Effect of Simvastatin on Cetuximab Resistance in Human
Effect of Simvastatin on Cetuximab Resistance in Human
Background Metastatic colorectal cancer (CRC) patients with v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations are resistant to treatment with cetuximab, a monoclonal antibody that targets the epidermal growth factor receptor. Statins have reported antitumor activity, but it is unknown whether simvastatin can reverse cetuximab resistance in KRAS mutant CRC.
Methods Human CRC cell lines with KRAS mutations (LS153, LS174T, DLD1, LoVo, SW403, SW480, SNU175, and LS1034) or with v-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutations (DiFi, SW48, HT29, and RKO) were used to test the effect of cetuximab, simvastatin, and cetuximab plus simvastatin on cell proliferation and apoptosis in vitro. Because BRAF mutant may be responsible for cetuximab resistance in KRAS wild-type cells, we measured the growth of xenograft tumors originating from KRAS mutant and BRAF mutant cells in mice treated with cetuximab alone or plus simvastatin (n = 5 mice per treatment group). We used immunoblot assays to study RAS-regulated activation of BRAF protein after simvastatin treatment. All statistical tests were two-sided.
Results Addition of simvastatin (0.2 μM) to cetuximab (0.03–1.0 μM) reduced cell proliferation of KRAS mutant (P < .001) but not of BRAF mutant CRC cells in vitro. Treatment of KRAS mutant cells with simvastatin reduced BRAF activity and induced apoptosis. Treatment with cetuximab and simvastatin reduced the growth of xenograft tumors originating from KRAS mutant cells compared with cetuximab alone (eg, for tumors originating from DLD1 cells, cetuximab vs cetuximab + simvastatin, mean tumor volume = 49.4 vs 20.2 cm, mean difference = 29.2 cm, 95% confidence interval = 19.7 to 38.5, P < .001); treatment with cetuximab alone or in combination with simvastatin had no effect on the growth of BRAF mutant tumors.
Conclusion Simvastatin may overcome cetuximab resistance in colon cancer cells with KRAS mutations by modulating BRAF activity and inducing apoptosis.
Monoclonal antibodies (mAbs) such as cetuximab and panitumumab that target the epidermal growth factor receptor (EGFR) have proven to be efficacious in terms of response rate and progression-free survival in combination with standard cytotoxic chemotherapy in metastatic colorectal cancer (CRC). Recently, mutations in the v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS), which occur in approximately 40% of CRC patients, have emerged as the major negative predictive factor for treatment response in patients receiving cetuximab. Currently, there is no effective treatment for metastatic CRC patients whose tumors harbor KRAS mutations and who have failed to respond to 5-fluorouracil combined with irinotecan or oxaliplatin chemotherapy. The benefit of adding cetuximab to first-line or second-line irinotecan-containing chemotherapy regimens has been addressed in two prospective trials—cetuximab combined with irinotecan in first-line therapy for metastatic CRC (CRYSTAL) (1) and oxaliplatin and cetuximab in first-line treatment of metastatic CRC (OPUS). In the CRYSTAL trial, 1198 metastatic CRC patients were randomly assigned to first-line FOLFIRI (irinotecan, infusional 5-fluorouracil, and leucovorin) treatment with or without cetuximab. Patients with wild-type KRAS who received FOLFIRI treatment with cetuximab showed statistically significantly increased overall survival compared with patients who received FOLFIRI treatment without cetuximab (median overall survival, 23.5 vs 20.0 months; P = .009). In the randomized phase II OPUS trial, addition of cetuximab to FOLFOX (leucovorin, 5-fluorouracil, and oxaliplatin) as first-line treatment was associated with an increased objective response rate compared with FOLFOX chemotherapy alone (cetuximab + FOLFOX vs FOLFOX, 61% vs 37%; odds ratio = 2.54; P = .011) in the KRAS wild-type subset of patients. Nevertheless, in the OPUS trial, there was no medial overall survival benefit in cetuximab plus FOLFOX group compared with FOLFOX group (cetuximab + FOLFOX vs FOLFOX, 22.8 vs 18.5 months, hazard ratio = 0.855; P = .385). However, in patients with KRAS mutations, there was no survival benefit with the addition of cetuximab to FOLFIRI or FOLFOX treatment in both trials. KRAS mutations may appear in many codons, but mutations in codons 12 and 13 of exon 2 and codon 61 of exon 3 are more frequent than others, especially in CRC.
Cetuximab binds to the extracellular domain of EGFR, leading to the inhibition of its downstream signaling pathways, including the RAS-RAF-mitogen-activated protein kinase 1 (MAPK1) axis that is mainly involved in cell proliferation and the v-akt murine thymoma viral oncogene homolog 1(AKT1) pathway that is mainly involved in cell survival and tumor invasion. It has been demonstrated that in the absence of KRAS mutations, resistance to cetuximab or panitumumab could be caused by alterations of other proteins of the RAS-RAF-MAPK1 pathway, such as v-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutation. We postulated that modulation of BRAF activity, which is downstream of RAS, may overcome the resistance to cetuximab or panitumumab in KRAS mutant CRC.
Statins are widely used as lipid-lowering agents to lower cardiovascular risk with a favorable safety profile. Statins, such as lovastatin, simvastatin, pravastatin, and atorvastatin, are 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, which lead to decreased production of farnesyl pyrophosphate and geranylgeranyl pyrophosphate. Farnesyl pyrophosphate and geranylgeranyl pyrophosphate are essential substrates for posttranslational modifications of RAS and ras homolog gene family, member A (RHOA), which play an important role in cellular proliferation. Based on the effect of statin on posttranscriptional modifications of RAS and RHOA, the antitumor effect of statins has been suggested in various cancer cell lines. Furthermore, more recent studies using cancer gene signatures have systematically screened an array of drugs for potential antitumor effect and have discovered statins as potential novel targeted anticancer agents. Given the cardiovascular therapeutic dose of lovastatin is 1 mg kg d, the equivalent dose of simvastatin in terms of potency is half of lovastatin, which is 0.5 mg kg d. Using this concentration, we have previously shown that simvastatin, at a concentration range of 0.1–0.2 μM, induced cell senescence or cytostatic effect of prostate cancer cells in vitro. In addition, we have shown the well tolerability and promising antitumor effect of a combination of 40 mg daily dose of simvastatin and standard FOLFIRI treatment in 49 metastatic CRC patients with disease control rate of 83.7% (95% confidence interval [CI] = 73.4% to 94.0%). In this study, we hypothesized that simvastatin may overcome cetuximab resistance in KRAS mutant CRC cells by modulating the RAS-BRAF pathway. We investigated whether the antitumor activity of cetuximab can be potentiated by simvastatin in colon cancer cell lines with or without KRAS mutations. Functional and biochemical studies were performed in vitro to test the effect of cetuximab, simvastatin, and cetuximab plus simvastatin on cell proliferation and apoptosis. The effect of drugs was tested on tumor growth in mouse xenograft models. We also investigated whether simvastatin has an effect on RAS signaling and modulation of BRAF activity in KRAS wild-type and KRAS mutant cell lines.
Abstract and Introduction
Abstract
Background Metastatic colorectal cancer (CRC) patients with v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations are resistant to treatment with cetuximab, a monoclonal antibody that targets the epidermal growth factor receptor. Statins have reported antitumor activity, but it is unknown whether simvastatin can reverse cetuximab resistance in KRAS mutant CRC.
Methods Human CRC cell lines with KRAS mutations (LS153, LS174T, DLD1, LoVo, SW403, SW480, SNU175, and LS1034) or with v-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutations (DiFi, SW48, HT29, and RKO) were used to test the effect of cetuximab, simvastatin, and cetuximab plus simvastatin on cell proliferation and apoptosis in vitro. Because BRAF mutant may be responsible for cetuximab resistance in KRAS wild-type cells, we measured the growth of xenograft tumors originating from KRAS mutant and BRAF mutant cells in mice treated with cetuximab alone or plus simvastatin (n = 5 mice per treatment group). We used immunoblot assays to study RAS-regulated activation of BRAF protein after simvastatin treatment. All statistical tests were two-sided.
Results Addition of simvastatin (0.2 μM) to cetuximab (0.03–1.0 μM) reduced cell proliferation of KRAS mutant (P < .001) but not of BRAF mutant CRC cells in vitro. Treatment of KRAS mutant cells with simvastatin reduced BRAF activity and induced apoptosis. Treatment with cetuximab and simvastatin reduced the growth of xenograft tumors originating from KRAS mutant cells compared with cetuximab alone (eg, for tumors originating from DLD1 cells, cetuximab vs cetuximab + simvastatin, mean tumor volume = 49.4 vs 20.2 cm, mean difference = 29.2 cm, 95% confidence interval = 19.7 to 38.5, P < .001); treatment with cetuximab alone or in combination with simvastatin had no effect on the growth of BRAF mutant tumors.
Conclusion Simvastatin may overcome cetuximab resistance in colon cancer cells with KRAS mutations by modulating BRAF activity and inducing apoptosis.
Introduction
Monoclonal antibodies (mAbs) such as cetuximab and panitumumab that target the epidermal growth factor receptor (EGFR) have proven to be efficacious in terms of response rate and progression-free survival in combination with standard cytotoxic chemotherapy in metastatic colorectal cancer (CRC). Recently, mutations in the v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS), which occur in approximately 40% of CRC patients, have emerged as the major negative predictive factor for treatment response in patients receiving cetuximab. Currently, there is no effective treatment for metastatic CRC patients whose tumors harbor KRAS mutations and who have failed to respond to 5-fluorouracil combined with irinotecan or oxaliplatin chemotherapy. The benefit of adding cetuximab to first-line or second-line irinotecan-containing chemotherapy regimens has been addressed in two prospective trials—cetuximab combined with irinotecan in first-line therapy for metastatic CRC (CRYSTAL) (1) and oxaliplatin and cetuximab in first-line treatment of metastatic CRC (OPUS). In the CRYSTAL trial, 1198 metastatic CRC patients were randomly assigned to first-line FOLFIRI (irinotecan, infusional 5-fluorouracil, and leucovorin) treatment with or without cetuximab. Patients with wild-type KRAS who received FOLFIRI treatment with cetuximab showed statistically significantly increased overall survival compared with patients who received FOLFIRI treatment without cetuximab (median overall survival, 23.5 vs 20.0 months; P = .009). In the randomized phase II OPUS trial, addition of cetuximab to FOLFOX (leucovorin, 5-fluorouracil, and oxaliplatin) as first-line treatment was associated with an increased objective response rate compared with FOLFOX chemotherapy alone (cetuximab + FOLFOX vs FOLFOX, 61% vs 37%; odds ratio = 2.54; P = .011) in the KRAS wild-type subset of patients. Nevertheless, in the OPUS trial, there was no medial overall survival benefit in cetuximab plus FOLFOX group compared with FOLFOX group (cetuximab + FOLFOX vs FOLFOX, 22.8 vs 18.5 months, hazard ratio = 0.855; P = .385). However, in patients with KRAS mutations, there was no survival benefit with the addition of cetuximab to FOLFIRI or FOLFOX treatment in both trials. KRAS mutations may appear in many codons, but mutations in codons 12 and 13 of exon 2 and codon 61 of exon 3 are more frequent than others, especially in CRC.
Cetuximab binds to the extracellular domain of EGFR, leading to the inhibition of its downstream signaling pathways, including the RAS-RAF-mitogen-activated protein kinase 1 (MAPK1) axis that is mainly involved in cell proliferation and the v-akt murine thymoma viral oncogene homolog 1(AKT1) pathway that is mainly involved in cell survival and tumor invasion. It has been demonstrated that in the absence of KRAS mutations, resistance to cetuximab or panitumumab could be caused by alterations of other proteins of the RAS-RAF-MAPK1 pathway, such as v-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutation. We postulated that modulation of BRAF activity, which is downstream of RAS, may overcome the resistance to cetuximab or panitumumab in KRAS mutant CRC.
Statins are widely used as lipid-lowering agents to lower cardiovascular risk with a favorable safety profile. Statins, such as lovastatin, simvastatin, pravastatin, and atorvastatin, are 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, which lead to decreased production of farnesyl pyrophosphate and geranylgeranyl pyrophosphate. Farnesyl pyrophosphate and geranylgeranyl pyrophosphate are essential substrates for posttranslational modifications of RAS and ras homolog gene family, member A (RHOA), which play an important role in cellular proliferation. Based on the effect of statin on posttranscriptional modifications of RAS and RHOA, the antitumor effect of statins has been suggested in various cancer cell lines. Furthermore, more recent studies using cancer gene signatures have systematically screened an array of drugs for potential antitumor effect and have discovered statins as potential novel targeted anticancer agents. Given the cardiovascular therapeutic dose of lovastatin is 1 mg kg d, the equivalent dose of simvastatin in terms of potency is half of lovastatin, which is 0.5 mg kg d. Using this concentration, we have previously shown that simvastatin, at a concentration range of 0.1–0.2 μM, induced cell senescence or cytostatic effect of prostate cancer cells in vitro. In addition, we have shown the well tolerability and promising antitumor effect of a combination of 40 mg daily dose of simvastatin and standard FOLFIRI treatment in 49 metastatic CRC patients with disease control rate of 83.7% (95% confidence interval [CI] = 73.4% to 94.0%). In this study, we hypothesized that simvastatin may overcome cetuximab resistance in KRAS mutant CRC cells by modulating the RAS-BRAF pathway. We investigated whether the antitumor activity of cetuximab can be potentiated by simvastatin in colon cancer cell lines with or without KRAS mutations. Functional and biochemical studies were performed in vitro to test the effect of cetuximab, simvastatin, and cetuximab plus simvastatin on cell proliferation and apoptosis. The effect of drugs was tested on tumor growth in mouse xenograft models. We also investigated whether simvastatin has an effect on RAS signaling and modulation of BRAF activity in KRAS wild-type and KRAS mutant cell lines.