Author Manuscript Published OnlineFirst on May 5, 2020; DOI: 10.1158/1535-7163.MCT-19-0762 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Rac signaling drives clear cell renal carcinoma tumor growth by priming the tumor microenvironment for an angiogenic switch Erik T. Goka1, Pallavi Chaturvedi1, Dayrelis T. Mesa Lopez1, Marc E. Lippman2 1Geneyus LLC, Miami Florida 33136 2Department of Oncology, Georgetown University, Washington, DC 20057 Keywords: Rac1, renal cell carcinoma, angiogenesis, VEGF *To whom correspondence should be addressed: Erik Goka, Ph.D. Geneyus LLC 1951 NW 7th Ave, Suite 300, Miami FL, 33136 email: [email protected] Disclosure of Potential Conflicts of interest E.T. Goka has ownership interest (including stock, patents, etc.) in patents, stock, and is an employee of Geneyus LLC. P. Chaturvedi is an employee of Geneyus. D.T.M. Lopez is an employee of Geneyus LLC. M.E. Lippman is a board member at Geneyus; and has Ownership Interest (including stock.patents, etc.) in Geneyus LLC. 1 Downloaded from mct.aacrjournals.org on September 24, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 5, 2020; DOI: 10.1158/1535-7163.MCT-19-0762 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract Clear cell renal cell carcinoma (ccRCC) remains a common cause of cancer mortality. Better understanding of ccRCC molecular drivers resulted in the development of anti-angiogenic therapies that block the blood vessels that supply tumors with nutrients for growth and metastasis. Unfortunately, most ccRCC patients eventually become resistant to initial treatments creating a need for alternative treatment options. We investigated the role of the small GTPase Rac1 in ccRCC. Analysis of ccRCC clinical samples indicates that Rac signaling drives disease progression and predicts patients with poorer outcomes. Investigation of Rac1 identifies multiple roles for Rac1 in the pathogenesis of ccRCC. Rac1 is overexpressed in renal cell carcinoma cell lines and drives proliferation and migratory/metastatic potential. Rac1 is also critical for endothelial cells to grow and form endothelial tubular networks potentiated by angiogenic factors. Importantly, Rac1 controls paracrine signaling of angiogenic factors including VEGF from renal carcinoma cells to surrounding blood vessels. A novel Rac1 inhibitor impaired the growth and migratory potential of both renal carcinoma cells and endothelial cells and reduced VEGF production by renal cell carcinoma cells thereby limiting paracrine signaling both in vitro and in vivo. Lastly, Rac1 was shown to be downstream of VEGF receptor (VEGFR) signaling and required for activation of MAPK signaling. In combination with VEGFR2 inhibitors, Rac inhibition provides enhanced suppression of angiogenesis. Therefore, targeting Rac in ccRCC has the potential to block the growth of tumor cells, endothelial cell recruitment, and paracrine signaling from tumor cells to other cells in the tumor microenvironment. 2 Downloaded from mct.aacrjournals.org on September 24, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 5, 2020; DOI: 10.1158/1535-7163.MCT-19-0762 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Introduction The incidence and mortality of renal cell carcinoma (RCC) increased worldwide to 403,000 new cases and 175,000 deaths estimated in 2018 (1). Clear cell renal cell carcinoma (ccRCC) accounts for 80% of all RCC (2). Studies of hereditary and sporadic ccRCCs identifies loss of the von Hippel-Lindau (VHL) tumor-suppressor gene as critical through a mechanism involving hypoxia- inducible factor (HIF)(3). VEGF, a HIF regulated gene, is a potent stimulator of new blood vessel formation (4). Angiogenesis is required for tumors to obtain oxygen and nutrients for growth (5). Pro-angiogenic growth factors such as VEGF and basic fibroblast growth factor (bFGF) bind to receptors on endothelial cells and entice vessel branching and growth toward concentration gradients within tumors (6). This led to the use of small-molecule inhibitors such as Sunitinib that target the VEGF Receptor family (VEGFR1-3)(7). Unfortunately, up to 20% of ccRCC patients show de novo resistance to VEGFR inhibitors and patients that do respond become resistant (8). Mechanisms of resistance have yet to be elucidated (9). Next-generation sequencing has provided insight into the genetic complexity of ccRCCs. Only 56% of ccRCC harbor VHL alterations which may explain why anti-angiogenic treatment fails (10). Elucidation of alternative drivers of ccRCC may allow novel therapeutic options for patient treatment. Rac1, a Rho family GTPase that act as a molecular switch, cycles between the active, GTP-bound, and inactive, GDP-bound state (11). Levels of activated Rac1 are tightly regulated by guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and protein stability through proteasomal degradation. In fact, HECT domain and ankyrin repeat containing E3 ubiquitin ligase 1 (HACE1), the E3 ubiquitin ligase for Rac1, was first discovered in Wilms’ tumor, a RCC of childhood (12). HACE1 is lost in multiple cancers resulting in the accumulation of activated Rac1 (13, 14). Rac1 is involved in numerous cellular functions such as migration and invasion, adhesion, proliferation, cellular metabolism, evasion of apoptosis, and reactive oxygen species (ROS) production (15). Hyperactivation of the Rac signaling pathway is common in human cancers and drives tumor initiation, progression, and metastatic dissemination. Overexpression/amplification of Rac1 has been observed in breast, prostate, ovarian, lung, nasopharyngeal carcinoma, leukemia, and gastric cancer (16-20). Hyperactivation of Rac signaling has been implicated in resistance to chemotherapy, radiotherapy, and therapies targeting the EGFR/HER2 family of receptors and BRAF (21-25). Lastly, Rac has been shown to play a role in resistance to bevacizumab, an anti-VEGF therapy (26), suggesting the combinational approach of combining VEGF/VEGFR-targeted therapies with a Rac inhibitor may improve the effectiveness of anti-angiogenic therapies. We investigated Rac signaling in ccRCC and found multiple aberrations of the signaling pathway leading to hyperactivation potentiating aggressive disease and poor patient outcomes. Using molecular and pharmaceutical based approaches, we show that ccRCC is highly susceptible to Rac1 blockade both in 3 Downloaded from mct.aacrjournals.org on September 24, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 5, 2020; DOI: 10.1158/1535-7163.MCT-19-0762 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. vitro and in vivo. We also show that Rac targeting attenuates angiogenesis by directly affecting endothelial cells. Lastly, we show that Rac1 plays a role in the production of angiogenic growth factors in ccRCC and that systemic treatment with a Rac inhibitor can indirectly attenuate the angiogenic response. 4 Downloaded from mct.aacrjournals.org on September 24, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 5, 2020; DOI: 10.1158/1535-7163.MCT-19-0762 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Material and Methods Cell Lines and cell culture. Caki-1 and 786-O cells were purchased from ATCC and cultured in McCoy’s 5A and 769-P cells purchased form ATCC and cultured in RPMI media. HEK293T cells were cultured in DMEM. Caki-1, 786-O, 769-P, and HEK293T cell media were supplemented with 5% (v/v) fetal bovine serum (Invitrogen, Carlsbad, CA). HUVECs were purchased from Lonza and grown in Endothelial Cell Growth Media 2. Cells were cultured at 37°C in a humidified atmosphere of 5% CO2. Mycoplasma testing (Lonza) were conducted every 5 cell passages. GYS32661(21) was manufactured by Patheon. Sunitinib was purchased from Selleckchem. Transfection. For transient knockdown of Rac1 SilencerValidated Human RAC1 siRNA oligos (Cat# AM51334) along with Negative control siRNA (Cat# AM4611) were purchased from Ambion. siRNAs were transfected using RNAiMax (Invitrogen). HACE1 was cloned in pLv105 lentiviaral vector (Genecopoeia) and pLv105 Empty Vector were used to generate stable cell lines. The vectors were cotransfected with plasmid psPAX and pMD2G plasmids into 293T cells using Lipofectamine 2000 (Invitrogen). Viral supernatant was collected 48 h post- transfection, filtered (0.45-μm pore size), and added to cells in the presence of 8 μg/mL polybrene (Sigma–Aldrich). Puromycin (2 μg/mL) was used for selection. Viral supernatant was added to the cells and stable clones were selected for puromycin resistance. The Cancer Genome Atlas data mining. The clear cell renal carcinoma dataset (KIRC) was accessed and mined through the Cancer Genome Atlas (TCGA) Research Network (Provisional 2017) (http://cancergenome.nih.gov/). Kaplan- Meier curves from TCGA-KIRC datasets were generated using KMPLOT (27). Immunoblot assays. Cell lysates were prepared in RIPA lysis buffer. Blots were probed with Rac1 (1:1000 Millipore, clone 23A8), HACE1 (1:1000 abcam, EPR7962), p44/42 MAPK (Erk1/2) (1:5000 Cell Signaling, #4695), Phospho- p44/42 MAPK (Erk1/2) (1:1000 Cell Signaling, #4370), Phospho-VEGF Receptor 2 (Tyr1175)