Teppei Morikawa

Tumour micro-environment elicits innate resistance to RAF inhibitors through HGF secretion

Drug resistance presents a challenge to the treatment of cancer patients. Many studies have focused on cell-autonomous mechanisms of drug resistance. By contrast, we proposed that the tumour micro-environment confers innate resistance to therapy. Here we developed a co-culture system to systematically assay the ability of 23 stromal cell types to influence the innate resistance of 45 cancer cell lines to 35 anticancer drugs. We found that stroma-mediated resistance is common, particularly to targeted agents. We characterized further the stroma-mediated resistance of BRAF-mutant melanoma to RAF inhibitors because most patients with this type of cancer show some degree of innate resistance. Proteomic analysis showed that stromal cell secretion of hepatocyte growth factor (HGF) resulted in activation of the HGF receptor MET, reactivation of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-OH kinase (PI(3)K)–AKT signalling pathways, and immediate resistance to RAF inhibition. Immunohistochemistry experiments confirmed stromal cell expression of HGF in patients with BRAF-mutant melanoma and showed a significant correlation between HGF expression by stromal cells and innate resistance to RAF inhibitor treatment. Dual inhibition of RAF and either HGF or MET resulted in reversal of drug resistance, suggesting RAF plus HGF or MET inhibitory combination therapy as a potential therapeutic strategy for BRAF-mutant melanoma. A similar resistance mechanism was uncovered in a subset of BRAF-mutant colorectal and glioblastoma cell lines. More generally, this study indicates that the systematic dissection of interactions between tumours and their micro-environment can uncover important mechanisms underlying drug resistance.Drug resistance presents a challenge to the treatment of cancer patients. Many studies have focused on cell-autonomous mechanisms of drug resistance. By contrast, we proposed that the tumour micro-environment confers innate resistance to therapy. Here we developed a co-culture system to systematically assay the ability of 23 stromal cell types to influence the innate resistance of 45 cancer cell lines to 35 anticancer drugs. We found that stroma-mediated resistance is common, particularly to targeted agents. We characterized further the stroma-mediated resistance of BRAF-mutant melanoma to RAF inhibitors because most patients with this type of cancer show some degree of innate resistance. Proteomic analysis showed that stromal cell secretion of hepatocyte growth factor (HGF) resulted in activation of the HGF receptor MET, reactivation of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-OH kinase (PI(3)K)-AKT signalling pathways, and immediate resistance to RAF inhibition. Immunohistochemistry experiments confirmed stromal cell expression of HGF in patients with BRAF-mutant melanoma and showed a significant correlation between HGF expression by stromal cells and innate resistance to RAF inhibitor treatment. Dual inhibition of RAF and either HGF or MET resulted in reversal of drug resistance, suggesting RAF plus HGF or MET inhibitory combination therapy as a potential therapeutic strategy for BRAF-mutant melanoma. A similar resistance mechanism was uncovered in a subset of BRAF-mutant colorectal and glioblastoma cell lines. More generally, this study indicates that the systematic dissection of interactions between tumours and their micro-environment can uncover important mechanisms underlying drug resistance.

Restriction of intestinal stem cell expansion and the regenerative response by YAP

A remarkable feature of regenerative processes is their ability to halt proliferation once an organ’s structure has been restored. The Wnt signalling pathway is the major driving force for homeostatic self-renewal and regeneration in the mammalian intestine. However, the mechanisms that counterbalance Wnt-driven proliferation are poorly understood. Here we demonstrate in mice and humans that yes-associated protein 1 (YAP; also known as YAP1)—a protein known for its powerful growth-inducing and oncogenic properties—has an unexpected growth-suppressive function, restricting Wnt signals during intestinal regeneration. Transgenic expression of YAP reduces Wnt target gene expression and results in the rapid loss of intestinal crypts. In addition, loss of YAP results in Wnt hypersensitivity during regeneration, leading to hyperplasia, expansion of intestinal stem cells and niche cells, and formation of ectopic crypts and microadenomas. We find that cytoplasmic YAP restricts elevated Wnt signalling independently of the AXIN–APC–GSK-3β complex partly by limiting the activity of dishevelled (DVL). DVL signals in the nucleus of intestinal stem cells, and its forced expression leads to enhanced Wnt signalling in crypts. YAP dampens Wnt signals by restricting DVL nuclear translocation during regenerative growth. Finally, we provide evidence that YAP is silenced in a subset of highly aggressive and undifferentiated human colorectal carcinomas, and that its expression can restrict the growth of colorectal carcinoma xenografts. Collectively, our work describes a novel mechanistic paradigm for how proliferative signals are counterbalanced in regenerating tissues. Additionally, our findings have important implications for the targeting of YAP in human malignancies.

Association of CTNNB1 (β-Catenin) Alterations, Body Mass Index, and Physical Activity with Survival in Patients with Colorectal Cancer

CONTEXTnAlterations of the WNT signaling pathway and cadherin-associated protein β 1 (CTNNB1 or β-catenin) have been implicated in colorectal carcinogenesis and metabolic diseases.nnnOBJECTIVEnTo test the hypothesis that CTNNB1 activation in colorectal cancer modifies prognostic associations of body mass index (BMI) and level of postdiagnosis physical activity.nnnDESIGN, SETTING, AND PATIENTSnTwo US prospective cohort studies (Nurses Health Study and the Health Professionals Follow-up Study) were used to evaluate CTNNB1 localization by immunohistochemistry in 955 patients with stage I, II, III, or IV colon and rectal cancer from 1980 through 2004. A Cox proportional hazards model was used to compute the hazard ratio (HR) for mortality, adjusting for clinical and tumor features, including microsatellite instability, CpG island methylator phenotype, level of long interspersed nucleotide element 1 methylation, mutations in KRAS, BRAF, or PIK3CA, and tumor protein p53.nnnMAIN OUTCOME MEASURESnColorectal cancer-specific mortality and overall mortality through June 30, 2009.nnnRESULTSnIn obese patients (BMI ≥30), positive status for nuclear CTNNB1 was associated with significantly better colorectal cancer-specific survival (adjusted HR, 0.24 [95% confidence interval {CI}, 0.12-0.49], P <.001 for interaction; 5-year survival: 0.85 for patients with positive nuclear CTNNB1 status vs 0.78 for those with negative status) and overall survival (adjusted HR, 0.56 [95% CI, 0.35-0.90], P = .03 for interaction; 5-year survival: 0.77 for patients with positive nuclear CTNNB1 status vs 0.74 for those with negative status), while CTNNB1 status was not associated with prognosis among nonobese patients (BMI <30). Among patients with negative status for nuclear CTNNB1 and cancer in stages I, II, or III, postdiagnosis physical activity was associated with better colorectal cancer-specific survival (adjusted HR, 0.33 [95% CI, 0.13-0.81], P = .05 for interaction; 5-year survival: 0.97 for ≥18 vs 0.89 for <18 metabolic equivalent task hours/week), while postdiagnosis physical activity was not associated with colorectal cancer-specific survival among patients with positive status for nuclear CTNNB1 (adjusted HR, 1.07 [95% CI, 0.50-2.30]).nnnCONCLUSIONSnAmong obese patients only, activation of CTNNB1 was associated with better colorectal cancer-specific survival and overall survival. Postdiagnosis physical activity was associated with better colorectal cancer-specific survival only among patients with negative status for nuclear CTNNB1. These molecular pathological epidemiology findings suggest that the effects of alterations in the WNT-CTNNB1 pathway on outcome are modified by BMI and physical activity.

STAT3 Expression, Molecular Features, Inflammation Patterns, and Prognosis in a Database of 724 Colorectal Cancers

Purpose: STAT3 is a transcription factor that is constitutively activated in some cancers. It seems to play crucial roles in cell proliferation and survival, angiogenesis, tumor-promoting inflammation, and suppression of antitumor host immune response in the tumor microenvironment. Although the STAT3 signaling pathway is a potential drug target, clinical, pathologic, molecular, or prognostic features of STAT3-activated colorectal cancer remain uncertain. Experimental Design: Utilizing a database of 724 colon and rectal cancer cases, we evaluated phosphorylated STAT3 (p-STAT3) expression by immunohistochemistry. The Cox proportional hazards model was used to compute mortality HR, adjusting for clinical, pathologic, and molecular features, including microsatellite instability (MSI), the CpG island methylator phenotype (CIMP), LINE-1 methylation, 18q LOH, TP53 (p53), CTNNB1 (β-catenin), JC virus T-antigen, and KRAS, BRAF, and PIK3CA mutations. Results: Among the 724 tumors, 131 (18%) showed high-level p-STAT3 expression (p-STAT3-high), 244 (34%) showed low-level expression (p-STAT3-low), and the remaining 349 (48%) were negative for p-STAT3. p-STAT3 overexpression was associated with significantly higher colorectal cancer–specific mortality [log-rank P = 0.0020; univariate HR (p-STAT3-high vs. p-STAT3-negative): 1.85, 95% CI: 1.30–2.63, Ptrend = 0.0005; multivariate HR: 1.61, 95% CI: 1.11–2.34, Ptrend = 0.015]. p-STAT3 expression was positively associated with peritumoral lymphocytic reaction (multivariate OR: 3.23; 95% CI: 1.89–5.53, P < 0.0001). p-STAT3 expression was not associated with MSI, CIMP, or LINE-1 hypomethylation. Conclusions: STAT3 activation in colorectal cancer is associated with adverse clinical outcome, supporting its potential roles as a prognostic biomarker and a chemoprevention and/or therapeutic target. Clin Cancer Res; 17(6); 1452–62. ©2011 AACR.

MGMT promoter methylation, loss of expression and prognosis in 855 colorectal cancers

ObjectiveO6-methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme. MGMT promoter hypermethylation and epigenetic silencing often occur as early events in carcinogenesis. However, prognostic significance of MGMT alterations in colorectal cancer remains uncertain.MethodsUtilizing a database of 855 colon and rectal cancers in two prospective cohort studies (the Nurses’ Health Study and the Health Professionals Follow-up Study), we detected MGMT promoter hypermethylation in 325 tumors (38%) by MethyLight and loss of MGMT expression in 37% (247/672) of tumors by immunohistochemistry. We assessed the CpG island methylator phenotype (CIMP) using eight methylation markers [CACNA1G, CDKN2A (p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1], and LINE-1 (L1) hypomethylation, TP53 (p53), and microsatellite instability (MSI).ResultsMGMT hypermethylation was not associated with colorectal cancer–specific mortality in univariate or multivariate Cox regression analysis [adjusted hazard ratio (HR)xa0=xa01.03; 95% confidence interval (CI), 0.79–1.36] that adjusted for clinical and tumor features, including CIMP, MSI, and BRAF mutation. Similarly, MGMT loss was not associated with patient survival. MGMT loss was associated with G>A mutations in KRAS (pxa0=xa00.019) and PIK3CA (pxa0=xa00.0031).ConclusionsDespite a well-established role of MGMT aberrations in carcinogenesis, neither MGMT promoter methylation nor MGMT loss serves as a prognostic biomarker in colorectal cancer.

Prognostic Significance and Molecular Associations of Tumor Growth Pattern in Colorectal Cancer

BackgroundInfiltrative growth pattern at the tumor margin has been associated with shorter patient survival. However, little is known about the prognostic significance of tumor growth pattern, independent of tumoral molecular alterations and other histologic features.MethodsUtilizing a database of 1139 colon and rectal cancer patients in two prospective cohort studies, histologic features including tumor growth pattern, tumor differentiation, lymphocytic reaction, mucinous component, and signet ring cell component were recorded by a single pathologist. Cox proportional hazard model was used to compute mortality hazard ratio, adjusting for clinical, pathologic, and tumor molecular features, including microsatellite instability, the CpG island methylator phenotype, long interspersed nucleotide element 1 (LINE-1) methylation, and KRAS,BRAF, and PIK3CA mutations.ResultsAmong 1139 colorectal cancers, we observed expansile growth pattern in 372 tumors (33%), intermediate growth pattern in 610 tumors (54%), and infiltrative growth pattern in 157 tumors (14%). Compared to patients with expansile growth pattern, those with infiltrative growth pattern experienced shorter cancer-specific survival (log rank Pxa0<xa00.0001; multivariate hazard ratio 1.74; 95% confidence interval 1.22–2.47) and overall survival (log rank Pxa0<xa00.0001; multivariate hazard ratio 1.78; 95% confidence interval 1.33–2.39). The prognostic association of infiltrative growth pattern was confined to patients with stage I–III disease (Pinteraction with stagexa0=xa00.0001).ConclusionsInfiltrative growth pattern was associated with worse prognosis among stage I–III colorectal cancer patients, independent of other clinical, pathologic, and molecular characteristics.

Tumor TP53 expression status, body mass index, and prognosis in colorectal cancer

Inactivation of the TP53 (p53) pathway by TP53 mutations is one of key steps in colorectal carcinogenesis. TP53 also plays an important role in cellular energy metabolism. We hypothesized that TP53‐altered tumor cells might behave aggressively independent of energy balance, while progression of TP53‐intact cells might depend on excess energy balance. Utilizing a database of 1,060 colon and rectal cancer patients in two prospective cohort studies, we evaluated TP53 expression by immunohistochemistry. Among 1,060 colorectal cancers, 457 (43%) tumors were positive for TP53. Cox proportional hazards model was used to compute mortality hazard ratio (HR), adjusting for clinical and tumoral features, including microsatellite instability, the CpG island methylator phenotype, LINE‐1 methylation, KRAS, BRAF and PIK3CA. TP53 positivity was not significantly associated with cancer‐specific survival in univariate analysis with HR of 1.16 [95% confidence interval (CI) = 0.92–1.45], which became significant after stage adjustment (multivariate HR = 1.30; 95% CI = 1.02–1.65). Notably, we found a possible modifying effect of patients body mass index (BMI) on tumor TP53. In non‐obese patients (BMI < 30 kg/m2), TP53 positivity was associated with shorter cancer‐specific survival (multivariate HR = 1.53; 95% CI = 1.17‐2.00), while TP53 positivity was not significantly associated with survival among obese patients (BMI ≥30 kg/m2). Effect of TP53 positivity on cancer‐specific survival significantly differed by BMI (pinteraction = 0.0051). The adverse effect of obesity on patient mortality was limited to TP53‐negative patients. These molecular pathological epidemiology data may support a dual role of TP53 alterations in cell‐cycle deregulation and cell autonomy with respect to energy balance status.

Insulin-like growth factor 2 messenger RNA binding protein 3 (IGF2BP3) is a marker of unfavourable prognosis in colorectal cancer

BACKGROUNDnEvidence suggests that insulin-like growth factor 2 messenger RNA binding protein 3 (IGF2BP3, also known as IMP3) represents a promising cancer biomarker. However, the clinical, pathological, molecular and prognostic features of IGF2BP3-positive colorectal cancers remain uncertain.nnnMATERIALS AND METHODSnWe evaluated IGF2BP3 expression by immunohistochemistry in 671 rectal and colon cancer cases that form part of a molecular pathological epidemiology database. Cox proportional hazards regression models were used to compute mortality hazard ratio (HR), adjusting for clinical, pathological and molecular features, including microsatellite instability, the CpG island methylator phenotype, LINE-1 methylation and KRAS, BRAF and PIK3CA mutations.nnnRESULTSnAmong 671 colorectal cancers, 234 (35%) tumours were positive for IGF2BP3. In contrast, normal colorectal epithelium was negative for IGF2BP3 in all 403 specimens of normal mucosa adjacent to carcinoma. IGF2BP3 positivity was associated with poor differentiation (p=0.0003), stage III-IV disease (p=0.0081), BRAF mutation (p=0.031), and LINE-1 hypomethylation (p=0.020). IGF2BP3 positivity was significantly associated with shorter colorectal cancer-specific [log-rank p<0.0001; multivariate HR, 1.37; 95% confidence interval (CI), 1.02-1.84] and overall survival (log-rank p=0.0004; multivariate HR, 1.32; 95% CI, 1.05-1.66).nnnCONCLUSIONSnIGF2BP3 expression in colorectal cancer is associated with adverse clinical outcome. Our findings support a role for IGF2BP3 as a diagnostic and/or prognostic biomarker in colorectal cancer.

TGFBR2 and BAX Mononucleotide Tract Mutations, Microsatellite Instability, and Prognosis in 1072 Colorectal Cancers

Background Mononucleotide tracts in the coding regions of the TGFBR2 and BAX genes are commonly mutated in microsatellite instability-high (MSI-high) colon cancers. The receptor TGFBR2 plays an important role in the TGFB1 (transforming growth factor-β, TGF-β) signaling pathway, and BAX plays a key role in apoptosis. However, a role of TGFBR2 or BAX mononucleotide mutation in colorectal cancer as a prognostic biomarker remains uncertain. Methodology/Principal Findings We utilized a database of 1072 rectal and colon cancers in two prospective cohort studies (the Nurses Health Study and the Health Professionals Follow-up Study). Cox proportional hazards model was used to compute mortality hazard ratio (HR), adjusted for clinical, pathological and molecular features including the CpG island methylator phenotype (CIMP), LINE-1 methylation, and KRAS, BRAF and PIK3CA mutations. MSI-high was observed in 15% (162/1072) of all colorectal cancers. TGFBR2 and BAX mononucleotide mutations were detected in 74% (117/159) and 30% (48/158) of MSI-high tumors, respectively. In Kaplan-Meier analysis as well as univariate and multivariate Cox regression analyses, compared to microsatellite stable (MSS)/MSI-low cases, MSI-high cases were associated with superior colorectal cancer-specific survival [adjusted HR, 0.34; 95% confidence interval (CI), 0.20–0.57] regardless of TGFBR2 or BAX mutation status. Among MSI-high tumors, TGFBR2 mononucleotide mutation was associated with CIMP-high independent of other variables [multivariate odds ratio, 3.57; 95% CI, 1.66–7.66; pu200a=u200a0.0011]. Conclusions TGFBR2 or BAX mononucleotide mutations are not associated with the patient survival outcome in MSI-high colorectal cancer. Our data do not support those mutations as prognostic biomarkers (beyond MSI) in colorectal carcinoma.

LIN28 cooperates with WNT signaling to drive invasive intestinal and colorectal adenocarcinoma in mice and humans

Colorectal cancer (CRC) remains a major contributor to cancer-related mortality. LIN28A and LIN28B are highly related RNA-binding protein paralogs that regulate biogenesis of let-7 microRNAs and influence development, metabolism, tissue regeneration, and oncogenesis. Here we demonstrate that overexpression of either LIN28 paralog cooperates with the Wnt pathway to promote invasive intestinal adenocarcinoma in murine models. When LIN28 alone is induced genetically, half of the resulting tumors harbor Ctnnb1 (β-catenin) mutation. When overexpressed in Apc(Min/+) mice, LIN28 accelerates tumor formation and enhances proliferation and invasiveness. In conditional genetic models, enforced expression of a LIN28-resistant form of the let-7 microRNA reduces LIN28-induced tumor burden, while silencing of LIN28 expression reduces tumor volume and increases tumor differentiation, indicating that LIN28 contributes to tumor maintenance. We detected aberrant expression of LIN28A and/or LIN28B in 38% of a large series of human CRC samples (n = 595), where LIN28 expression levels were associated with invasive tumor growth. Our late-stage CRC murine models and analysis of primary human tumors demonstrate prominent roles for both LIN28 paralogs in promoting CRC growth and progression and implicate the LIN28/let-7 pathway as a therapeutic target.