Resistance to chemotherapy represents a major limitation in the treatment of colorectal cancer. Novel strategies to circumvent resistance are critical to prolonging patient survival. Rac1b, a constitutively activated isoform of the small GTPase Rac1, is upregulated with disease progression and promotes cell proliferation and inhibits apoptosis by activation of NF-kB signaling. Here, we show that Rac1b overexpression correlates with cancer stage and confirmed Rac1b expression is associated with increased growth through enhancing NF-kB activity. Rac1b knockdown reduced cellular proliferation and reduced NF-kB activity. Surprisingly, Rac1b expression and NF-kB activity were upregulated in cells treated with chemotherapeutics, suggesting that Rac1b facilitates chemo-resistance through activation of NF-kB signaling. Knockdown of Rac1b or Rac inhibition increases the sensitivity of the cells to oxaliplatin. When used in combination, inhibition of Rac prevents the increase in NF-kB activity associated with chemotherapy treatment and increases the sensitivity of the cells to oxaliplatin. Although Rac inhibition or oxaliplatin treatment alone reduces the growth of colorectal cancer in vivo, combination therapy results in improved outcomes compared with single agents alone. We provide the first evidence that Rac1b expression confers resistance to chemotherapy in colorectal cancer. Additionally, we show that the use of a Rac inhibitor prevents chemoresistance by blocking activation of chemotherapy induced NF-kB signaling, providing a novel strategy to overcome resistance to chemotherapy in colorectal cancer.

Clear cell renal cell carcinoma (ccRCC) remains a common cause of cancer mortality. Better understanding of ccRCC molecular drivers resulted in the development of antiangiogenic 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 RCC 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 RCC 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.

Prostate cancer remains a common cause of cancer mortality in men. Initially, cancers are dependent of androgens for growth and survival. First line therapies reduce levels of circulating androgens or target the androgen receptor (AR) directly. Although most patients show durable responses, many patients eventually progress to castration-resistant prostate cancer (CRPC) creating a need for alternative treatment options. The Rac1 signaling pathway has previously been implicated as a driver of cancer initiation and disease progression. We investigated the role of HACE1, the E3 ubiquitin ligase for Rac1, in prostate cancer and found that HACE1 is commonly lost resulting in hyperactive Rac signaling leading to enhanced cellular proliferation, motility and viability. Importantly, we show that a Rac inhibitor can attenuate the growth and survival of prostate cancer cells. Rac signaling was also found to be critical in prostate cancers that express the AR. Rac inhibition in androgen dependent cells resulted in reduction of AR target gene expression suggesting that targeting Rac1 may be an alternative method for blocking the AR signaling axis. Finally, when used in combination with AR antagonists, Rac inhibition enhanced the suppression of AR target gene expression. Therefore, targeting Rac in prostate cancer has the potential to enhance the efficacy of approved AR therapies.

<p>Supplementary Figure 1. A) Rac1 DNA copy number in blood, normal prostate, and prostate adenocarcinoma from patients in the TCGA PRAD dataset B) Rac1 mRNA expression of normal prostate and prostate adenocarcinoma from the TCGA PRAD dataset.</p>

<p>Figure S6. (A) Five different colorectal cancer cells showing the sensitivity for GYS32661, KRAS and BRAF mutation and Rac1b expression. (B) HCT116-EV and HCT116-Rac1b cells stably expressing the NFκÎ' reporter was treated with different dose of GYS32661. The cells were lysed in reporter lysis buffer and NFκB reporter activity was measured. The results, presented as the NF-kB-mediated luciferase activity normalized to the protein concentration are the means {plus minus} SEM from three independent experiments. (C) Mice body weight following treatment with GYS32661.</p>