Dipyridamole Enhances the Cytotoxicities of Trametinib Against

Published OnlineFirst September 25, 2019; DOI: 10.1158/1535-7163.MCT-19-0413

MOLECULAR CANCER THERAPEUTICS | SMALL MOLECULE THERAPEUTICS

Dipyridamole Enhances the Cytotoxicities of Trametinib against Colon Cancer Cells through Combined Targeting of HMGCS1 and MEK Pathway Sheng Zhou, Huanji Xu, Qiulin Tang, Hongwei Xia, and Feng Bi

ABSTRACT ◥ Both the MAPK pathway and mevalonate (MVA) signaling induced by EGF treatment, while combination with HMGCS1 pathway play an increasingly significant role in the carcinogenesis knockdown could completely reverse the upregulation of HMGCS1 of colorectal carcinoma, whereas the cross-talk between these two induced by EGF treatment and increase the sensitivity of colon pathways and its implication in targeted therapy remains unclear in cancer cells to trametinib. Finally, we combined trametinib and colorectal carcinoma. Here, we identified that HMGCS1 (3-hydroxy- dipyridamole, a common clinically used drug that could suppress the 3-methylglutaryl-CoA synthase 1), the rate-limiting enzyme of the activity of SREBF2 (sterol regulatory element-binding transcription MVA pathway, is overexpressed in colon cancer tissues and posi- factor 2), a transcription factor regulating HMGCS1 expression, and tively regulates the cell proliferation, migration, and invasion of colon identified its synergistic effect in inhibiting the proliferation and cancer cells. In addition, HMGCS1 could enhance the activity of survival of colon cancer cells in vitro as well as the in vivo tumorigenic pERK independent of the MVA pathway, and the suppression of potential of colon cancer cells. Together, the current data indicated HMGCS1 could completely reduce the EGF-induced proliferation of that HMGCS1 may be a novel biomarker, and the combination of colon cancer cells. Furthermore, we found that trametinib, a MEK targeting HMGCS1 and MEK might be a promising therapeutic inhibitor, could only partially abolish the upregulation of HMGCS1 strategy for patients with colon cancer.

Introduction HMGCR, which is an intermediate of MVA (8–9). Recent large- scale genomic studies using cBioPortal have identified that Colorectal cancer is the third most common carcinoma in the world, HMGCS1 genomic amplifications and mutations across various tumor with the 5-year survival of 12%, resulting in about 700,000 deaths types, especially in breast and lung cancers (10). As the intermediate of annually (1–2). Currently, chemotherapy and targeted therapy are still MVA, HMG-CoA could also be catalyzed by HMGCL (3-hydroxy-3- the key strategies for the treatment of metastatic colon cancers. It is methylglutaryl-CoA lyase) to produce ketone bodies. It was reported estimated that over 50% of colon cancers are driven by mutations in the that HMGCS1 could also act as a key ketogenic enzyme to be a RAS gene (3). In addition, RAS oncogenic protein expression connects “synthetic lethal” partner of BRAFV600E in melanoma. There are growth factor signaling to multiple downstream pathways, including two isoforms of HMGCS, cytosolic HMGCS1 and mitochondrial the MAPK pathway and PI3K/AKT signaling pathway (4). Targeting HMGCS2 (11). Interestingly, HMGCS2 is not selectively important the MAPK pathway is thought to be a crucial strategy for colorectal for BRAFV600E cancer cell proliferation. In addition, HMGCS1 is an carcinoma (5). Many drug development efforts have been focused on important gene that could promote cell proliferation and tumor the kinases downstream of RAS, including MEK. In addition, many growth in BRAFV600E-positive melanoma by enhancing pharmaceutical companies have developed MEK kinase inhibitors, but BRAFV600E-dependent MEK1 activation through the HMGCL– the clinical benefit has been disappointing up to now (6–7). Thus, acetoacetate axis (12). However, the role of HMGCS1 signaling in recognizing predictive biomarkers for the MEK inhibitor response and EGF-mediated malignancies and its implication in targeted therapy in finding an effective combination treatment method therapy is essential colon cancer cells remain elusive. for the clinical use in the future. SREBF2 is a transcription factor that participates in the regulation of HMGCS1 is the rate-limiting enzyme of the mevalonate (MVA) lipid metabolism. In response to sterol depletion, SREBF2 could be pathway, catalyzing the synthesis of HMG-CoA, the substrate of cleaved and become activated, resulting in the transcriptional induc- tion of HMGCS1 (13). It was reported that dipyridamole could inhibit the nuclear accumulation of SREBF2 cleavage to decrease the mRNA – Department of Abdominal Oncology, Cancer Center and Laboratory of Molec- expression of HMGCS1 (14 15). Here, we will investigate the regu- ular Targeted Therapy in Oncology, West China Hospital, Sichuan University, lation mechanism of HMGCS1 in colorectal carcinoma and its impli- Chengdu, Sichuan Province, China. cations in targeted therapy. Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). Materials and Methods S. Zhou and H. Xu are co-first authors of this article. Cell culture and reagents Corresponding Authors: Feng Bi, Sichuan University, Chengdu 610041, Sichuan Province, China. Phone: 8628-8516-4044; Fax: 8628-8542-3203, E-mail: Human cancer cell lines originated from the State Key Laboratory of [email protected]; and Hongwei Xia, [email protected] Biotherapy, West China Hospital, Sichuan University. And all the cells were tested and authenticated by an AmpFlSTR Identifiler PCR assays Mol Cancer Ther 2020;19:135–46 in the year of 2019 in TSINGKE Biological Technology Company. The doi: 10.1158/1535-7163.MCT-19-0413 results showed that SW480 (KRAS G12V) and SW1116 (KRASG12A) 2019 American Association for Cancer Research. were 100% matched, and HCT116 (KRAS G13D) was 98% matched.

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All the cell lines were confirmed to be Mycoplasma negative via a RNAi was maintained at least for 1 week. Both nontargeting control, PCR method and directly thawed from the liquid nitrogen jar. HCT siNC, and target-specific siRNA were obtained from GenePharma: si- 116 (KRAS G13D), SW480 (KRAS G12V), and SW1116 (KRASG12A) HMGCS1#1 and shHMGCS1: 50-GCCACAGGAAATGCTAGACC- were grown in DMEM supplemented with 10% FBS (Gibco), 100 TAC-30; si-HMGCS1#2: 50-CAGAGACAAUCAUCGACAATT-30; si- m 0 0 mU/mL of penicillin, and 100 g/mL of streptomycin in a 5% CO2 SREBF2: 5 -GCCCUCUAUUGGAUGAUGC-3 . atmosphere at 37C. All the cell lines used were negative for mycoplasma. Trametinib (GSK1120212) and dipyridamole (HY-B0312) Immunoblotting were from MedChemExpress. Trametinib was prepared at 1 mmol/L For HMGCS1, c-Myc, Cyclin D1, pERK, and ERK protein detection, and dipyridamole was prepared as 50 mmol/L stock solutions in 100% cells were lysed using boiling SDS lysis buffer (1.1% SDS, 11% glycerol, DMSO. Animal-free Recombinant Human EGF (AF-100-15) was 0.1 mol/L Tris at pH 6.8 with 10% b-mercaptoethanol). In addition, the obtained from Peprotech. Tentivirus containing the HMGCS1 protein concentration was determined using the Bradford Protein gene (pHBLV-U6-ZsGreen-Puro-shHMGCS1) was purchased from Assay Kit (BioRad). The proteins were separated by SDS-PAGE and Hanbio Biotechnology Co., Ltd. The following antibodies were used were immunoblotted and transferred to polyvinyl difluoride (PVDF) for WB and IHC staining: Anti-c-Myc, anti-cyclin D1, anti-pERK, and membranes (Millipore) according to standard protocols. Finally, the anti-ERK were from Abcam. Anti-HMGCS1 (A6) and anti-GAPDH blotted proteins were detected and quantified using the ODYSSEY mAb were obtained from Santa Cruz Biotechnology. Infrared Imaging System (LI-COR Biosciences). The following primary antibodies were used: HMGCS1 (1:500), c-Myc (1:2,000), Cyclin CCK-8 assay and colony formation assay D1 (1:10,000), pERK (1:2,000), and ERK (1:2,000). For cell proliferation assays, cells were seeded into 96-well plates at a density of 6 103 cells per well. After treatment with the indicated IHC drugs, 10 mL of CCK-8 solution (Dojindo) was added to each well, and The HMGCS1 levels were examined by IHC in 66 pairs of colon then the plates were incubated at 37C for 2 hours. The optical density cancer patients represented on tissue microarrays that were prepared (OD) was recorded at 450 nm. For the clonogenic assays, cells (1.5 by Shanghai Outdo Biotech Co., Ltd.. The human colon cancer tissues 103) were seeded into 35-mm dishes and cultured for 2 days before were provided by the Ethics Committee of Taizhou Hospital of treatment. The cells were then treated with the drug and cultured for Zhejiang Province. The tissues of xenograft tumors were fixed over- 5 days. So, cells were transfected with siRNA at the time point of 0 and night in 4% paraformaldehyde, embedded in paraffin, and cut into 5- 7 days. At last, remaining cells were fixed with methanol (1%) and mm sections. The sections were subjected to hematoxylin and eosin formaldehyde (1%), stained with 0.5% crystal violet, and photo- staining, as well as IHC staining, following standard protocols. The graphed using a digital scanner. tissue staining intensity and percentage were scored. Staining intensity was scored as 0 (no staining), 1 (weak staining), 2 (moderate staining), EdU assay or 3 (strong staining). The percentage of positive cells was scored as 0 Cell proliferation was also determined by the 5-ethynyl-20-deox- (<1%), 1 (1%–25%), 2 (26%–50%), 3 (51%–75%), or 4 (>75%). And yuridine assay using a Cell-Light EdU Apollo488 In Vitro Imaging Kit comprehensive score ¼ staining percentage intensity. The follow- (Guangzhou RiboBio Co., Ltd.). The cells were then visualized under a ing primary antibodies were used: HMGCS1 (1:100), c-Myc (1:200), fluorescence microscopy (20 10). At last, the ratio of EdU-stained and Cyclin D1 (1:200). cells (with red fluorescence) to Hoechst-stained cells (with blue fluorescence) was calculated. Wound-healing assay Confluent cultures of primary cancer cells were scratched using a Drug combination studies 200-mL pipette tip after overnight starvation. Then, cells were incu- Synergy between trametinib and dipyridamole was evaluated using bated at 37C with the indicated treatments. the combination index (CI; ref. 16). Dose–response curves were generated for trametinib and dipyridamole alone and in combination Cell invasion assay at a constant ratio following compound exposure for 72 hours and For cell invasion assay, 5 104 cells were resuspended in serum-free assessment by the CCK8 assay. CalcuSyn software (Biosoft) was used medium and were added to the upper inserts. Next, 500 mL of medium to evaluate synergy using the median-effects model. supplemented with 10% FBS was added to the lower chamber as a chemoattractant. After 48 hours, the cells migrating to the bottom of Establishment of a trametinib-resistant colon cancer cell line the filters were stained with a three-step stain set (Thermo Fisher The human colon cancer cell line SW480 is sensitive to trametinib Scientific), and the number of cells was counted under a microscope. in vitro. SW480 cells were continuously exposed to gradually increasing concentrations (10, 25, 50, 100, 500, and 1,000 nmol/L) of Tumor xenografting trametinib. The IC50 of SW480 cells to trametinib was initially The animals were handled according to the guidelines of the China determined, and SW480 cells were cultured in medium containing Animal Welfare Legislation, as provided by the Committee on Ethics trametinib at concentrations just below their respective IC50 values. in the Care and Use of Laboratory Animals of Sichuan University West Then, SW480 cells cultured for a further 2 weeks with trametinib at the China Hospital. HCT116 xenografts were generated via injection of 1 next higher concentration. After 5 to 6 months, SW480 was cultured in 106 cells into the flanks of 4- to 5-week-old female NOD/SCID mice medium containing 1 mmol/L trametinib for use in the current study. obtained from Beijing Huafukang Bioscience Co. Inc. The mice (n ¼ 5 animals for each group) were randomized to 6 treatment groups once siRNA transfection the tumors reached an average size of 100 mm3. Trametinib was Transfection was performed with Lipofectamine RNAiMAX (Invi- administered daily at 1 mg/kg via oral gavage. Dipyridamole was trogen) according to the manufacturer's protocol. After 24 hours, the administered daily at 70 mg/kg via intraperitoneal injection. pHBLV- transfected cells were used for subsequent experiments. The effect of U6-ZsGreen-Puro-shHMGCS1 was administered on the first and

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seventh days at 25 mL(1 108 TU/mL) via intratumor injection. The sistent with this finding, HMGCS1 overexpression could also increase vehicle used for trametinib was PBS and that for dipyridamole was pERK expression, but not the total ERK (Fig. 2B). However, inhibition corn oil. The tumor volume was measured using an electronic caliper of the MVA pathway by simvastatin could instead increase HMGCS1 every 3 to 4 days and was calculated using the following formula: length and pERK expression (Supplementary Fig. S2A), in contrast to the (width2) 0.5. effect of HMGCS1 knockdown, indicating HMGCS1 regulates ERK activity independent on the MVA pathway. Previous studies have Statistical analysis found that HMGCS1 could upregulate HMGCL (3-hydroxy-3- Student t test (two-tailed) was used for comparisons between groups methylglutaryl-CoA lyase) and its product acetoacetate (AA), which in cell proliferation assays and gene expression analysis by GraphPad enhances BRAF V600E–dependent activation of MEK1 (19). These Prism 5. Significance was presented as P value <0.05 (), <0.01 (), and results are consistent with our observation that LiAA (lithium acet- <0.001 (); nonsignificant differences were presented as NS. In the oacetate, which can be used as an acetoacetate standard) could also clinical specimen study, c2 test or Fisher test was used for the elevate the expression of pERK (Fig. 2C). Dehydroacetic acid (DAA), associations of HMGCS1 expression with categorical variables. an inhibitory homolog of acetoacetate, could reverse the activating effect of acetoacetate on colon cancer cells (Supplementary Fig. S2B). Further mechanistic studies revealed that DAA treatment decreased Results the expression of pERK (Fig. 2D; Supplementary Fig. S2C), and HMGCS1 is a key oncogene in colon cancer cells effectively inhibited the HMGCS1-induced overexpression of pERK We analyzed the cancer genomics datasets using cBioPortal (17–18), in HCT116 and SW480 cells (Fig. 2E). Similarly, DAA could also showing that the frequency of amplification and mRNA upregulation decrease the upregulation of pERK induced by simvastatin in HCT116 of HMGCS1 was 8% in colon cancers (Fig. 1A), and the transcript and SW480 cells (Fig. 2F). In addition, the pERK upregulation levels of HMGCS1 were robustly higher in patients with colon cancer observed with simvastatin treatment was decreased upon treatment than in normal patients (Fig. 1B). In addition, IHC from the path- with the simvastatin–siHMGCS1 combination (Fig. 2G). These results ologic tissues of 66 patients with colon adenocarcinoma showed that demonstrated that HMGCS1 signals acted through increasing the the expression levels of HMGCS1 in colon tumor tissues were signif- product acetoacetate to promote pERK expression, and this effect icantly higher than that of normal tissue (Fig. 1C). The mean could be reversed by dehydroacetic acid, an inhibitory homolog of immunoreactivity scores were 2.969 and 9.106, respectively acetoacetate. (Fig. 1D), in the normal and cancer tissues. For HMGCS1, the immunoreactivity scores of most of the tumor tissues were 5 to 12 Suppression of HMGCS1 completely reduces EGF-induced cell (high staining), and the percentage was 83.3%, while most of the proliferation and survival of colon cancer cells adjacent normal tissues had a score of 0 to 4 (low staining), and the Activation of the EGFR–RAS–RAF–MEK–MAPK signaling path- percentage was 83.3% (Fig. 1E). Statistical analysis showed that the way could promote cancer cell proliferation, survival, angiogenesis, HMGCS1 protein level was not affected by the patient's age, gender, and migration, and EGF is a crucial ligand of EGFR (20–23). In our and lymph node status (P > 0.05), but was associated with poor study, we found that EGF treatment upregulated the expression levels pathology grade (P < 0.01; Supplementary Table S1). Western blotting of HMGCS1, p-ERK, c-Myc, and Cyclin D1 in HCT116, SW480, and in 18 pairs of colon cancer tissue samples further showed that the SW1116 cell lines by Western blot analysis (Fig. 3A). Functional expression of HMGCS1 was higher in carcinoma region than in distal studies demonstrated that the activated EGFR signaling also promoted normal region (Fig. 1F). Next, we investigated the effect of HMGCS1 cell growth and multiplication by the Cell Counting Kit-8 assay and suppression on the malignant phenotypes of colon cancer cells. Results colony formation assay. Then, we also found that the inhibition of showed that cellular growth was strongly inhibited in colon cancer cells HMGCS1 by si-HMGCS1#1 completely reversed the EGF treatment– with si-HMGCS1#1 and si-HMGCS1#2 transfection compared with induced cell proliferation (Fig. 3B and C; Supplementary Fig. S3A– the cells transfected with the control siRNAs (Fig. 1G–I). Figure 1P–R S3C) and the expression of HMGCS1 and c-Myc (Fig. 3D). Together, and Supplementary Fig. S1A showed the results of Western blot all of the data indicated that activated EGFR–MAPK signaling acted analysis to verify whether si-HMGCS1 performs its inhibition function through increasing HMGCS1 to promote the survival and prolifera- properly. And we concluded that HMGCS1 inhibitory effect was tion of colon cancer cells. stronger than HMGCS2; Edu assays and colony formation assays also indicated that si-HMGCS1#1 could significantly inhibited the prolif- Expression of HMGCS1 determines the response of colon cancer eration and growth of colon cancer cells (Fig. 1J–L; Supplementary cells to trametinib Fig. S1B and S1C; Fig. 1M–O). In addition, si-HMGCS1#1 treatment Trametinib is a clinically used adenosine triphosphate- could decrease the expression of c-Myc and cyclin D1, the prolifer- noncompetitive and MEK inhibitor (20, 24). Here, we found that ation-related proteins (Fig. 1P–R). With the transwell assay and trametinib decreased the expression of HMGCS1 and c-Myc in wound-healing assay, we also detected that the colon cancer cells HCT116 and SW480 cancer cells (Fig. 4A and B) but did not decrease transfected with si-HMGCS1#1 displayed fewer migrated cells than the expression of HMGCS1 in SW1116 cells (Supplementary the vector control (Supplementary Fig. S1D–E, S1F–G). Collectively, Fig. S3D). Next, we screened several colon cancer cell lines for their the results suggested that HMGCS1 could regulate the proliferation response to trametinib and detected the HMGCS1 protein level in and migration of colon cancer cells and served as an oncogene in colon colon cancer cell lines. On the basis of the response to trametinib, we > cancer. selected 4 sensitive (IC50 50 nmol/L) cell lines and 1 resistant (IC50 10 mmol/L) cell line (Fig. 4G). As indicated in Fig. 4H, the SW480, HMGCS1 downregulation reduces pERK level in colon cancer HCT116, COLO320, and RKO cell lines were significantly more cells independent of the MVA pathway sensitive to trametinib than SW1116 cells with the highest expression We found that HMGCS1 knockdown attenuated the phosphory- of HMGCS1. Next, we determined whether HMGCS1 protein also lation of ERK (pERK) in SW480 and HCT116 cells (Fig. 2A). Con- mediated acquired drug resistance of trametinib by inducing the

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Figure 1. HMG-CoA synthase 1 is a key oncogene in colon cancer cells. A, HMGCS1 status in a colon cancer study obtained from http://www.cbioportal.org. B, Transcript levels of HMGCS1 in patients with colon cancer and normal patients obtained from http://ualcan.path.uab.edu/index.html. C, Tissue arrays were stained with H&E. Representative IHC staining results for HMGCS1 in human colon tumor tissue and distal normal tissue. D and E, The immunoreactivity scores of cancer and distal normal tissue samples are represented by black closed circles. Frequency distribution of HMGCS1 staining scores for tumor tissues and normal tissues(0–4: low expression; 5–12: high expression). F, HMGCS1 was identiﬁed and conﬁrmed by Western blot analysis in 18 pairs of colon cancer tissues (F: distal normal tissues; C: colon cancer tissues). G–I, Cell proliferation assays at day 3 after HCT116, SW480, and SW1116 cells were transfected with si-HMGCS1#1 and si-HMGCS1#2 using the CCK8 assay. J–L, Suppression of HMGCS1#1 expression attenuated the proliferation of colon cancer cells by the EdU assay. M–O, Quantitative analysis of clonogenic assays transfected with si-HMGCS1#1 at day 7 in HCT116, SW480, and SW1116 cells. P–R, si-HMGCS1 were introduced into HCT116, SW480, and SW1116 cells by transfection. The lysates of control and HMGCS1 knockdown HCT116, SW480, and SW1116 cells were subjected to Western blotting to detect HMGCS1, c-Myc,and cyclin D1. GAPDH served as a control.

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Figure 2. HMGCS1 downregulation reduces pERK level in colon cancer cells independent of the MVA pathway. A, WB for HMGCS1, pERK, and ERK in HCT116 and SW480 cells treated with si-NC or si-HMGCS1. B, WB for HMGCS1, pERK, and ERK in HCT116 and SW480 cells treated with pcDNA3.1 or pHMGCS1. C, WB for pERK and ERK in HCT116 and SW480 cells treated with the indicated concentrations of LiAA (0–5 mmol/L or 0–0.8 mmol/L) or DMSO for 48 hours. D, WB for pERK and ERK in HCT116 and SW480 cells treated with the indicated concentrations of DAA (0–0.8 mmol/L) for 48 hours. E, WB for HMGCS1, pERK, and ERK in colon cancer cells treated with pHMGCS1 (and/or DAA) for 72 hours. F, WB for HMGCS1, pERK, and ERK in colon cancer cells treated with DAA (or/and simvastatin) for 48 hours. G, WB for HMGCS1, pERK, and ERK in colon cancer cells treated with si-HMGCS1#1 (and/or simvastatin) for 72 hours. trametinib-resistant cell line SW480R, which is 4-fold more resistant to formation (Fig. 5D–F). The Western blotting analysis also showed the trametinib than the parent SW480S cell line (Supplementary Fig. S2E). combination group inhibited HMGCS1, c-Myc, and cyclin D1 more Western blotting demonstrated that the HMGCS1 protein level was thoroughly than the single group (Fig. 5G). significantly elevated in SW480R, the trametinib-resistant cell line In vivo, the combination group of trametinib and shHMGCS1 compared with that in the parent SW480S cell line (Supplementary showed a statistically significant reduction in the tumor volume Fig. S2F). HMGCS1 protein overexpression in both primary (SW1116) compared with the vehicle-treated controls or monotherapy and acquired (SW480R) resistant cell lines suggested the possible role groups in the HCT116 xenografts (Fig. 5I and J). However, there of HMGCS1 in the resistance to trametinib. Further investigations was no significant difference in the body weight (Supplementary demonstrated that trametinib just partially reversed the elevated cell Fig. S2I). The knockdown efficiency of pHBLV-U6-ZsGreen-Puro- number and colony formation induced by EGF stimulation (Fig. 4C shHMGCS1 at the protein level was determined in the HCT116, and D; Supplementary Fig. S2G and S2H). In addition, we also SW480, and SW1116 cell lines using Western blot assay (Fig. 5H). detected that trametinib did not entirely reverse the upregulation of The combination of trametinib and shHMGCS1 significantly HMGCS1, c-Myc, and cyclin D1 induced by EGF treatment compared reduced the expression levels of c-Myc and Cyclin D1 compared with trametinib treatment alone by Western blot assay (Fig. 4E and F). with the monotherapy groups (Fig. 5K). Furthermore, we detected These results suggested that HMGCS1 mediated primary or acquired HMGCS1, c-Myc, and Cyclin D1 expression by immunostaining resistance of trametinib in colon cancer cells. pathologic tissue sections of xenograft tumors. And the overall protein expression levels of these genes were significantly Suppression of HMGCS1 enhances the antitumor property of decreased in the combination group (Fig. 5L–Q). Although tra- trametinib in vitro and in vivo metinib could effectively restrain the survival and growth of colon The combination of HMGCS1 knockdown and trametinib was cancer cells, it did not achieve adequate cytotoxicity and inhibited more effective at restraining cell proliferation (Fig. 5A–C) and colony the overexpression of HMGCS1 induced by EGF, implying that the

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Figure 3. Suppression of HMGCS1 completely reduces EGF-induced cell proliferation and survival of colon cancer cells. A, WB for HMGCS1, c-Myc, Cyclin D1, pERK, and ERK in HCT116, SW480, and SW1116 cells treated with EGF (20 ng/mL) at the indicated times (0.5, 2, 4, 6, and 8 hours) in serum-free medium. B, CCK-8 assay for HCT116, SW480, and SW1116 cells cultured with si-NC or si-HMGCS1#1 (and/or 20 ng/mL EGF) for 3 days (, P < 0.05; , P < 0.01; , P < 0.001). The data are presented as means SD of experiments performed in triplicate. C, Clonogenic assays of HCT116, SW480, and SW1116 cells cultured with si-NC or si-HMGCS1#1 (or/and 20 ng/mL EGF) at day 7 (, P < 0.05; , P < 0.01; , P < 0.001). The data are presented as means SD of the experiments performed in triplicate. D, WB for HMGCS1 and c-Myc in HCT116, SW480, and SW1116 cells treated with si-NC or si-HMGCS1#1 (and/or 20 ng/mL EGF) in serum-free medium for 48 hours.

coinhibition of HMGCS1 and MEK could achieve more dramat- idamole could suppress HMGCS1 through inhibition of cleavage of ically efﬁciency. the transcription factor sterol regulatory element-binding transcription factor 2 (SREBF2; ref. 14). In addition, we found that Antitumor effect of a combination of trametinib and dipyridamole decreased the expression of HMGCS1, c-Myc, and dipyridamole cyclin D1 in the HCT116, SW480, and SW1116 cell lines (Sup- To investigate whether there are FDA-approved inhibitors that plementary Fig. S4A–S4C). Furthermore, CCK8 assays and Edu could interdict the activity ofHMGCS1andinhibitHMGCS1 assays indicated that dipyridamole signiﬁcantly enhanced the formation, we performed a literature review and found that dipyr- cytotoxicity of trametinib in colon cancer cells (Fig. 6A–C, G;

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Figure 4. Expression of HMGCS1 determines the response of colon cancer cells to trametinib. A and B, WB for HMGCS1, c-Myc, pERK, and ERK in HCT116 and SW480 cells treated with the indicated concentrations of trametinib (0–200 nmol/L) or DMSO for 48 hours. C, CCK-8 assay for HCT116 and SW480 cells treated with DMSO or trametinib (10 nmol/L; and/or 20 ng/mL EGF) for 3 days. D, Qualitative analysis of clonogenic assays of HCT116 and SW480 cells cultured with DMSO or 10 nmol/L trametinib (and/or 20 ng/mL EGF) at day 7 (, P < 0.05; , P < 0.01; , P < 0.001). E and F, WB for HMGCS1, c-Myc, and Cyclin D1 in HCT116 and SW480 cells treated with DMSO or 10 nmol/L trametinib for 48 hours (and/or 20 ng/mL EGF) for 2 days. G, CCK-8 assay of SW1116, SW480, HCT116, COLO320, and RKO cells treated with trametinib at 5 nmol/L to 5 mmol/L for 3 days. H, WB to detect HMGCS1 in SW1116, SW480, HCT116, COLO320, and RKO cells. GAPDH served as a control.

Supplementary Fig. S4D). Colony formation assays demonstrated colon cancer cells (Fig. 6D–F). Next, we evaluated whether the that dipyridamole, combined with trametinib, inhibited cell sur- effect of the trametinib–dipyridamole combination was synergistic. vival more signiﬁcantly than trametinib or dipyridamole alone in We disposed cells with increasing concentrations of trametinib and

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Figure 5. Suppression of HMGCS1 enhances the antitumor property of trametinib in vitro and in vivo. A–C, CCK-8 assay for HCT116, SW480, and SW1116 cells treated with si- HMGCS1#1 (and/or 25 nmol/L trametinib) for 3 days. D–F, Clonogenic assays and quantitative analysis of HCT116, SW480, and SW1116 cells cultured with DMSO or 25 nmol/L trametinib (and/or si-HMGCS1#1) at day 7. G, WB for HMGCS1, c-Myc, and Cyclin D1 in HCT116, SW480, and SW1116 cells treated with si-HMGCS1#1 (and/or 25 nmol/L trametinib) for 2 days. H, The relative expression level of HMGCS1 protein in the pHBLV-U6-ZsGreen-Puro-shHMGCS1 group was signiﬁcantly lower than that in the shNC group by the WB assay. I, Tumor formation assays in nude mice subcutaneously injected with HCT116 cells (1 106). When the tumors reached 3 mm in diameter, the mice were given intratumor injection with DMSO, pHBLV-U6-ZsGreen-Puro-shHMGCS1 (1 108 TU/ml), and/or trametinib (1 mg/kg) orally administered daily. J, The tumor sizes were measured after 2 weeks. The graph shows the relative tumor volume of each group (n ¼ 5 animals for each group). Data, means SD. , P < 0.05, , P < 0.01, , P < 0.001 using Student t test (two-tailed). K, The tumors were analyzed for HMGCS1 and proliferation (c-Myc, Cyclin D1) proteins using an immunoblot assay. GAPDH was used as the protein loading control. L, N,andP, Representative IHC staining results for HMGCS1, c-Myc, and Cyclin D1 in xenograft tumor tissues. M, O, and Q, The graph shows the immunoreactivity scores of HMGCS1, c-Myc, and Cyclin D1 in each group (n ¼ 5 animals for each group).

dipyridamole in combination and alone. And CI was used to cell lines (Supplementary Fig. S4E). Western blot analysis revealed evaluate the synergy at multiple effects (16). The combination of that dipyridamole synergized with trametinib to dramatically trametinib with dipyridamole synergistically decreased CCK8 reduce the expression of HMGCS1 and proliferation-related genes, activity at multiple effective concentrations in HCT116 and SW480 c-Myc and cyclin D1 (Fig. 6H). In vivo,asshowninFig. 6I and J,

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Figure 6. Antitumor effect of a combination of trametinib and dipyridamole. A–C, Cell proliferation assays at day 3 for the HCT116, SW480, and SW1116 cells cultured with dipyridamole (50 mmol/L) or DMSO in the presence or absence of 25 nmol/L trametinib. D–F, Clonogenic assays and quantitative analysis of HCT116, SW480, and SW1116 cells cultured with DMSO or 50 mmol/L dipyridamole (and/or 25 nmol/L trametinib) at day 7. G, The combination of trametinib and dipyridamole attenuated the proliferation of colon cancer cells by the EdU assay. H, WB for HMGCS1, c-Myc, and Cyclin D1 in HCT116, SW480, and SW1116 cells treated with 50 mmol/L dipyridamole (and/or 25 nmol/L trametinib). I, Tumor formation assays in nude mice subcutaneously injected with HCT116 cells (1 106). When the tumors reached 3 mm in diameter, the mice were intraperitoneal injection of DMSO, dipyridamole (70 mg/kg), and/or trametinib (1 mg/kg) orally administered daily. J, The graph shows the relative tumor volume of each group (n ¼ 5 animals for each group). K, The tumors were analyzed for HMGCS1 and proliferation (c-Myc, Cyclin D1) proteins using an immunoblot assay. GAPDH was used as the protein loading control. L, N,andP, Representative IHC staining results for HMGCS1, c-Myc, and Cyclin D1 in xenograft tumor tissues. M, O,andQ, The graph shows the immunoreactivity scores of HMGCS1, c-Myc, and Cyclin D1 in each group (n ¼ 5 animals for each group). the combination group of trametinib and dipyridamole showed a IHC to show that the overall protein expression levels of HMGCS1, quite dramatic reduction in the tumor volume compared with c-Myc, and Cyclin D1 were remarkably reduced in the combina- monotherapy groups. Western blotting assays also indicated that tion group (Fig. 6L–Q). Altogether, our ﬁndings unveiled that the combination group signiﬁcantly downregulated the expression combined trametinib with dipyridamole produced synthetic lethal- levels of HMGCS1, c-Myc, and cyclin D1 (Fig. 6K). At last, we used ity in vivo.

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Discussion The HMGCS1 gene, which encodes HMG-CoA synthase 1, is a key rate-limiting enzyme in the cholesterol and isoprenoid biosynthesis Mutations in the RAS/RAF gene are the most common event in pathway (9). Previous studies have demonstrated that HMGCS1 also colon cancer, comprising approximately 39% to 53% of all colo- acts as a key ketogenic enzyme and is specifically important for rectal carcinoma patients (25). Therefore, targeting the down- BRAFV600E-dependent transformation signaling in human melano- stream substrate MEK in KRAS-, BRAF-, or NRAS-mutant tumors ma cells (12). In addition, we used cBioPortal for the analysis of cancer is a major and potential strategy in colon cancer (26). Given the genomics datasets and found HMGCS1 is amplified in various cancers, limited effects MEK inhibitors as monotherapeutic agents, com- including breast and lung cancers (10). In our study, we presented the bination regimens to overcome MEK inhibitor resistance is a highly first evidence that HMGCS1 acts as an oncogene in colon cancer cells possible treatment. In fact, the proposed methods of simultaneous and positively regulates the cell proliferation, migration, and invasion inhibition of the MAPK and PI3K/AKT/mTOR pathways are of colon cancer cells, and the suppression of HMGCS1 by siRNA or potentially useful in various types of cancer (27). Martinelli and dipyridamole could enhance the antitumor property of trametinib in colleagues have reported that compared with monotherapy, the colon cancer cells. MEK1/2 inhibitor pimasertib combined with sorafenib or with the Dipyridamole has been clinically approved for the prevention of PI3K/mTOR inhibitor BEZ235 effectively inhibited tumor growth cerebral ischemia. It is well known that dipyridamole inhibited the and increased survival in mice (28). Halilovic and colleagues (29) cellular reuptake of adenosine into platelets and endothelial cells, observed that the combined therapy of MEK and AKT inhibitors giving rise to increased extracellular concentrations of adeno- increased antitumor activity in KRAS/PIK3CA double-mutant sine (42, 43). Many studies have demonstrated its antitumor activity HCT15 cells. Flanigan and colleagues (30) revealed the synergistic in several cancer types (44–46). Recently, dipyridamole was iden- effects of OSI-906, an (IGF1R)/insulin receptor (IR) tyrosine kinase tified to blunt the expression of rising HMGCS1 following statin inhibitor (TKI) combined with selumetinib and U0126 in vitro and treatment by inhibiting the cleavage of sterol regulatory element- in vivo. With the phenomenon of trametinib resistance, Xue and binding transcription factor 2 (SREBF2; refs. 12–13,47).Tofurther colleagues (4) predicted that the inhibitors of MAP3K1 and investigate whether SREBF2 suppression enhanced the cytotoxicity MAP2K4 showed a synergy effect with MEK inhibition in various of trametinib against colon cancer, we still discovered that the cancers. Manchado and colleagues (31) demonstrated that FGFR1 combination of trametinib and si-SREBF2 was more effective in mediated adaptive resistance to trametinib and validated a com- restraining cell proliferation (Supplementary Fig. S5A and S5B) and binatorial approach to treat KRAS-mutant lung cancer. Levine and colony formation (Supplementary Fig. S5C and S5D). To evaluate Cagan (32) identified that trametinib plus statin led to synergistic whether si-SREBF2 performed its inhibiting function on HMGCS1 suppression of tumor formation of A549 lung adenocarcinoma activity appropriately, Western blot analysis was used to test the cells. effect of si-SREBF2 treatment on the protein level of HMGCS1 MVA pathway metabolites are essential for cancer cell survival (Supplementary Fig. S5E–S5H). The above results were consistent and growth (33). Statins are inhibitors of the HMGCR with with those reported by Ali Talebi1 (48). They found that fatostatin, anticancer efficacy (34, 35), the rate-limiting enzyme of MVA an SREBF inhibitor, could enhance the efficacy of a BRAFV600E pathway. However, in recent years, the clinical studies have shown inhibitor (vemurafenib) both in vitro and in a preclinical PDX that low-dose or high-dose statins and their combination with in vivo model of melanoma. chemical drugs or MAPK-targeted drugs such as afatinib, erlotinib, In summary, we have identified that the HMGCS1 gene acted as and cetuximab demonstrated different efficacies in patients with an oncogene in colon cancer cells and as a novel resistance varioustumors(36–41). To go a step further and validate that biomarker for the MEK inhibitor trametinib. The results in vitro simvastatin acted through increasing the expression of HMGCS1 to and in vivo demonstrated that the suppression of HMGCS1 by upregulate pERK, we used si-SREBF2 and fatostatin (an SREBF siRNA and dipyridamole enhanced the antitumor properties of inhibitor) to decrease the expression of HMGCS1, which could also trametinib in colon cancer cells (Supplementary Fig. S6A–S6B). prevent the increased pERK level by simvastatin treatment alone Notably, our data also provided a novel therapeutic strategy (Supplementary Fig. S4F–S4H). Taken together, we suspected that through combined targeting of HMGCS1 and MEK in colon cancer the feedback activation of HMGCS1 was supposed to be the cells, and further investigations should be carried out to validate this resistance mechanism for statins, which could upregulate the strategy in clinical trials. expression of pERK. As Hegardt reported (11), there may also be other functions of HMGCS1 that may be crucial for the survival of colon cancer; thus, targeting HMGCS1, the upstream of HMGCR, Disclosure of Potential Conflicts of Interest may be a better choice. No potential conflicts of interest were disclosed. In our report, we can draw the conclusion that the overexpression of HMGCS1 might be a novel biomarker of colon cancer cells and Authors’ Contributions mediated trametinib cellular resistance. Because we found that the Conception and design: S. Zhou, H. Xu, Q. Tang, H. Xia, F. Bi HMGCS1 protein levels in trametinib-resistant cell lines were signif- Development of methodology: S. Zhou, F. Bi icantly elevated and suppression of HMGCS1 by siRNA enhanced the Acquisition of data (provided animals, acquired and managed patients, provided susceptibility of primary trametinib resistance cell line (SW1116) and facilities, etc.): S. Zhou an acquired resistance cell line (SW480R), SW480R was established as Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): S. Zhou, H. Xu a model for acquired resistance using a sensitive cell line SW480. Our Writing, review, and/or revision of the manuscript: S. Zhou, H. Xu study results might provide a novel therapeutic strategy for colon Administrative, technical, or material support (i.e., reporting or organizing data, cancer by combined targeting of HMGCS1 and MEK in vitro and constructing databases): S. Zhou in vivo. Study supervision: S. Zhou

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HMGCS1 and MEK Inhibitor Combination for Colon Cancers

Acknowledgments The costs of publication of this article were defrayed in part by the payment of page advertisement We really appreciate the technician Zhang Yi (Core Facility of West China Hospital) charges. This article must therefore be hereby marked in accordance for assistance with experiments and equipment in the pathological analyses. This study with 18 U.S.C. Section 1734 solely to indicate this fact. was supported by the National Natural Science Foundation of China (F. Bi, grant no. 81872020), Sichuan Science and Technology Program (H.W. Xia, grant no. 2019YJ0056), Received April 16, 2019; revised July 19, 2019; accepted September 18, 2019; and Sichuan Science and Technology Program (F. Bi, grant no. 2019YJ0056) published ﬁrst September 25, 2019.

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Dipyridamole Enhances the Cytotoxicities of Trametinib against Colon Cancer Cells through Combined Targeting of HMGCS1 and MEK Pathway

Sheng Zhou, Huanji Xu, Qiulin Tang, et al.

Mol Cancer Ther 2020;19:135-146. Published OnlineFirst September 25, 2019.

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