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Experimental and computational studies pinpoint rate-limiting step(s) in metastasis governed by Rac1. Using ovarian cancer cell and animal models, Rac1 expression was manipulated, and quantitative measurements of cell-cell and cell-substrate adhesion, cell invasion, mesothelial clearance, and peritoneal tumor growth discriminated the tumor behaviors most highly influenced by Rac1. The experimental data were used to parameterize an agent-based computational model simulating peritoneal niche colonization, intravasation, and hematogenous metastasis to distant organs. Increased ovarian cancer cell survival afforded by the more rapid adhesion and intravasation upon Rac1 overexpression is predicted to increase the numbers of and the rates at which tumor cells are disseminated to distant sites. Surprisingly, crowding of cancer cells along the blood vessel was found to decrease the numbers of cells reaching a distant niche irrespective of Rac1 overexpression or knockdown, suggesting that sites for tumor cell intravasation are rate limiting and become accessible if cells intravasate rapidly or are displaced due to diminished viability. Modeling predictions were confirmed through animal studies of Rac1-dependent metastasis to the lung. Collectively, the experimental and modeling approaches identify cell adhesion, rapid intravasation, and survival in the blood as parameters in the ovarian metastatic cascade that are most critically dependent on Rac1.

Epidemiologic studies report improved breast cancer survival in women who receive ketorolac (Toradol) for postoperative pain relief compared with other analgesic agents. Ketorolac is a racemic drug. The S-enantiomer inhibits cyclooxygenases; R-ketorolac is a selective inhibitor of the small GTPases Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42), which are signaling molecules up-regulated during breast cancer progression and metastasis. The goal of this study was to determine whether R-ketorolac altered breast cancer development in the mouse mammary tumor virus-polyoma middle T-antigen model. Mice were administered ketorolac orally at 1 mg/kg twice daily to approximate the typical human dose. Mammary glands were analyzed for tumor number and immunohistochemical markers of proliferation and differentiation. R-ketorolac treatment significantly reduced mammary epithelial proliferation, based on Ki67 staining, and suppressed tumor development. Proliferative mammary epithelium from R-ketorolac–treated mice displayed greater differentiation, based on significantly higher total E-cadherin and decreased keratin 5 staining than epithelium of placebo-treated mice. No differences were detected in estrogen receptor, progesterone receptor, β-catenin, or vimentin expression between placebo and R-ketorolac treatment groups. These findings indicate that R-ketorolac treatment slows tumor progression in an aggressive model of breast cancer. R-ketorolac may thus represent a novel therapeutic approach for breast cancer prevention or treatment based on its pharmacologic activity as a Rac1 and Cdc42 inhibitor.

Aberrant Rac1 signaling contributes to cancer growth and metastasis, therefore, we investigated the effect of altering Rac1 expression and activity to pinpoint which metastatic processes and tumor microenvironment immune factors are influenced by Rac1 signaling. We have identified that the R- enantiomer of ketorolac, a drug given for pain management, is a Rac1 and Cdc42 selective inhibitor. In a retrospective study, we found that treatment with racemic ketorolac significantly increased the survival probability of ovarian cancer patients. RNA seq analyses of ascites tumor cell samples from ovarian cancer patients in a prospective study receiving racemic ketorolac for clinically indicated use in pain relief, showed significant downregulation of genes involved in endocytosis, regulation of actin cytoskeleton, ER protein processing, TNF and NOD signaling. To further distinguish the activities most directly regulated by Rac1 and important in ovarian cancer, we tested the impact of manipulating the expression of Rac1 in ovarian cancer cells on adhesive and invasive cell behaviors, as well as the impacts of gene expression relative to ketorolac treatment. Using electric cell-substrate impedance sensing (ECIS), Rac1 overexpression resulted in increased cell-cell barrier formation and cell adhesion. Whereas, CRISPR-CAS9 mediated knockdown of Rac1 resulted in decreased cell adhesion. Using Matrigel invasion chambers and mesothelial clearance assays, Rac1 overexpression led to increased ovarian cancer cell invasion when compared to parental and Rac1 knockdown cell lines. Furthermore, intraperitoneal growth of ovarian cancer cells overexpression Rac1 relative to parental control lines increased tumor burden in vivo. Conversely, with knockdown of Rac1, tumor burden and omental adhesion was decreased, as quantified using IVIS bioluminescence imaging. RNA seq analyses of tumors manipulated for Rac1 as compared to R-ketorolac will be presented. The composite data indicate that targeting Rac1 in ovarian cancer with R-ketorolac or otherwise has therapeutic benefit in decreasing tumor cell dissemination and metastasis. Citation Format: Melanie Rivera, Martha M. Grimes, Laurie G. Hudson, Angela Wandinger-Ness. Rac1 as a driver and therapeutic target in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2898.

The small GTPase Rac1 (Ras-related C3 botulinum toxin substrate 1) is commonly overexpressed in cancer and high Rac1 levels are associated with poor patient outcomes in ovarian cancer. Rac1 drives multiple hallmarks of cancer such as proliferation, quiescence, epithelial-to-mesenchymal transition (EMT), and transcriptional programs relevant to tumor development, progression, and chemoresistance. EMT promotes metastatic dissemination, chemoresistance, and cancer stem cell properties. We find that modest overexpression of Rac1 in ovarian tumor cells causes morphologic changes consistent with EMT. Elevated Rac1 (4- fold) expression decreases colony number and increases colony area when compared to vector control cells. In addition, cell invasion through an artificial basement membrane is increased by 10-fold in cells overexpressing Rac1. An increase in mesenchymal markers (e.g., N-cadherin) and decrease in epithelial markers (e.g., E-cadherin) was observed as a consequence of Rac1 expression. Rac1 overexpression was associated with selective upregulation of Slug/Snai2 (8-fold) and Zeb2 (100-fold) mRNA levels with little impact on Snai1, Twist, or Zeb1. Slug/Snai2 expression is reportedly induced by oxidative stress and treatment of six ovarian tumor cell lines with tert-butyl hydroperoxide (TBHP) to generate oxidative stress increased the expression Slug/Snai2, Snai1, and heme oxygenase-1, an indicator of oxidative stress response. This finding confirms that elevated oxidative stress regulates certain EMT transcription factors in various ovarian cancer cells. Rac1 is an essential factor for activation of NADPH oxidases 1, 2, and 3 and generation of reactive oxygen species (ROS). Basal level of ROS was elevated in cells overexpressing Rac1 compared to vector control cells as detected by the fluorescent probe Dihydroethidium (DHE). Similarly, elevated Rac1 increased expression of the oxidative stress response genes heme oxygenase-1 (17-fold) and superoxide dismutase-1 (12-fold), further supporting that Rac1 stimulated ROS signaling in these cells. We further tested the impact of Rac1 inhibition in vivo using R-ketorolac. Analysis of tumor tissue from mice showed decreased expression of heme-oxygenase-1 and Slug/Snai2 in mice treated with R-ketorolac compared to placebo controls. R-ketorolac also decreased invasive implantation and expansion of ovarian cancer xenografts. Taken together, these findings illustrate the functional impact of Rac1 overexpression in ovarian cancer cells and suggest that Rac1 may be a viable target for further drug development to attenuate EMT in ovarian tumors. Citation Format: Martha M. Grimes, Dayna R. Dominquez, Michaela L. Granados, Alejandra Rosales, Melanie Rivera, Angela Wandinger-Ness, Laurie G. Hudson. Rac1 overexpression promotes epithelial-to-mesenchymal transition in ovarian cancer cells [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B59.

Rac1 is a high-value therapeutic target for cancer based on its tumor-promoting activities, yet clinical applications targeting Rac1 are in their infancy. High expression and hyperactivation of Rac1 in ovarian cancer, along with our identification of R-ketorolac as a novel Rac1 and Cdc42 selective inhibitor with translational potential, prompt us to test the hypothesis that targeting Rac1 has therapeutic utility for ovarian cancer. Ascites tumor cell samples from ovarian cancer patients in a prospective study receiving racemic ketorolac for clinically indicated use in pain relief were previously reported to show time-dependent reduction of Rac1 and Cdc42 activities post-treatment. New RNA seq data of these patient samples reveals significant downregulation of genes involved in endocytosis, regulation of actin cytoskeleton, ER protein processing, TNF and NOD signaling. Conversely, the identified downregulated genes were overexpressed and associated with worse survival in ovarian cancer patients analyzed through The Cancer Genome Atlas (TCGA). Among the downregulated genes in the NOD pathway are chemokines and proinflammatory cytokines. Follow-up cytokine panels from patients confirm that racemic ketorolac treatment reduces the levels of immunosuppressive cytokines IL-6, IL-10, and RANTES in ascites fluids. Together, these data indicate there may be a benefit to the anti-inflammatory activity of the S- enantiomer, as well as the GTPase inhibitory activity of the R-enantiomer of ketorolac for ovarian cancer treatment. Citation Format: Melanie Rivera, Martha M. Grimes, Dayna Dominguez, S. Ray Kenney, Yuna Guo, Elsa Romero, Kathryn J. Brayer, Yan Guo, Scott A. Ness, Sarah F. Adams, Carolyn Muller, Laurie G. Hudson, Angela Wandinger-Ness. Rac1 as a therapeutic target in ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B70.

International audience; The addition of slightly (CaCO3) and highly soluble (Na2CO3) carbonate salts is expected to favor the formation of carboaluminate phases in hydrated calcium aluminate cements (CACs). A multi-method approach including X-ray diffraction, thermogravimetric analysis, and thermodynamic calculations is applied to highlight that the “conversion phenomena” in CACs cannot be mitigated by the formation of carboaluminate phases (monocarboaluminate: Mc and hemicarboaluminate: Hc) which are anticipated to form following the addition of carbonate salts. Here, carboaluminate phase formation is shown to depend on three factors: (1) water availability, (2) carbonate content of the salts, and their ability to mobilize CO3 2− species in solution, and (3) lime content associated with the carbonate salt. The latter two factors are linked to the composition and solubility of the carbonate agent. It is concluded that limestone (CaCO3), despite being a source of calcium and carbonate species, contributes only slightly to carboaluminate phase formation due to its low solubility and slow dissolution rate. Soluble carbonate salts (Na2CO3) fail to boost carboaluminate phase formation as the availability of Ca2+ ions and water are limiting. Detailed thermodynamic calculations are used to elucidate conditions that affect the formation of carboaluminate phases.

The tumor microenvironment for epithelial ovarian cancer is complex and rich in bioactive molecules that modulate cell-cell interactions and stimulate numerous signal transduction cascades. These signals ultimately modulate all aspects of tumor behavior including progression, metastasis and therapeutic response. Many of the signaling pathways converge on the small GTPase Ras-related C3 botulinum toxin substrate (Rac)1. In addition to regulating actin cytoskeleton remodeling necessary for tumor cell adhesion, migration and invasion, Rac1 through its downstream effectors, regulates cancer cell survival, tumor angiogenesis, phenotypic plasticity, quiescence, and resistance to therapeutics. In this review we discuss evidence for Rac1 activation within the ovarian tumor microenvironment, mechanisms of Rac1 dysregulation as they apply to ovarian cancer, and the potential benefits of targeting aberrant Rac1 activity in this disease. The potential for Rac1 contribution to extraperitoneal dissemination of ovarian cancer is addressed.

Five-year ovarian cancer patient survival has not improved for decades, largely due to disease recurrence. Therefore, drivers of relapse need to be identified. We first reported that Rac1 and Cdc42 GTPases are highly overexpressed in serous ovarian cancer using immunohistochemistry. To correlate GTPase expression with survival outcomes, we analyzed gene expression data in The Cancer Genome Atlas, revealing that ovarian cancer patients with the highest RAC1 RNA levels had significantly worse age-adjusted survival than those with low RAC1. Quantitative analyses of Rac1b, a constitutively active Rac1 splice variant, show dramatic overexpression in tumor epithelia relative to stroma and normal epithelia. Since high Rac1 and Cdc42 levels drive cancer cell adhesion, invasion and metastasis, we used multiple assays to assess the dependence of ovarian cancer cell behaviors on Rac1 and Cdc42. Using Matrigel invasion chambers, treatment of multiple ovarian cancer cell lines with specific Rac1 and Cdc42 inhibitors resulted in significantly decreased cellular invasion. Conversely, with overexpression of Rac1, ovarian cancer cells demonstrated increased invasion. Furthermore, IP injection of ovarian cancer cells overexpressing Rac1 resulted in increased adhesion, invasion and metastasis in the omentum and peritoneum in vivo. These data support Rac1 and Cdc42 as important drivers of invasive and metastatic ovarian cancer and as novel high-value therapeutic targets with potential impact in reducing disease metastasis. Citation Format: Melanie Rivera, Dayna Dominguez, Christine Pauken, Elsa Romero, S. Ray Kenney, Yang Shi, Ji-Hyun Lee, Jennifer Gillette, Laurie G. Hudson, Angela Wandinger-Ness. Rac1 and Cdc42 as drivers in ovarian cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3159.

PURPOSE OF THE STUDY: The Ras-homologous (Rho) family GTPases Rac1 and Cdc42 contribute to metastatic dissemination through regulation of actin reorganization, cell motility, cell-cell and cell-extracellular matrix adhesion. The purpose of the study was to investigate the contributions of these GTPases in ovarian cancer progression. EXPERIMENTAL PROCEDURES: GTPase target expression and activity was determined by analysis of The Cancer Genome Atlas (TCGA), immunohistochemistry of ovarian cancer tissues and enzymatic assays of patient samples. The effects Rac1 and Cdc42 expression were investigated by comparing cell behaviors in vitro and in vivo in engineered cell lines and use of inhibitors of Rac1, Cdc42 or dual inhibition by the R-enantiomer of ketorolac. SUMMARY OF THE DATA: Expression of the constitutively active Rac1 splice variant Rac1b and elevation of Cdc42 protein were detected in advanced ovarian cancer specimens thus providing the first evidence for dysregulation of these GTPase targets in ovarian cancer. To confirm the importance of Rac1, RNA-seq gene expression data from 305 Stage III or IV ovarian cancer samples were downloaded from The Cancer Genome Atlas (TCGA) and evaluated for RAC1 gene expression. The samples with the highest Rac1 RNA levels had significantly worse (p=0.039) age adjusted survival than the patients with the lowest Rac1 levels. Additional analyses identified more than 4,000 genes that were differentially expressed (FDR corrected p CONCLUSIONS: These findings indicate that high expression of Rac1 is associated with late stage disease and/or poor patient survival and with genes involved in extracellular matrix, cell proliferation and cell migration. These functions of Rac1 were confirmed in experimental models and inhibition of protein activity provided anti-tumor benefit in vivo. Together, these data suggest that Rac1 and/or Cdc42 function as drivers of ovarian cancer and may represent novel therapeutic targets to improve ovarian cancer outcome. Citation Format: Hudson LG, Kenney SR, Rivera, M., Weisend, J. Luo, L., Ness, S., Gillette, JM, Wandinger-Ness A. RAC 1 AND CDC42 GTPASES AS REGULATORS OF OVARIAN CANCER PROGRESSION AND NICHE INTERACTIONS [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr MIP-061.