosphorylation in response to chemoattractant stimulation in metastatic carcinoma cells.
• Wang, W., Mouneimne, G., Sidani, M., Wyckoff, J., Chen, X., Makris, A., Goswami, S., Bresnick, A. R., & Condeelis, J. S. (2006). The activity status of cofilin is directly related to invasion, intravasation, and metastasis of mammary tumors. The Journal of cell biology, 173(3), 395-404.
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  Understanding the mechanisms controlling cancer cell invasion and metastasis constitutes a fundamental step in setting new strategies for diagnosis, prognosis, and therapy of metastatic cancers. LIM kinase1 (LIMK1) is a member of a novel class of serine-threonine protein kinases. Cofilin, a LIMK1 substrate, is essential for the regulation of actin polymerization and depolymerization during cell migration. Previous studies have made opposite conclusions as to the role of LIMK1 in tumor cell motility and metastasis, claiming either an increase or decrease in cell motility and metastasis as a result of LIMK1 over expression (Zebda, N., O. Bernard, M. Bailly, S. Welti, D.S. Lawrence, and J.S. Condeelis. 2000. J. Cell Biol. 151:1119-1128; Davila, M., A.R. Frost, W.E. Grizzle, and R. Chakrabarti. 2003. J. Biol. Chem. 278:36868-36875; Yoshioka, K., V. Foletta, O. Bernard, and K. Itoh. 2003. Proc. Natl. Acad. Sci. USA. 100:7247-7252; Nishita, M., C. Tomizawa, M. Yamamoto, Y. Horita, K. Ohashi, and K. Mizuno. 2005. J. Cell Biol. 171:349-359). We resolve this paradox by showing that the effects of LIMK1 expression on migration, intravasation, and metastasis of cancer cells can be most simply explained by its regulation of the output of the cofilin pathway. LIMK1-mediated decreases or increases in the activity of the cofilin pathway are shown to cause proportional decreases or increases in motility, intravasation, and metastasis of tumor cells.
• Soon, L., Mouneimne, G., Segall, J., Wyckoff, J., & Condeelis, J. (2005). Description and characterization of a chamber for viewing and quantifying cancer cell chemotaxis. Cell motility and the cytoskeleton, 62(1), 27-34.
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  Direct observations of cancer cell invasion underscore the importance of chemotaxis in invasion and metastasis. Yet, there is to date, no established method for real-time imaging of cancer chemotaxis towards factors clinically correlated with metastasis. A chamber has been designed and tested, called the Soon chamber, which allows the direct observation and quantification of cancer cell chemotaxis. The premise for the design of the Soon chamber is the incorporation of a dam, which creates a steep gradient while retaining stability associated with a pressure-driven system. The design is based on the characteristics of cancer cell motility such as relatively low speeds, and slower motility responses to stimuli compared to classical amoeboid cells like neutrophils and Dictyostelium. We tested MTLn3 breast carcinoma cells in the Soon chamber in the presence of an EGF gradient, obtaining hour-long time-lapses of chemotaxis. MTLn3 cells migrated further, more linearly, and at greater speeds within an EGF gradient compared to buffer controls. Computation of the degree of orientation towards the EGF/buffer source showed that MTLn3 cells were significantly more directional toward the EGF gradient compared to buffer controls. Analysis of the time-lapse data obtained during chemotaxis demonstrated that two populations of cancer cells were present. One population exhibited oscillations in directionality occurring at average intervals of 12 min while the second population exhibited sustained high levels of directionality toward the source of EGF. This result suggests that polarized cancer cells can avoid the need for oscillatory path corrections during chemotaxis.
• Ghosh, M., Song, X., Mouneimne, G., Sidani, M., Lawrence, D. S., & Condeelis, J. S. (2004). Cofilin promotes actin polymerization and defines the direction of cell motility. Science (New York, N.Y.), 304(5671), 743-6.
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  A general caging method for proteins that are regulated by phosphorylation was used to study the in vivo biochemical action of cofilin and the subsequent cellular response. By acute and local activation of a chemically engineered, light-sensitive phosphocofilin mimic, we demonstrate that cofilin polymerizes actin, generates protrusions, and determines the direction of cell migration. We propose a role for cofilin that is distinct from its role as an actin-depolymerizing factor.
• Mouneimne, G., Soon, L., DesMarais, V., Sidani, M., Song, X., Yip, S., Ghosh, M., Eddy, R., Backer, J. M., & Condeelis, J. (2004). Phospholipase C and cofilin are required for carcinoma cell directionality in response to EGF stimulation. The Journal of cell biology, 166(5), 697-708.
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  The epidermal growth factor (EGF)-induced increase in free barbed ends, resulting in actin polymerization at the leading edge of the lamellipodium in carcinoma cells, occurs as two transients: an early one at 1 min and a late one at 3 min. Our results reveal that phospholipase (PLC) is required for triggering the early barbed end transient. Phosphoinositide-3 kinase selectively regulates the late barbed end transient. Inhibition of PLC inhibits cofilin activity in cells during the early transient, delays the initiation of protrusions, and inhibits the ability of cells to sense a gradient of EGF. Suppression of cofilin, using either small interfering RNA silencing or function-blocking antibodies, selectively inhibits the early transient. Therefore, our results demonstrate that the early PLC and cofilin-dependent barbed end transient is required for the initiation of protrusions and is involved in setting the direction of cell movement in response to EGF.
• Safieh-Garabedian, B., Mouneimne, G. M., El-Jouni, W., Khattar, M., & Talhouk, R. (2004). The effect of endotoxin on functional parameters of mammary CID-9 cells. Reproduction (Cambridge, England), 127(3), 397-406.
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  The effect of endotoxin on mammary CID-9 cells, which differentiate in culture and express beta-casein, was investigated. Cells in culture supplemented with lactogenic hormones and dripped with EMS-Matrix (EMS-drip), were treated daily with endotoxin (0.5-500 microg/ml). Endotoxin at concentrations of less or equal to 10 microg/ml did not affect cell growth and viability up to 5 days post endotoxin treatment. Endotoxin (0.01-10 microg/ml) was added to the culture medium, upon confluence, and functional parameters were examined within 48 h post endotoxin treatment. Nuclear factor-kappaB (NF-kappaB) (p52) increased in nuclear extracts from endotoxin-stimulated cells within 1 h of treatment, while beta-casein mRNA and protein expression decreased in a concentration-dependent manner at 24 and 48 h post treatment. Zymography showed that the 72 and 92 kDa gelatinase activity increased in cells at 24 and 48 h post endotoxin treatment at 10 and 50 microg/ml. At the latter concentration, the active form of 72 kDa gelatinase was induced at 48 h. Interleukin-6 and tumor necrosis factor-alpha levels increased at 1-3 h post endotoxin treatment and peaked at 6 h in cells on plastic and EHS-drip. Nerve growth factor (NGF) levels increased in control and endotoxin-treated cells in a time-dependent manner, and endotoxin increased NGF levels in culture at 6 and 9 h post endotoxin treatment. This study shows that endotoxin activated NF-kappaB, suppressed beta-casein expression and upregulated gelatinases, cytokines and NGF. This model could be used to investigate the role of mammary cells in initiating and propagating inflammation and to test candidate molecules for potential anti-inflammatory properties.
• Sukumvanich, P., DesMarais, V., Sarmiento, C. V., Wang, Y., Ichetovkin, I., Mouneimne, G., Almo, S., & Condeelis, J. (2004). Cellular localization of activated N-WASP using a conformation-sensitive antibody. Cell motility and the cytoskeleton, 59(2), 141-52.
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  The main regulators of Arp2/3 activity appear to be N-WASP and the other members of the Scar/WAVE family of proteins. We show here that after EGF stimulation, N-WASP is recruited to the nucleation zone of the dynamic leading edge compartment of carcinoma cells, with maximal recruitment of N-WASP within 1 min after EGF stimulation. The timing of N-WASP recruitment mirrors the timing of barbed-end formation at the leading edge. To determine the cellular activation of N-WASP after EGF stimulation, we made a conformation-sensitive antibody (CSA) against the CRIB domain of N-WASP that is predicted to recognize N-WASP in its open, active conformation, but not in its closed, inactive conformation. The ability of CSA to detect only active N-WASP was demonstrated by in vitro experiments using immunoprecipitation of active N-WASP from EGF-stimulated cells and Cdc42 activation of N-WASP activity. In cell staining experiments, N-WASP is maximally accessible to CSA 40 sec after EGF stimulation and this activated N-WASP is in the nucleation zone. These results indicate that active N-WASP is present at the leading edge of lamellipods, an unexpected finding given its reported involvement in filopod formation. This work establishes the feasibility of using antibodies directed against specific conformations or epitopes with changing accessibilities as a window on the status and localization of activity.
• Talhouk, R. S., Saadé, N. E., Mouneimne, G., Masaad, C. A., & Safieh-Garabedian, B. (2004). Growth hormone releasing hormone reverses endotoxin-induced localized inflammatory hyperalgesia without reducing the upregulated cytokines, nerve growth factor and gelatinase activity. Progress in neuro-psychopharmacology & biological psychiatry, 28(4), 625-31.
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  During inflammatory processes, the hypothalamic-pituitary axis is activated which can subsequently result in analgesia. For example, hypothalamic corticotrophin-releasing hormone (CRH) that is released during such activation has been attributed with analgesic actions. It is believed that the somatotrophic axis is also activated during inflammation. The aim of this study was to determine the analgesic actions of growth hormone-releasing hormone (GHRH), in a rat model of localized inflammatory hyperalgesia, induced by intraplantar (i.pl.) endotoxin (ET) injections. Pretreatment with intraperitoneal (i.p.) injections of GHRH (2, 5, 10 microg kg(-1)) 30 min before i.pl. ET injection (1.25 microg in 50 microl saline) prevented, in a dose-dependent manner, both mechanical hyperalgesia determined by the paw pressure (PP) test and thermal hyperalgesia determined by the hot plate (HP) and paw immersion (PI) tests. Pretreatment with GHRH had no significant effect on the elevated levels of the inflammatory mediators, interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, IL-6 and nerve growth factor (NGF) due to i.pl. ET injection. No significant effect was obtained by pretreatment with GHRH, on the increased expression of gelatinase B due to ET injection. In conclusion, GHRH reverses inflammatory hyperalgesia in the rat without affecting the upregulated inflammatory mediators and these actions may be clinically important.
• Talhouk, R. S., Hajjar, L., Abou-Gergi, R., Simaa'n, C. J., Mouneimne, G., Saade', N. E., & Safieh-Garabedian, B. (2000). Functional interplay between gelatinases and hyperalgesia in endotoxin-induced localized inflammatory pain. Pain, 84(2-3), 397-405.
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  The role of ECM-degrading proteinases in normal developmental processes and in pathological conditions is extensively studied. However, few reports describe the role ECM-degrading proteinases play in modulating hyperalgesia. The goal of this study is to describe the regulation of gelatinases during endotoxin mediated local inflammation, induced by intra plantar endotoxin (ET; 1.25 microg/50 microl) injection in Balb/c mice, and to correlate that with hyperalgesia. ET injections induced hyperalgesia, as determined by hot plate and paw pressure tests, which peaked by 24 h and recovered by 48 h post-injection. Contralateral paw of ET injected mice and saline injected paws in control mice elicited no hyperalgesia. Zymography showed that ET and saline injected paws elicited increased gelatinase activity by 9 h after injection. However, only the former maintained high levels of expression of a 90 kD gelatinase up to at least 96 h post ET injection, while in the latter gelatinase expression was down regulated by 24 h. Interestingly, the 90-kD gelatinase was upregulated in the contralateral paw of the ET-injected mice beyond 48 h post injection. Saline injection in that paw, during a time when gelatinases are upregulated, induced hyperalgesia. Intraperitoneal injection of either ZnCl(2) (100 microM), thymulin (5 microg/100 microl), or morphine (2 mg/kg/100 microl) reversed the ET-induced hyperalgesia and suppressed gelatinase activity. Furthermore, intraperitoneal injection of MPI, an ECM-degrading proteinase inhibitor, reversed ET induced hyperalgesia. Taken together, the above suggests that a functional interplay exists between gelatinase upregulation triggered by ET injections and hyperalgesia. The exact mechanism underlying such correlation remains to be determined.

Presentations

• Langlais, P. R., Mouneimne, G., Parker, S., Krantz, J., & Barker, N. (2018, June/Summer). Characterization of the G2L1 Interactome Leads to the Discovery that CLIP2, G2L1, and EB1 Undergo Insulin-Stimulated Phosphorylation. American Diabetes Association. Orlando, Florida.
• Mouneimne, G. (2015, January). Distinct Modes of Actin Cytoskeletal Remodeling Control Cancer Cell Invasion.. Invited Speaker. Burlington, Vermont: University of Vermont.

Poster Presentations

• Mouneimne, G., Langlais, P. R., Parker, S. S., Lee, N. Y., Krantz, J., Deer, C. G., Kwak, E., Barker, N. K., Barker, N. K., Kwak, E., Deer, C. G., Krantz, J., Parker, S. S., Lee, N. Y., Langlais, P. R., & Mouneimne, G. (2019, June/Summer). What’s on the Tube? Microtubule-Associated Proteins, Microtubule Stabilization, and Insulin Action.. Federation of American Societies For Experimental Biology - The Regulation of Glucose Metabolism Conference.