rac1b overexpression confers resistance to chemotherapy ... · 3/29/2019 · 1 rac1b...

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RAC1b overexpression confers resistance to chemotherapy

treatment in colorectal cancer

Erik T. Goka1*, Pallavi Chaturvedi1*, Dayrelis T. Mesa Lopez1, Adriana De La Garza1, Marc E. Lippman2

1 Geneyus, LLC, Miami, Florida 33136

2 Department of Oncology, Georgetown University, Washington, DC 20057

* Equal contribution

Running Title: RAC1b confers resistance to chemotherapy in colon cancer

Keywords: Rac1b, Rac1, Oxaliplatin, Colorectal cancer

*To whom correspondence should be addressed:

Erik Goka, Ph.D.

Geneyus, LLC

1951 NW 7th Ave, Suite 300 Miami, FL 33136.

Email:[email protected]

Phone: (858) 204-4652

Disclosure of Potential Conflicts of Interest

The authors are employed by Geneyus, LLC and have received salary compensation for time, effort, and hold financial interest in the company. All patents related to the work presented here are held by Geneyus, LLC.

on March 18, 2021. © 2019 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on March 29, 2019; DOI: 10.1158/1535-7163.MCT-18-0955

http://mct.aacrjournals.org/

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Abstract

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 NFκΒ signaling. Here, we show that Rac1b overexpression correlates with cancer stage and confirmed Rac1b expression is associated with increased growth through enhancing NFκB activity. Rac1b knockdown reduced cellular proliferation and reduced NFκB activity. Surprisingly, Rac1b expression and NFκB activity were upregulated in cells treated with chemotherapeutics, suggesting that Rac1b facilitates chemo-resistance through activation of NFκB signaling. Knockdown of Rac1b or Rac inhibtion increases the sensitivity of the cells to oxaliplatin. When used in combination, inhibition of Rac prevents the increase in NFκB activity associated with chemotherapy treatment and increases the sensitivity of the cells to oxaliplatin. While Rac inhibition or oxaliplatin treatment alone reduces the growth of colorectal cancer in vivo, combination therapy results in improved outcomes compared to 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 NFκB signaling providing a novel strategy to overcome resistance to chemotherapy in colorectal cancer.




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Introduction

Colorectal cancer (CRC) is the third most common malignancy worldwide and continues to be a major health concern with over one million men and women being affected annually(1,2). 50% of patients progress to metastatic disease. The average 5-year survival rate for CRC is only 50-60%, resulting in close to 700,000 deaths worldwide in 2012 alone. Although colonoscopy has been effective in reducing mortality by detecting earlier stage disease, the identification of predictive biomarkers of disease progression remains limited.

CRC patients with late stage (stage III and IV) colon cancer are routinely treated with a chemotherapeutic regimen consisting of the combination of oxaliplatin (OXA) and 5-fluorouracil (5FU). Oxaliplatin, a third-generation platinum-based chemotherapy drug (3), has a major impact on the management and outcome of colorectal cancer patients in combination with 5-FU and leucovorin (FOLFOX regimen) or capecitabine (CapeOx regimen) for the adjuvant treatment of stage III (4). Other combined treatments are also in use in stage IV. However, the major obstacle in treating CRC is overcoming acquired resistance to chemotherapy. Targeted therapies have also been used to treat colorectal cancers. However, only a few of these targeted agents have shown activity in CRC (5,6). Therefore, there remains an unmet need for additional treatment options for CRC patients that become resistant to first-line therapy options (7). The discovery of novel drivers of CRC and the development of new compounds targeting these drivers may overcome mechanisms of resistance and improve upon the outcomes currently achieved.

The Rho family of GTPases has been widely studied in numerous types of cancer, including CRC. Identification of a novel splice variant of the Rho GTPase Rac1: Rac1b has piqued interest in Rac signaling in CRC. Rac1b is a naturally occurring splice variant, preferentially expressed in colon and breast tumors (8,9). Rac1b is characterized by the insertion of 19 amino acid residues immediately C-terminal to the “switch II” domain. This insertion greatly reduces the intrinsic GTPase activity of Rac1b and impairs sequestration by RhoGDIs, resulting in Rac1b being preferentially found in a GTP-bound, active form (10). While Rac1 activation strongly activates conical downstream signaling through p21-activated kinases (PAKs) or Jun N-terminal Kinase (JNK), Rac1b has been shown to poorly activate PAK and JNK(11). Instead, Rac1b has been shown to facilitate the proliferation of lung and colon cancer cells, via NF-kB pathway, and inducing Cyclin D1 expression(12,13). The expression of Rac1b in fibroblasts stimulated cell-cycle progression and survival under conditions of serum starvation(14). It has been recently shown that Rac1b mediates an MMP-3-epithelial to mesenchymal transition (EMT) in cultured cells through the induction of Reactive Oxygen Species (ROS)(15). It has also been recently reported that Rac1b promotes canonical Wnt signaling, a pathway often deregulated in CRC(14).

In the present study we show that Rac1b is overexpressed at both the RNA and protein level in CRC patients compared to normal colon tissue. Similar to previous findings, we observe that the overexpression of Rac1b in colon cancer cells enhanced the proliferation of cancer cells via NFκB pathway activation. Additionally, the overexpression




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of Rac1b in HCT116 cells confers resistance to 5FU and OXA while the knock down of Rac1b in HT29 cells sensitizes the cells to 5FU or OXA. Moreover, we observe a significant upregulation of Rac1b protein upon treatment of either 5FU or OXA suggesting that Rac1b up-regulation is part of a stress response of CRC cells to the treatment with these DNA-damaging drugs facilitating resistance through NFκB signaling. Targeting Rac1b with a small molecule Rac inhibitor reduced the growth of colon cancer cells in vitro and in vivo. Importantly, the addition of a Rac inhibitor in combination with 5FU or OXA enhanced the anti-tumoral effects of either agent alone in vitro and in vivo. Our combinatorial approach introduces a strategy to enhance the efficacy of DNA-damaging therapies for the treatment of cancer. Therefore, increased Rac1b levels contribute to the development of chemoresistance to these drugs in CRC.




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Material and Methods

Cell Lines and cell culture. Colon cancer cells HCT116, HT29 cells were cultured in McCoy’s 5A and SW48 and SW480 cells were cultured in Leibovitz’s L-15 medium. Colo320DM cells were cultured in RPMI media. All cell lines were purchased from ATCC. Cells were tested for Mycoplasma (Lonza) every 10 passages. Media were supplemented with 5% (v/v) fetal bovine serum (Invitrogen, Carlsbad, CA) and cultured at 37°C in a humidified atmosphere of 5% CO2. Oxaliplatin (OXA), 5FU, and JSH-23 were purchased

from Sigma-Aldrich.

Transfection. For stable knockdown of Rac1b the siRNA sequences, 5-GAAACGUACGGUAAGGAUA-3 and 5-GGCAAAGACAAGCCGAUUG-3 were cloned in a psi-LVRH1GH lentiviral vector. The vectors were cotransfected with plasmid pCMV-dR8.91 and plasmid encoding vesicular stomatitis virus G protein into 293T cells using Lipofectamine 2000. 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. For Rac1b overexpression, the Rac1b cDNA was cloned in pLv105 lentiviral vector. The transfection was done as mentioned above. Viral supernatant was added to cells and selected for with puromycin.

The Cancer Genome Atlas data mining. The colorectal cancer dataset was accessed and mined through the Cancer Genome Atlas (TCGA) Research Network (Provisional 2017) (http://cancergenome.nih.gov/). Tumor samples with corresponding RNAseq (475 patients) were used for analysis. TCGA data was also obtained using the OncomineTM platform (Thermo Fisher).

Immunoblot assays. Cell lysates were prepared in RIPA lysis buffer. Blots were probed with Rac1b (1:1000 Millipore# 09-27 Rac1 (1:1000 Millipore, clone 23A8), IκΒα, phospho IκΒα and Phospho P65 (1:1000 Cell signaling) Cyclin D1 (1:5000 Abcam, EPR2241) antibodies. Fluorescent tag anti-rabbit (Licor) and anti-mouse (Licor) secondary antibodies were used. Signal was detected using Licor. Blots were re-probed with an anti-actin (Abcam) antibody.

NFκB luciferase assay. Cells were transduced with NFκB luciferase reporter lentiviral particles (Qiagen) per manufacture’s protocol. Constructs were added as indicated in figures. Assays of luciferase activity were done as described previously(16). HCT116 and HT29 cells expressing NFκB reporter were grown to a subconfluent density and treated either with OXA, GYS32661 or with the combination of both. Cell lysates were prepared and reporter activity was measured using the luciferase reporter assay system (Promega).

Rac pull-down experiments. GTP-bound Rac1 and Rac1b levels were determined using a Rac1 activation assay kit according to the manufacturer’s protocol (Pierce Biotechnology, Rockford, IL). Cells were washed in cold PBS and lysed on ice in 600 μl lysis buffer. Total lysates were cleared by centrifugation. Equal amount of lysate was incubated for 30 min at 4°C with PAK binding domain (PBD) agarose beads. Precipitated complexes were washed thrice with lysis buffer. After the final wash, the supernatant was discarded and 40 μl of 2x




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Laemmli buffer were added to the beads. Total lysates and precipitates were then analyzed by Western blot. In vitro pull down experiments were conducted as previously described(17).

Proliferation assay. 1x 103 cells were cultured in 6 well cell culture plates in triplicate. The cells were grown in McCoy’s media supplemented either with 2.5% FBS or 5% FBS. The cells were trypsinized and counted with hemocytometer for 5 consecutive days.

Migration Assays. Cells were seeded in the upper compartment of a 24-well Boyden chamber (8-mm pore size; Costar) and allowed to migrate for 16 h in response to CM in the lower compartment. The cells that migrated to the underside of the filter were stained with crystal violet and counted under bright-field microscopy.

Clonogenic assay (2D colony formation assay). 500 cells per well were seeded in six-well plates, and treated with or without the indicated drugs. At 2–3 weeks after seeding, the colonies were stained with crystal violet.

Soft-agar assay. The soft-agar colony-forming assay was performed in 6-well plates with a base of 2 ml of medium containing 5% FBS with 0.6% agar (Amresco). Cells were seeded in 2 ml of medium containing 5% FBS with 0.35% agar at 1 × 103 (for HCT116 and HT29 cells) cells/well and layered onto the base. 2 ml of DMEM with 10% FBS was covered on the top of agar gel. The photographs of colonies growing in the plates were taken and scored using ImageJ.

Cytotoxicity assays. Cells were plated in 96-well plates (5,000 cells per well), and 24 hours later, various concentrations of chemotherapeutic drugs or GYS32661 were added and incubated for 3 days. Cytotoxicity was measured using a standard PrestoBlue dye (Invitrogen).

Tumor growth. Adult (8–10 weeks of age) SCID mice were used for xenograft studies. HT29 cells or HCT116 (1x106 cells) were injected subcutaneously into the left and right flank of the mice. When the tumors reached approximately 100 mm3, mice were divided into five groups (N=5) for treatment with saline, GYS32661, OXA, and the appropriate combinations of GYS32661 and OXA. The OXA treatment group was injected intraperitoneally with 10 mg/kg OXA twice per week for 3 weeks. The GYS32661 treatment group was injected intraperitoneally 30 mg/kg for 5 consecutive days for 3 weeks. The combination treatment groups received OXA (twice per week, 10 mg/kg), and GYS32661 (5 days, 30 mg/kg). The control group received saline intraperitoneally. Tumor volume and body weight were measured every 3 days. Tumor volume was calculated using the formula V= (AB2)/2, where A is the largest diameter and B is the smallest diameter. These experiments have been conducted in accordance with and approved by the University of Miami Institutional Animal Care and Use Committee. Immunohistochemistry. Immunohistochemical staining for Rac1b was performed on colorectal cancer TMA (US Biomax). Slides were dewaxed with xylene and then hydrated in an ethanol series. Antigen retrieval was done by using citrate buffer solution (pH 6.0) for 30 minutes at 95°C. The endogenous peroxidase activity was blocked using hydrogen




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peroxide. The tissues were blocked with Avidin-biotin blocking system (Vector laboratories, Burlingame, CA). After blocking, slides were incubated with rabbit anti-Rac1b antibody (Millipore, diluted 1:1000) for 1h at room temperature. After incubation with secondary antibody (LSAB Kit, DakoCytomation) the slides were developed with diaminobenzidine tetrahydrochloride (DAB, Sigma-Aldrich, St Louis, MO, USA). Slides were counterstained with hematoxylin. The immunohistochemical slides were scanned into high-resolution images using the Olympus VS120. All digital images obtained in .svs format were visualized with Olympus software. Each TMA spot was examined by two independent reviewers who assigned a score of 0 (no staining), 1 (<10% of malignant cells staining), 2 (10%-50% of malignant cells staining), or 3 (>50% of malignant cells staining) within carcinomatous areas.

Statistical analysis. Data are expressed as mean ± SEM. Differences between 2 groups and multiple groups were analyzed by 2-tailed Student’s t test and 1-way ANOVA, respectively. P-value less than 0.05 was considered significant.




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Results Rac1b is overexpressed in colorectal adenocarcinoma Rac1b has been shown to be upregulated in colon and breast cancers, suggesting an oncogenic role for Rac1b (8,18). To confirm Rac1b overexpression occurs in clinical samples, Rac1b transcripts were queried from The Cancer Genome Atlas (TCGA) CRC (19) patient dataset containing data from 475 CRC patients and 41 normal samples. RNAseq analysis from patients identified all Rac1 transcripts including the transcripts for Rac1 and Rac1b. The level of Rac1b transcript was analyzed from the total Rac1 levels (20) and the ratio of Rac1b over all Rac1 transcripts was calculated (20). Rac1b transcripts were significantly higher (p< 0.0001) in CRC patients as compared to normal colon (Fig. 1A). To determine if Rac1 played a role in CRC progression, Rac1b transcript levels were analyzed in the different stages of CRC. While we observed a trend that higher Rac1b expression correlated with higher colorectal cancer staging, only stage IV (metastatic) CRC was significantly elevated (Fig. 1B). While Rac1 has been observed to be overexpressed in many cancer types(21), transcript levels of Rac1 were not significantly elevated compared to normal colon (Supplementary Figure 1) indicating that Rac1b, and not Rac1, drives colorectal cancer progression. To confirm that Rac1b was elevated at the protein level in CRC patient samples, immunohistochemistry for Rac1b was performed on tissue microarrays containing samples from stage IV metastatic CRC and normal tissue (Fig. 1C). Rac1b staining was observed in normal colon epithelial cells. However, Rac1b staining was significantly higher in CRC metastasis confirming that Rac1b is elevated upon progression to carcinoma and metastatic disease. IHC analysis of Rac1b also confirmed a correlation between Rac1b and CRC stage (Fig. 1D and Supplementary Table S1). Multiple colorectal cancer cell lines obtained from metastatic sites also express high levels of Rac1b protein (Fig 1E). Therefore, an oncogenic splice variant of Rac1, Rac1b, is overexpressed upon progression to colon adenocarcinoma and metastatic disease. Rac1b controls cell proliferation and cell viability through NFκB

Rac1 is canonically involved in cytoskeletal rearrangements, cell cycle progression, cell survival, migration and invasion (22,23). Rac1b however, has been shown to preferentially activate AKT signaling and NFκB resulting in increased CyclinD1 and progression through the cell cycle (24) . To further investigate the role of Rac1b in CRC, Rac1b was stably overexpressed in HCT116 colon adenocarcinoma cells. Rac1b overexpression resulted in increased CyclinD1 expression (Fig. 2A). To confirm the activity of exogenous expression of Rac1b in the HCT116 cells, cellular proliferation assays were carried out on HC116-EV and HCT116-Rac1b overexpressing cells in growth media containing 2.5% and 5% concentrations of serum (Fig 2B and 2C). Rac1b overexpression resulted in a significant increase in cellular proliferation in media containing both 2.5% and 5% serum conditions (Fig 2B and 2C). To test the effects of Rac1b overexpression on growth in three-dimensions, HCT116-EV and HCT116-Rac1b cells were seeded in soft agar (Fig 2D). Rac1b overexpression resulted in a greater than two-fold increase in colony formation in soft agar. Together, these data support the role of Rac1b in enhancing the




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proliferative potential of CRC. To confirm that increased proliferation in Rac1b overexpressing cells was attributed to Rac1b overexpression, HCT116-EV and HCT116-Rac1b cells were engineered to stably express NFκB luciferase reporter. The over expression of Rac1b in HCT116 cells resulted in a greater than 3-fold increase in NFκB reporter activity compared to the empty vector control (Fig 2E). To confirm that Rac1b overexpression was elevating NFκB activity, the addition of JSH-23, a NFκB translocation inhibitor(25,26) was used to block the activation of NFκB activity caused by Rac1b overexpression (Fig. 2E). As expected, JSH-23 attenuated NFκB signaling at previously reported doses(25) (Supplementary Figure 2). Therefore, Rac1b overexpression is sufficient to enhance NFκB activity in colorectal cancer cells resulting in enhanced cellular proliferation. In order to study the dependency of Rac1b in colorectal cells, Rac1b was stably knocked down using a Rac1b specific shRNA. Rac1b shRNA expressing HT29 cells showed ~80% reduction in Rac1b levels compared to a non-silencing shRNA control as determined by Western blot (Fig. 2F). Rac1 levels were not altered, validating the specificity of the shRNA for Rac1b (Fig. 2F). Knockdown of Rac1b in HT29 cells reduced CyclinD1 levels further supporting the role of Rac1 as a determinate of colorectal cancer cell proliferation (Fig. 2A and 2F). To test if Rac1b knockdown effects cell growth, proliferation assays were conducted on shRac1b and shNSC cells. Rac1b knockdown resulted in a significant reduction in proliferation of HT29 cells grown in both 2.5% serum (Fig. 2G) and 5% serum concentrations (Fig. 2H). Knockdown of Rac1b resulted in significant suppression of anchorage-independent growth in soft agar (Fig 2I). To confirm that the molecular knockdown of Rac1b resulted in reduced NFκB activity, shRac1b and shNSC HT29 cells were engineered to express a NFκB luciferase reporter. Similar to previous studies, the knockdown of Rac1b resulted in suppression of NFκB activity (Fig. 2J) 16. Together, these data showed that Rac1b enhances CRC growth and survival by activating NFκB signaling resulting in enhanced CyclinD1 and cellular proliferation. Rac1b expression confers resistance to chemotherapy

5-Fluorouracil (5FU) in combination with oxaliplatin are components of the standard of care regimen for colorectal cancer (27),(28). Previous studies have shown that the NFκB transcription factor becomes activated upon resistance to oxaliplatin (29). Given that Rac1b expression activates NFκB activity, we queried whether Rac1b modulation in colorectal cells changes the sensitivity to chemotherapy. To do so, cellular viability assays were conducted on HCT116 EV and Rac1b overexpressing cells exposed to varying concentrations of OXA (Fig 3A). Rac1b overexpression in HCT116 shifted the sensitivity of cells to OXA compared to control cells with IC50 values of 1.1 μM and 570 nM, respectively (Fig 3A). Similar results were observed when the cells were treated with 5FU (Supplementary Figure 3). To further observe the effects of Rac1b overexpression on cells treated with clinically relevant concentrations of OXA, a 2D colony formation assay was conducted on HCT116-EV and Rac1b cells treated with 500 nM and 1 μM OXA (Fig 3B). To confirm that the growth and survival benefit was attributed to the enhanced NFκB activity, cells were treated with 500 nM OXA along with the NFκB inhibitor JSH-23 (Fig 3C). As expected, cells that overexpressed Rac1b were resistant to the OXA. However, the addition of the NFκB inhibitor re-sensitized the cells to the OXA. These findings were




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recapitulated in a 3D soft agar colony formation assay where Rac1b overexpression resulted in anchorage independent growth in the presence of OXA whereas growth in control cells was diminished (Fig 3D). As observed in the 2D colony formation assay, the treatment of cells with JSH-23 rescued the enhanced growth and survival phenotype observed by Rac1b overexpression (Fig 3E). Therefore, cells that overexpress Rac1b are capable of sustained growth in the continual presence of clinically relevant levels of oxaliplatin due to enhanced activation of NFκB signaling. To determine if the knockdown of Rac1b could sensitize colorectal cells to OXA, shNSC and shRac1b HT29 cells were treated with varying concentrations of OXA and a cellular viability assay was performed. Knockdown of Rac1b resulted in a 2.5 fold increase in sensitivity to OXA shifting the IC50 from 3.4 μM in the control cells down to 1.2 μM in the shRac1b cells (Fig 3F). Similar data was observed with 5FU (Supplemental Figure 4). The Rac1b knock down cells grown under anchorage-independent conditions showed significantly increased sensitivity to OXA compared to control cells (Fig 3G). These results suggest that expression of Rac1b in colon cancer confers resistance to OXA whereas suppression of Rac1b by molecular knockdown can shift the sensitivity of CRC cells to OXA. Together, these data strongly support that Rac1b expression confers resistance to oxaliplatin.

While many patients with colorectal cancer respond initially to treatment, a major reason for the failure of advanced colorectal cancer treatment is the occurrence of chemoresistance to oxaliplatin-based chemotherapy (7). It has become clear that a well-established mechanism of chemoresistance to oxaliplatin-based chemotherapy is the hyper-activation of NFκB pathway (7,30). While treating HCT116 colorectal cancer cells with OXA, we observed a dramatic increase of Rac1b protein expression in HCT116 EV control cells (Fig. 3H). Moreover, a pronounced increase in Rac1b protein was also observed in the HCT116-Rac1b cells that were treated with OXA for the same time period (Fig. 3H). Therefore OXA treatment resulted in an increase in both endogenous as well as exogenous levels of Rac1b. Interestingly, Rac1 levels did not seem to fluctuate upon OXA treatment suggesting that Rac1b is selectively upregulated in response to OXA treatment. Since we have previously shown that increased Rac1b expression enhances NFκB signaling, we performed NFκB luciferase assays on HCT116-control and HCT116-Rac1b cells treated with OXA for 72 hours (Fig. 3I). Consistent with previous observations, OXA increased Rac1b protein expression in both the control HCT116 cells and Rac1b overexpressing cells.

As expected, the HCT116-Rac1b cells had higher basal NFκB activity as compared to HCT116-EV cells. Upon OXA treatment, the HCT116-control cells significantly enhanced NFκB activity as well as the HCT116-Rac1b overexpressing cells. Thus, upregulation of Rac1b activates NFκB signaling and occurs when HCT116 colorectal cancer cells are treated with OXA. Upregulation of Rac1b upon OXA was also observed in HT29 cells indicating that this is not a cell type specific phenomenon (Fig. 3J). Rac1b was also upregulated in a similar manner upon treatment of 5-FU, indicating that upregulation of Rac1b may be a common mechanism for overcoming resistance to chemotherapeutics (Supplementary Figure 5).

To test the effects of Rac1b expression on chemoresistance of colorectal cells in vivo, HCT116-EV control and HCT116-Rac1b overexpressing cells were inoculated subcutaneously into nude mice. Once tumors reached 100 mm3, mice were randomized to




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vehicle or treatment arms. The HCT116-EV tumors treated with the OXA grew significantly slower compared to vehicle treated group (Fig. 3K) However no difference in the tumor volume was observed in HCT116-Rac1b overexpressing cells treated with OXA as compared to vehicle treated group (Fig.3K). The average tumor weight was significantly smaller for HCT116-EV OXA treated as compared to vehicle, but no difference was seen between HCT116-Rac1b treated with OXA as compared to vehicle treated group (Fig. 3L). From these findings we conclude that Rac1b is upregulated upon treatment of colorectal cancer cells with chemotherapeutic agents and provides resistance to chemotherapy. The Rac1b mediated acquired resistance is mediated by activation of the NFκB pathway.

GYS32661 inhibits both Rac1 and Rac1b in colorectal cells

While Rac1 and Rac1b are established drivers of cancer in a number of different malignancies (8),(31),(24), clinically available compounds have yet to be developed. Rac1 inhibitors such as NSC23766 and EHT1864 have moderate effects in a number of different diseases, including cancer (32-35). However, these compounds lack efficacy in vivo. Therefore, there exists a need for inhibitors with improved pharmacologic properties. Modification of the central heterocyclic ring structure to an imidazole while maintaining the quinolone and morphaline moieties improved both the activity of the compound while driving down metabolic clearance as measured by human liver microsomes (hCLint) (Fig. 4A and Fig. 4B). To demonstrate that GYS32661 inhibits Rac1, in vitro pull-down assay using the PAK binding domain (PBD) was conducted with recombinant Rac1 protein (Fig. 4C). Pull-down results confirmed that GYS32661 at IC50 1.18 μM inhibited activated Rac1 (Fig. 4C)

To confirm that GYS32661 is capable of inhibiting endogenous Rac1 and Rac1b in CRC cells, Rac1 and Rac1b in vivo activation assays were conducted using GYS32661 in colorectal cancers cells (Fig. 4D). GYS32661 treatment showed significant inhibition of Rac1 and Rac1b activity at 5 μM in HCT116 colorectal cancer cells (Fig. 4D). Therefore, compound GYS32661 is a potent Rac inhibitor capable of inhibiting both Rac1 and Rac1b in vivo. To investigate the effects of Rac1 and Rac1b inhibition on CRC cell lines, cellular viability assays were performed on a panel of CRC cell lines (Supplementary Figure 6A). All of the CRC cell lines were sensitive to Rac inhibition with IC50 ranging from 2.9 μM to 8.2 μM. (Supplementary Figure 6A). Canonical Rac signaling results in enhanced cell migration and is a prerequisite for tumor invasion and metastasis (36,37). To determine if Rac inhibition would reduce cellular motility, Boyden chamber migration assays were conducted on HCT116 cells. Rac inhibition significantly reduced the migratory ability of HCT116 cells (Fig. 4E). Rac inhibition was able to significantly reduce the number of colonies in the soft agar (Fig. 4F). To confirm that GYS32661 was capable of attenuating NFκB signaling, luciferase assays were conducted on HCT116 cells. Cells were stimulated with TNFα, a potent activator of NFκB signaling in colorectal cancer (38), and incubated with increasing concentrations of GYS32661 (Fig. 4G). Rac1b overexpression in HCT116 cells showed enhanced NFκB reporter activity (Fig. 2E). To study whether GYS32661 would overcome the Rac1b mediated enhanced NFκB reporter activity, HCT116-EV and HCT116-Rac1b cells expressing the NFκΒ reporter was treated with different doses of

GYS32661. 10 M GYS32661 significantly blocked Rac1b mediated NFκB activity (Supplementary Figure 6B). Rac inhibition with GYS32661 significantly attenuated TNFα-




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stimulated NFκB activity. Under conditions when NFκB signaling is inactive, IκBα sequesters NFκB (RelA/p65) in the cytosol. Upon stimulation with TNFα, IKBα, becomes phoshorylated targeting it for proteasomal degradation (39),(40). Once released, RelA/p65 becomes phosphorylated and forms an active dimer with the p50 NFκB subunit. Once the subunits dimerize, NFκB translocates to the nucleus and turns on target gene transcription (39),(40). Additional confirmation of the ability of GYS32661 to inhibit NFκB signaling was performed by observing activation of upstream IKBα as well as phosphorylation of the RelA/p65 subunit by western blot (Fig. 4H). HCT116 cells stimulated with TNFα resulted in phosphorylation of IKBα and P65. However, treatment with GYS32661 attenuated phosphorylation of both IKBα and RelA/p65 (Fig. 4H). These data confirm that inhibition of Rac with GYS32661 inhibits downstream activation of NFκB signaling in colorectal cancer.

To test the effects of Rac inhibition of colorectal cells in vivo, HT29 colorectal cancer cells were inoculated subcutaneously into the flanks of nude mice. Once tumors reached 100 mm3, mice were randomized to treatment groups. The tumors treated with the Rac inhibitor grew significantly slower and the average tumor weight was significantly smaller compared to the vehicle control group (Fig 4I). Moreover, no change in the body weight was observed at the end of the experiment indicating that systemic treatment with a Rac inhibitor is tolerable (Supplemental Figure 6C). Altogether, Rac inhibition resulted in reduced cellular viability, migration, and CRC cell growth both in vitro and in vivo.

Inhibition of Rac sensitizes colorectal cancer cells to Chemotherapy

As we showed above that Rac1b is upregulated in response to oxaliplatin and 5FU treatment resulting in elevated NFκB signaling, we wanted to study whether treatment with the Rac inhibitor will re-sensitize colorectal cancer cells to oxaliplatin. To do so, a colony formation and soft agar assays were conducted on HCT116 cells in the presence of OXA, GYS32661, or the combination of both drugs (Fig 5A and Fig 5B). For colony formation assay the single agent treatment of HCT116 cells with OXA or GYS32661 resulted in a 60% and 55% reduction, respectively. When the two drugs were added in combination however, cellular viability dropped to 95% (Fig 5A). Similarly in soft agar HCT116 cells treated with OXA resulted in a 60% reduction and GYS32661 resulted in a 55% reduction in growth in soft agar. However, the combination of OXA and GYS32661 resulted in 85% reduction in growth in soft agar (Fig. 5B).

To study whether combination treatment overcomes the oxaliplatin-mediated increase in NFκB signaling, HCT116 and HT29 cells expressing the NFκB reporter were treated either with DMSO, GYS32661, OXA or combination of both. As shown above OXA treatment increased the NFκB reporter activity, which was drastically reduced when the cells were treated with OXA and Rac inhibitor (Fig 5C and 5D). These results were in concordance with activation of NFκB signaling pathway, where treatment with OXA or 5FU showed increased IκB and p65 phosphorylation and combining with GYS32661 results in down-regulation of phospho-IκB and phospho-p65 in HT29 colon cancer cell (Fig 5E). Similar results were observed in HCT116 cells were OXA mediated IκB and p65 phosphorylation was attenuated by combining Rac inhibitor GYS32661 (Supplementary Figure 7). To study the in vivo effect of the combination of Rac inhibitor GYS32661 with OXA, we subcutaneously inoculated 1 x 106 HCT116 cells in immunocompromised mice. When the tumors grew 100 mm3, the mice were separated into 4 groups- vehicle, OXA (10 mg/kg), GYS32661 (30 mg/kg) and OXA + GYS32661. The tumors treated with the




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combination grew significantly slower compared to OXA and GYS32661 alone treated tumors (Fig 5F and 5G). Together, we show that combining Rac inhibition with oxaliplatin sensitizes CRC to oxaliplatin by inhibiting the NFκB pathway.




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Discussion In the present study, we demonstrate for the first time that Rac1b plays a critical role in conferring chemoresistance in CRC. In colon cancer cells, expression of Rac1b is upregulated during treatment with the chemotherepeutic drugs 5-FU and oxaliplatin as part of a cellular stress response. The overexpression of Rac1b results in enhanced NFκB signaling driving both cellular growth and survival of colorectal cancer cells (Fig 5H). These results implicate Rac1b as playing a significant role in the development of chemotherapeutic drug resistance in CRC.

The proto-oncogene Rac1 has been reported to drive progression and metastasis of numerous cancers, including CRC. Rac1b, an alternatively spliced variant of Rac1 that is constitutively active, has been previously reported to be upregulated in primary colorectal tumors (18). High Rac1b expression is also reported in breast and lung cancer (8) (10). In a large sample cohort of patient data, our present study showed a similar observation of high Rac1b expression in colorectal adenocarcinoma patients. The expression of Rac1b protein was significantly high in patients with metastasis compared with non-metastatic CRC patients having localized disease and healthy controls. Rac1b has been associated with poor prognosis in KRAS/BRAF WT metastatic CRC patients treated with first-line FOLFOX/XELOX therapy (41). Previous studies demonstrate that Rac1b stimulates cell proliferation by enhancing the NFκΒ mediated signaling in colorectal and thyroid cancer cells(13) (12,24,42). In our study, we also showed that Rac1b expression provides a proliferative advantage to colorectal cancer cells via activation of the NFκΒ pathway. Treatment of unresectable colorectal cancer is empirical and most patients are candidates for FOLFOX therapy as a first-line treatment (43,44). However, approximately 40% of CRC become refractory to FOLFOX. The development of resistance occurs by several processes, like enhanced DNA damage repair, altered expression of oncogenes, reduced apoptosis, decreased drug import, and enhanced tolerance to platinum adduct accumulation (3), (45). One of the major clinical challenges in treating CRC is identifying the subset of patients who will respond to chemotherapy or alternatively, identifying patients that will be resistant to chemotherapy. Studies looking at gene expression signatures of CRC patients have suggested that biomarkers can identify patients that may respond poorly to conventional therapy(46). However, biomarkers of resistance that also guide subsequent treatment options for clinicians are still needed. Overexpression of Rac1b has been associated with poor prognosis in KRAS/BRAF WT metastatic colorectal cancer patients treated with FOLFOX therapy (41). In our studies, we observed that Rac1b expression was upregulated upon 5-FU and oxaliplatin treatment of CRC cells. Expression of Rac1 has been linked to chemo and radioresistance in head and neck squamous cell carcinomas (HNSCC) where Rac1 expression was increased after treatment with cisplatin or ionizing radiation (47). Inhibition of Rac1 in HNSCC cells restores anoiksis, decreases cell motility and enhances cell sensitivity to cisplatin and ionizing radiation (47). Similar results were shown in doxorubicin resistant squamous cell carcinoma (SCC), were pharmacological inhibition of Rac1 restores doxorubicin sensitivity (48). Rac1 expression is also implicated in radioresistance in different cancer cell types such as breast cancer, glioblastoma, pancreatic and cervical cancers (49) (50,51) (52).




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It has now been well established that the sensitivity of CRC to oxaliplatin-induced death is adversely affected by elevated NFκB activity (53,54). Colorectal cancer cell lines with acquired resistance to oxaliplatin showed increased activation of NFκB in comparison to their matched sensitive parental cells, implicating NFκB as a significant mediator of oxaliplatin resistance in these cells(53,54). Our data and others have shown that Rac1b expression activates the NFκB signaling pathway in colon, lung and thyroid cancer (12,13,24). Interestingly, we showed that oxaliplatin treatment results in upregulation of Rac1b, which leads to further enhanced NFκB-mediated signaling and a significant shift in the sensitivity to chemotherapeutic agents. This increase in Rac1b was observed in both endogenous and exogenous Rac1b suggesting that the increase in Rac1b may be regulated in part at the protein level and merits furthers investigation. Nevertheless, the increase in NFκB signaling and subsequent chemoresistance caused by Rac1b upregulation can be reversed through the use of a small molecule inhibitor targeting Rac1 and Rac1b both in vitro and in vivo. Importantly, combining the Rac inhibitor with oxaliplatin resensitizes CRC cells to oxaliplatin and was capable of significantly reducing tumor growth compared to either agent alone. The results suggest the combination of oxaliplatin and a Rac inhibitor may translate into improved outcomes in the clinic.

Overall, in this study we showed that Rac1b overexpression plays an important role in colon cancer progression from normal colon epithelium to colorectal adenocarcinoma and eventually metastatic disease. As Rac1b activates pro-survival pathways such as AKT and NFκB, it is conceivable that Rac1b may be a critical component in the establishment of distal metastases allowing cancer cells to proliferate in a foreign environment and resist the effect of chemotherapeutic drugs. Therefore, Rac1b may be a useful clinical prognostic molecular biomarker for both disease progression as well as a marker for chemo resistance. Lastly, we show evidence that the use of a small molecule inhibitor is both safe and effective in treating colorectal cancer alone or in combination with standard of care therapy.

Acknowledgments We would like to thank Lucas Outcault for his technical assistance.




16

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20

Figure Legends Figure 1. Rac1b is overexpressed in colorectal cancer. (A) Ratio of Rac1b and total Rac1 mRNA transcripts in normal colon and colorectal adenocarcinoma patients. Results are shown as mean ± SEM, **** P < 0.0001. (B) Ratio of Rac1b and total Rac1 mRNA transcripts in normal colon and colorectal adenocarcinoma patients by stage. Results are shown as mean ± SEM, * P < 0.05. (C) Immunohistochemistry of normal colon and metastatic colorectal adenocarcinoma in a tissue microarray stained with Rac1b antibody (D) Immunohistochemistry staining of Rac1b from Stage I, II, and III colorectal adenocarcinoma patient samples. (E) Rac1b and Rac1 protein levels in a panel of colorectal cancer cell lines.




21

Figure 2. Rac1b controls cell proliferation and cell viability through NFκΒ signaling. (A) Western blot analysis on HCT116 cells transduced with empty vector expressing lentivirus (HCT116-EV) and HCT116 transduced with Rac1b expressing lentivirus (HCT116-Rac1b) cells to determine Rac1b expression. Actin was used as a loading control. (B, C) Growth kinetics of HCT116 cells overexpressing either vector (HCT116-EV) or Rac1b (HCT116-Rac1b) grown in 2.5% serum (B) and 5% serum (C) Results are shown as mean ± SEM from three independent experiments. (D) HCT116-EV and HCT116-Rac1b were grown in soft agar to determine the anchorage independent growth. (E) NFκΒ reporter activity as measured by luciferase assay in HCT116-EV and HCT116-Rac1b cells starved and stimulated with 10 ng TNF and treated with JSH-23. Results are shown as mean ± SEM from four replicates. (F) Western blot analysis on the cell lysates isolated from HT29 cells expressing non-silencing shRNA (HT29-shNSC) or Rac1b specific shRNA (HT29-shRac1b) to determine Rac1b, Rac1 and Cyclin D1 expression. (G-H) Growth kinetics of HT29-shNSC or HT29-shRac1b in 2.5% serum (G) and 5% serum (H) Results are shown as mean ± SEM from three independent experiments. (I) Soft agar assay in HT29-shNSC and HT29-shRac1b cells. Representative images (left panel) and results are shown as mean ± SEM from three independent experiments (right panel). (J) NFκΒ reporter activity as measured by luciferase assay in HT29-shNSC and HT29-shRac1b cells. Results are shown as mean ± SEM from three independent experiments. Statistics are reported as: * P < 0.05, ** P < 0.01 and *** P < 0.001.




22

Figure 3. Rac1b expression confers resistance to chemotherapy. (A) 1x 103 HCT116 cells expressing empty vector and Rac1b were plated into 96 well plates and treated with different doses of OXA for 10 days. Error bars represent 95% CI. (B,C) 500 HCT116 empty vector and Rac1b cells were seeded in 12 well plates and treated with different doses of OXA (500 nM or 1 µM) and allowed to grow for 10 days (B) or the combination of OXA and JSH-23 for 10 days. Data is represented at the average and SEM of triplicates (C). Data is represented at the average and SEM of triplicates. (D,E) 1x 103 cells were plated in 0.7% agarose, treated with different doses of OXA (D) or the combination of OXA and JSH-23 (E) and monitored for the formation of colonies. Data is represented at the average and SEM of triplicates. (F) 1x 103 HT29 cells expressing shRNA against scramble control or Rac1b were plated in a 96 well plate and treated with different doses of OXA for 10 days and assayed for cellular viability. Error bars represent 95% CI. (G) 1x 103 cells were plated in 0.7% agarose, treated with different dose of OXA and monitored for the formation of colonies. Triplicate plates were prepared for each line (averages shown) and counted following crystal violet staining. (H) Cells were treated with different doses of OXA (1.25, 2.5, 5.0, 10 μM) for 72 hrs. SDS-PAGE was conducted followed by Western Blotting. The membrane was probed with Rac1b, Rac1 and tubulin antibodies. (I) HCT116EV and HCT116-Rac1b cells stably expressing the NFκΒ reporter were treated with different doses of OXA (0.5, 1.0, 1.5 μM) for 72 hrs and NFκB reporter activity was measured (left panel). SDS-PAGE followed by Western blot was ran in parallel. The membrane was probed with Rac1b, Rac1 and tubulin antibodies (right panel). The results, presented as the NFκB-mediated luciferase activity normalized to the protein concentration are the means ± SEM from three independent experiments. (J) HT29 cells stably expressing the NFκΒ reporter were treated with different doses of OXA (0.5, 1.0, 1.5 μM) for 72hrs (lower panel). SDS-PAGE followed by Western blot was ran in parallel. The membrane was probed with Rac1b, Rac1 and tubulin antibodies (upper panel). The results, presented as the NFκB-mediated luciferase activity normalized to the protein concentration are the means ± SEM from three independent experiments. (K) Tumor volume and (L) Tumor weight established from HCT116 colorectal cancer cells. 1x106

HCT116 EV and HCT116-Rac1b overexpressing cells were injected subcutaneously. Mice were treated either with vehicle or OXA (10 mg/kg/twice weekly) Results are shown as mean ± SEM (n = 4-6). Statistics are reported as: * P < 0.05, ** P < 0.01 and *** P < 0.001.




23

Figure 4. Rac1/Rac1b inhibition in colorectal cells. (A) Structure comparison of EHT1864 and GYS32661. The GYS32661 was derived from the EHT1864 backbone with key modifications (B) Comparison of biochemical properties such as hCLint, FP assay and Alpha Screen of EHT1864, and GYS32661. (C) Increasing concentrations of GYS32661 were incubated with full-length Rac1, in the presence of GTP, followed by pull-down of activated Rac1 using GST-PAK1. Rac1-GTP was visualized by immunoblotting. Densitometry measurements are graphed in lower panel. (D) HCT116 cells were incubated with 5, 10, 20 μM of GYS32661 compound for 1h. The GTP bound active Rac1 was pulled down by GST-PAK1. Activated Rac1 and Rac1b in the samples were analyzed by Western blotting. Normalized densitometry ratios of Pull-down to total cell lysates are below each set of data. (E) Representative image of migrated cells stained with crystal violet. Migration of HCT116 in the presence of 10 μM of GYS32661. Results are shown as mean ± SEM from three independent experiments ** P < 0.01 (F) Representative image of HCT116 colonies grew in soft agar assay in the presence of 6.25 μM of GYS32661. ** P < 0.01 (G) HCT116 cells stably expressing the NFκΒ reporter was treated with different dose of GYS32661. The cells were serum starved for 24h, treated with different dose for GYS32661 for 1h and then stimulated with TNFα for 15 mins. 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 ± SEM from three independent experiments, *** P < 0.001 (H) Cell lysates isolated from HCT116 stimulated with TNFα in the presence of 25 μM of GYS32661. Phospho Iκβα, Phospho P65 and Iκβα were analyzed by western blotting (I) Growth curves of tumors established from HT29 colorectal cancer cells. 1x106 HT29 cells were injected subcutaneously. Mice were treated either with vehicle or GYS32661 (30 mg/kg/day) Results are shown as mean ± SEM (n = 4-6); *, P < 0.05.




24

Figure 5. Rac inhibition sensitizes colorectal cancer cells to Chemotherapy. (A) 1x103

HCT116 cells were seeded for 2D colony formation assay. The cells were either treated with DMSO control, OXA 1.5 μM, GYS32661 5.5 μM and the combination of both. After two weeks the colonies were fixed and stained with crystal violet. Results are shown as mean ± SEM from three independent experiments, * P < 0.05, ** P < 0.01 and *** P < 0.001. (B) HCT116 seeded on soft agar and treated with DMSO control, OXA 1.5 μM, GYS32661 5.5 μM and the combination of both. After three weeks the colonies were fixed and stained with crystal violet. Results are shown as mean ± SEM from three independent experiments * P < 0.05, ** P < 0.01 and *** P < 0.001. (C) HCT116 and (D) HT29 cells stably expressing NFκΒ reporter were treated with OXA (1 μM), 32661 (20 μM) and combination of OXA and GYS32661. The NFkB-mediated luciferase activity was measured as mentioned above. * P < 0.05, ** P < 0.01, *** P < 0.001. (E) HT29 cells were treated with 5FU (25 μM), OXA (5 μM) and GYS32661 (25 μM) and the combination of two, stimulated with TNFα for 10 mins. 50 μg of protein was resolved on SDS-PAGE gel, transferred on the nitrocellulose member. The membrane was probed with phosho IκΒα, total IκΒα, and phospho P65 antibodies. (F) Tumor volume and (G) Tumor weight established from HCT116 colorectal cancer cells. 1x106 HCT116 cells were injected subcutaneously. Mice were treated with vehicle, GYS32661 (30 mg/kg/day), OXA (10 mg/kg twice a week) and combination of two. Results are shown as mean ± SEM (n = 4-6); *, P < 0.05, **P < 0.01. (H) In colon cancer cells Rac1b is upregulated during treatment with the DNA-damaging anti-cancer drugs 5-FU and OXA, as part of the cellular stress response. The overexpression of Rac1b results in enhanced NFκB signaling driving both cellular growth and survival of colorectal cancer cells and provides resistance to chemotherapy. Combining the Rac inhibitor GYS32661 with OXA overcomes this chemoresistance and resensitizes the colorectal cancer cells to chemotherapeutics drugs.




A

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Chemosensitive

Figure 5




Published OnlineFirst March 29, 2019.Mol Cancer Ther Erik T Goka, Pallavi Chaturvedi, Dayrelis T.Mesa Lopez, et al. treatment in colorectal cancerRAC1b overexpression confers resistance to chemotherapy

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rac1b overexpression confers resistance to chemotherapy ... · 3/29/2019 · 1 rac1b...

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