Sinicrope, Frank A.

AbstractBackground.Preclinical and epidemiological data suggest that metformin might have antineoplastic properties against colon cancer (CC). However, the effect of metformin use on patient survival in stage III CC after curative resection is unknown. The survival outcomes were comparable regardless of the duration of metformin use.Patients and Methods.Before randomization to FOLFOX (folinic acid, 5-fluorouracil, oxaliplatin) with or without cetuximab, 1,958 patients with stage III CC enrolled in the N0147 study completed a questionnaire with information on diabetes mellitus (DM) and metformin use. Cox models were used to assess the association between metformin use and disease-free survival (DFS), overall survival (OS), and the time to recurrence (TTR), adjusting for clinical and/or pathological factors.Results.Of the 1,958 patients, 1,691 (86%) reported no history of DM, 115 reported DM with metformin use (6%), and 152 reported DM without metformin use (8%). The adjuvant treatment arms were pooled, because metformin use showed homogeneous effects on outcomes across the two arms. Among the patients with DM (n = 267), DFS (adjusted hazard ratio [aHR], 0.90; 95% confidence interval [CI], 0.59–1.35; p = .60), OS (aHR, 0.99; 95% CI, 0.65–1.49; p = .95), and TTR (aHR, 0.87; 95% CI, 0.56–1.35; p = .53) were not different for the metformin users compared with the nonusers after adjusting for tumor and patient factors. The survival outcomes were comparable regardless of the duration of metformin use (<1, 1–5, 6–10, ≥11 years) before randomization (ptrend = .64 for DFS, ptrend = .84 for OS, and ptrend = .87 for TTR). No interaction effects were observed between metformin use and KRAS, BRAF mutation status, tumor site, T/N stage, gender, or age.Conclusions.Patients with stage III CC undergoing adjuvant chemotherapy who used metformin before the diagnosis of CC experienced DFS, OS, and TTR similar to those for non-DM patients and DM patients without metformin use.

Abstract Oncogenic BRAFV600E mutations activate MAPK signaling and are associated with treatment resistance and poor prognosis in patients with colorectal cancer. In BRAFV600E-mutant colorectal cancers, treatment failure may be related to BRAFV600E-mediated apoptosis resistance that occurs by an as yet undefined mechanism. We found that BRAFV600E can upregulate anti-apoptotic MCL-1 in a gene dose-dependent manner using colorectal cancer cell lines isogenic for BRAF. BRAFV600E-induced MCL-1 upregulation was confirmed by ectopic BRAFV600E expression that activated MEK/ERK signaling to phosphorylate (MCL-1Thr163) and stabilize MCL-1. Upregulation of MCL-1 was mediated by MEK/ERK shown by the ability of ERK siRNA to suppress MCL-1. Stabilization of MCL-1 by phosphorylation was shown by a phosphorylation-mimicking mutant and an unphosphorylated MCL-1 mutant that decreased or increased MCL-1 protein turnover, respectively. MEK/ERK inhibition by cobimetinib suppressed MCL-1 expression/phosphorylation and induced proapoptotic BIM to a greater extent than did vemurafenib in BRAFV600E cell lines. MCL-1 knockdown versus control shRNA significantly enhanced cobimetinib-induced apoptosis in vitro and in HT29 colon cancer xenografts. The small-molecule MCL-1 inhibitor, A-1210477, also enhanced cobimetinib-induced apoptosis in vitro that was due to disruption of the interaction of MCL-1 with proapoptotic BAK and BIM. Knockdown of BIM attenuated BAX, but not BAK, activation by cobimetinib plus A-1210477. In summary, BRAFV600E-mediated MEK/ERK activation can upregulate MCL-1 by phosphorylation/stabilization to confer apoptosis resistance that can be reversed by MCL-1 antagonism combined with cobimetinib, suggesting a novel therapeutic strategy against BRAFV600E-mutant CRCs. Mol Cancer Ther; 15(12); 3015–27. ©2016 AACR.

Abstract Background. Oncogenic BRAFV600E mutations in colorectal cancer (CRC) are associated with poor prognosis and treatment resistance. BRAF mutations activate MAP kinase signaling and may confer apoptosis resistance by upregulating anti-apoptotic BCL-2/BCL-XL/MCL-1 proteins. Results. Ectopic expression of mutant BRAFV600E was shown to potently activate MEK/ ERK that mediated the phosphorylation (T163) and upregulation of MCL-1, but not BCL-2 or BCL-XL. MEK/ERK-mediated phosphorylation of MCL-1 (T163) increased its protein stability, shown by an MCL-1 phospho-mimic mutant. In human colon cancers, mutant vs wild-type BRAF was associated with increased MCL-1 expression by IHC. MEK/ERK inhibition by cobimetinib induced pro-apoptotic BIM in BRAF mutant CRC cell lines (HT-29, RKO), and suppressed phospho-MCL-1/MCL-1 proteins as did ERK siRNA. Suppression of MCL-1 by shRNA or using a small molecule MCL-1 inhibitor (A-1210477) synergistically enhanced cobimetinib-induced apoptosis (Table) that was associated with BAK/BAX activation. A-1210477 antagonized MCL-1 by disrupting its interaction with BAK and BIM proteins, shown by immunoprecipitation. Conclusion. BRAF/MEK/ERK induces upregulation of MCL-1 via phosphorylation/stabilization to confer apoptosis resistance that can be overcome by combined ERK and MCL-1 inhibition, suggesting a novel therapeutic strategy against BRAF mutant CRCs. Drug-induced apoptosis (annexin V) in CRC cell linesDrugsHT-29 % apoptosisRKO % apoptosisDMSO16.842.11A-1210477 (2.5 μM)29.5222.88Cobimetinib(5 μM)28.0421.05Combination53.2250.93 Citation Format: Hisato Kawakami, Shengbing Huang, Frank A. Sinicrope. MEK/ERK and MCL-1 inhibition synergistically reverse apoptosis resistance in colon cancer cells with BRAF(V600E)-mediated MCL-1-upregulation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2115.

Although HER2‐positive breast cancers demonstrate a propensity for central nervous system (CNS) metastasis, it is unknown whether other HER2‐positive tumors, including adenocarcinomas of the esophagus/gastroesophageal junction (EAC), share this characteristic. Insight into this association may inform the development of HER2‐targeted therapies that penetrate the blood‐brain barrier. We examined HER2 overexpression and gene amplification in 708 patients with EAC who underwent curative‐intent surgery during a time period (1980–1997) when no patient received HER2‐targeted therapy. We identified patients whose site of first cancer recurrence was CNS and those who had a CNS relapse at any time. After a median follow‐up of 61.2 months, 3.4% (24/708) of patients developed CNS relapse (all involved the brain). Patients with HER2‐positive (vs ‐negative) primary tumors showed a higher 5‐year cumulative incidence of CNS relapse as first recurrence (5.8% vs. 1.2%; p = 0.0058) and at any time (8.3% vs. 2.4%; p = 0.0062). In a multivariable model that included covariates previously associated with HER2 or with CNS relapse in breast cancer, HER2 positivity was the only variable that was statistically significantly associated with shorter time to CNS relapse as first recurrence (p = 0.0026) or at any time (hazard ratio 4.3 [95% confidence interval 1.8 to 10.3]; p = 0.001). These are the first data in a non‐breast cancer to demonstrate an association between HER2 positivity and higher CNS relapse risk after surgery, and suggest that HER2‐positive EACs have a predilection for CNS metastases.

Alcohol consumption is associated with a modest increased risk of colon cancer, but its relationship with colon cancer survival has not been elucidated. Using data from a phase III randomized adjuvant trial, we assessed the association of alcohol consumption with colon cancer outcomes. Patients completed a risk factor questionnaire before randomization to FOLFOX or FOLFOX + cetuximab (N = 1984). Information was collected on lifestyle factors, including smoking, physical activity and consumption of different types of alcohol. Cox models assessed the association between alcohol consumption and outcomes of disease‐free survival (DFS), time‐to‐recurrence (TTR) and overall survival (OS), adjusting for age, sex, study arm, body mass, smoking, physical activity and performance status. No statistically significant difference in outcomes between ever and never drinkers were noted [hazard ratio (HR)DFS = 0.86, HRTTR = 0.87, HROS = 0.86, p‐values = 0.11–0.17]. However, when considering alcohol type, ever consumers of red wine (n = 628) had significantly better outcomes than never consumers (HRDFS = 0.80, HRTTR = 0.81, HROS = 0.78, p‐values = 0.01–0.02). Favorable outcomes were confirmed in patients who consumed 1‐30 glasses/month of red wine (n = 601, HR = 0.80–0.83, p‐values = 0.03–0.049); there was a suggestion of more favorable outcomes in patients who consumed >30 glasses/month of red wine (n = 27, HR = 0.33–0.38, p‐values = 0.05–0.06). Beer and liquor consumption were not associated with outcomes. Although alcohol consumption was not associated with colon cancer outcomes overall, mild to moderate red wine consumption was suggestively associated with longer OS, DFS and TTR in stage III colon cancer patients.

Abstract Background. Oncogenic KRAS mutations occur in nearly one-half of colorectal cancers (CRC) where they contribute to de novo apoptosis resistance and treatment failure. Inhibition of MEK/ERK is a promising strategy to sensitize KRAS mutant tumors to apoptosis that may involve upregulation of pro-apoptotic BH3-only proteins, including BIM. However, BH3-only proteins can be neutralized by anti-apoptotic Bcl-xL that we found was upregulated in isogenic KRAS mutant compared to wild-type HCT116 and DLD1 CRC cells. Aims. We determined the ability and mechanism by which a novel MEK/ERK inhibitor, GDC-0623, can induce BIM to promote apoptotic signaling. A strategy of concurrent induction of BH3-only proteins and antagonism of Bcl-xL was studied to overcome mutant KRAS-mediated apoptosis resistance. Results. Using isogenic KRAS HCT116 cells and KRAS mutant SW620 cells, we found that GDC-0623 can potently induce the BH3-only protein BIM, and to a lesser extent BIK, in both KRAS wild-type and mutant cells. Only KRAS mutant cells remained resistant to GDC-0623-induced apoptosis. Knockdown of Bcl-xL or use of a BH3 mimetic drug, ABT-263, sensitized KRAS mutant cells to GDC-0623-mediated apoptosis. GDC-0623 and ABT-263 were shown to interact synergistically to promote apoptosis in KRAS mutant cells. Dependence on BIM or BIK for synergy between GDC-0623 and ABT-263 was shown by shRNA knockdown that each attenuated drug-induced apoptosis. Mechanistically, ABT-263 treatment was shown to release BIM from its binding to Bcl-xL using immunoprecipitation. The ability of GDC-0623 to induce apoptosis occurred via BIM that was phosphorylated by MEK/ERK activation leading to BIM degradation. GDC-0623 was shown to dephosphorylate BIM at serine 69 in cells with wild-type BIM or a phospho-mimic mutation (S69E). However, GDC-0623's ability to dephosphorylate BIM was lost in cells containing a phosphorylation-defective BIM mutation (S69G) that showed delayed BIM degradation. Conclusion. GDC-0623 was shown to dephosphorylate BIM to enhance its accumulation and to promote apoptosis. Concurrent induction of BIM/BIK and antagonism of Bcl-xL can overcome mutant KRAS-mediated apoptosis resistance, suggesting a promising therapeutic strategy against KRAS mutant CRCs. Note: This abstract was not presented at the meeting. Citation Format: Aziz Zaanan, Koichi Okamoto, Hisato Kawakami, Shengbing Huang, Frank Sinicrope. MEK/ERK inhibitor GDC-0623 dephosphorylates and accumulates BIM that enables a synergistic apoptosis with the Bcl-xL antagonist, ABT-263, in mutant KRAS colorectal cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2937. doi:10.1158/1538-7445.AM2015-2937

Abstract Purpose: To determine the frequency and prognostic association of molecular markers by anatomic tumor site in patients with stage III colon carcinomas. Experimental Design: In a randomized trial of adjuvant FOLFOX ± cetuximab, BRAFV600E and KRAS (exon 2) mutations and DNA mismatch repair (MMR) proteins were analyzed in tumors (N = 3,018) in relationship to tumor location, including subsite. Cox models were used to assess clinical outcome, including overall survival (OS). Results: KRAS codon 12 mutations were most frequent at the splenic flexure and cecum; codon 13 mutations were evenly distributed. BRAF mutation frequency sharply increased from transverse colon to cecum in parallel with deficient (d) MMR. Nonmutated BRAF and KRAS tumors progressively decreased from sigmoid to transverse (all P < 0.0001). Significantly, poorer OS was found for mutant KRAS in distal [HR, 1.98; 95% confidence interval (CI), 1.49–2.63; P < 0.0001] versus proximal (1.25; 95% CI, 0.97–1.60; P = 0.079) cancers. BRAF status and outcome were not significantly associated with tumor site. Proximal versus distal dMMR tumors had significantly better outcome. An interaction test was significant for tumor site by KRAS (Padjusted = 0.043) and MMR (Padjusted = 0.010) for OS. Significant prognostic differences for biomarkers by tumor site were maintained in the FOLFOX arm. Tumor site was independently prognostic with a stepwise improvement from cecum to sigmoid (OS: Padjusted = 0.001). Conclusions: Mutation in BRAF or KRAS codon 12 was enriched in proximal cancers whereas nonmutated BRAF/KRAS was increased in distal tumors. Significant differences in outcome for KRAS mutations and dMMR were found by tumor site, indicating that their interpretation should occur in the context of tumor location. Clin Cancer Res; 21(23); 5294–304. ©2015 AACR.