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STAT3 (signal transducer and activator of transcription 3) is a transcription factor that is constitutively activated in some cancers. STAT3 appears to play crucial roles in cell proliferation and survival, angiogenesis, tumor-promoting inflammation and suppression of anti-tumor 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.
Utilizing a database of 724 colon and rectal cancer cases, we evaluated phosphorylated STAT3 (p-STAT3) expression by immunohistochemistry. Cox proportional hazards model was used to compute mortality hazard ratio (HR), adjusting for clinical, pathologic and molecular features, including microsatellite instability (MSI), the CpG island methylator phenotype (CIMP), LINE-1 methylation, 18q loss of heterozygosity, TP53 (p53), CTNNB1 (β-catenin), JC virus T-antigen, and KRAS, BRAF, and PIK3CA mutations.
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% confidence interval (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 odds ratio 3.23; 95% CI, 1.89–5.53; p<0.0001). p-STAT3 expression was not associated with MSI, CIMP, or LINE-1 hypomethylation.
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.
STAT3 (the HUGO-approved official symbol for signal transducer and activator of transcription 3) is a transcription factor that is activated in response to the binding of a large number of cytokines, hormones, and growth factors to their receptors as well as by activation of intracellular kinases (1–3). STAT3 is constitutively activated in a variety of human cancers including colorectal cancer and plays crucial roles in cancer cell proliferation, survival, metastasis and angiogenesis (1–3). In addition, STAT3 signaling is a major intrinsic pathway for cancer-related inflammation in the tumor microenvironment; STAT3 has been reported to induce cancer-promoting inflammation and inhibit anti-tumor immunity (4–8). Thus, accumulating evidence has implicated STAT3 as a promising target for cancer therapy and chemoprevention (9–15), and better understanding of the mechanism and consequence of STAT3 activation in human cancer is needed. Nonetheless, prognostic significance of STAT3 activation in colorectal cancer remains uncertain due to small sample sizes (N<130) of all of the previous studies on STAT3 and colorectal cancer prognosis (16–19). Given experimental data suggesting a tumor-promoting role of STAT3 in colorectal cancer (16, 20–23), we hypothesized that phosphorylated-STAT3 (p-STAT3) expression (i.e., STAT3 activation) might mark colorectal cancer with aggressive biological behavior.
To test this hypothesis, we examined the prognostic role of p-STAT3 expression, utilizing a database of 724 colorectal cancers in two prospective cohort studies with adequate follow-up data and clinical, pathological and molecular annotations. Because our database contains information on tumor variables including lymphocytic reaction patterns, KRAS, BRAF, and PIK3CA mutations, microsatellite instability (MSI), the CpG island methylator phenotype (CIMP) and LINE-1 methylation, we could robustly evaluate the relations between p-STAT3 and those variables as well as the prognostic effect of STAT3 activation independent of those potential confounders.
We utilized the databases of two prospective cohort studies; the Nurses’ Health Study (N=121,701 women followed since 1976; ref. 24) and the Health Professional Follow-up Study (N=51,529 men followed since 1986; ref. 24). A subset of the cohort participants developed colorectal cancers during prospective follow-up. Our study physicians reviewed medical record and obtained information on disease stage and tumor location. Information on body mass index (BMI) and family history of colorectal cancer was obtained prospectively through questionnaire before colorectal cancer developed. We collected paraffin-embedded tissue blocks from hospitals where patients underwent tumor resections. We excluded cases for which preoperative treatment was administered. Based on availability of adequate tissue specimens and follow-up data, a total of 724 colorectal cancers (diagnosed up to 2004) were included (Table 1). Those 724 patients were treated at hospitals throughout the U.S., in 48 States except for North Dakota and Alaska, and thus more representative colorectal cancer cases in the general U.S. population than patients in a few academic hospitals. Among our cohort studies, there was no significant difference in demographic features between cases with and without available tissue (24). Patients were observed until death or June 30, 2009, whichever came first, with median follow-up of 129 months for those who were censored. This current analysis represents a new analysis of p-STAT3 on the existing colorectal cancer database that has been previously characterized for CIMP, MSI, KRAS, BRAF, PIK3CA, LINE-1 methylation and clinical outcome (24–26). However, in any of our previous studies, we have neither examined p-STAT3 expression, nor the relationship between p-STAT3 and any of the other variables. Informed consent was obtained from all study subjects. Tissue collection and analyses were approved by the Harvard School of Public Health and Brigham and Women’s Hospital Institutional Review Boards.
Tissue sections from all colorectal cancer cases were reviewed by a pathologist (S.O.) unaware of other data. Tumor grade was categorized as high vs. low (<50% vs. ≥50% glandular area). Four components of lymphocytic reactions [Crohn’s-like lymphoid reaction, peritumoral lymphocytic reaction, intratumoral periglandular reaction, and tumor infiltrating lymphocytes (TIL)] were examined as previously described (27). Crohn’s-like reaction was defined as transmural lymphoid reaction. Peritumoral lymphocytic reaction was defined as discrete lymphoid reactions surrounding tumor. Intratumoral periglandular reaction was defined as lymphocytic reaction in tumor stroma within tumor mass. TIL was defined as lymphocytes infiltrating neoplastic epithelial cells.
DNA was extracted from tumor and PCR and Pyrosequencing targeted for KRAS (codons 12 and 13) (28), BRAF (codon 600) (26) and PIK3CA (exons 9 and 20) (29) were performed. MSI analysis was performed, using D2S123, D5S346, D17S250, BAT25, BAT26, BAT40, D18S55, D18S56, D18S67 and D18S487 (25). MSI-high was defined as the presence of instability in ≥30% of the markers, and MSI-low/microsatellite stability (MSS) as instability in <30% of the markers (25). For 18q LOH analysis using microsatellite markers (D18S55, D18S56, D18S67, D18S487), LOH at each locus was defined as ≥40% reduction of 1 of 2 allele peaks in tumor DNA relative to normal DNA (30). 18q LOH positivity was defined as the presence of LOH at any of the 18q markers, and 18q LOH negativity as the presence of ≥2 informative markers and the absence of LOH (30).
Using validated bisulfite DNA treatment and real-time PCR (MethyLight), we quantified DNA methylation in 8 CIMP-specific promoters [CACNA1G, CDKN2A (p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3 and SOCS1; refs. 25, 31]. CIMP-high was defined as the presence of ≥6/8 methylated promoters, CIMP-low as 1/8–5/8 methylated promoters, and CIMP-0 as the absence (0/8) of methylated promoters, according to the previously established criteria (25). In order to accurately quantify relatively high methylation levels in LINE-1 repetitive elements, we utilized Pyrosequencing (32).
Tissue microarrays were constructed as previously described (33). Two 0.6-mm tissue cores each from tumor and normal colonic mucosa were placed in each TMA block. Methods of immunohistochemistry have previously been described for TP53 (33), CTNNB1 (the HUGO-approved official gene symbol for β-catenin) (34), and JC virus T-antigen (JCVT) (35). STAT3 is activated by phosphorylation at Thy705, which induces dimerization, nuclear translocation, and DNA binding. In our current study, we utilized p-STAT3 antibody [Rabbit polyclonal anti-Phospho-Stat3 (Thr705), 1:70 dilution; Cell Signaling Technology, Boston, MA] which detects endogenous levels of STAT3 only when phosphorylated at Thr705. This antibody has been used for immunohistochemistry using formalin-fixed paraffin-embedded specimens (36–38) as well as for western blotting (38–41) in previous studies, and the specificity of the antibody was shown by western blotting.
For p-STAT3 staining, deparaffinized tissue sections in Antigen Retrieval Citra Solution (Biogenex Laboratories, San Ramon, CA) were treated with microwave in a pressure cooker (25 min). Tissue sections were incubated with 5% normal goat serum (Vector Laboratories, Burlingame, CA) in phosphate-buffered saline (30 min). Primary antibody against p-STAT3 (1:70 dilution) was applied, and the slides were maintained at 4°C for overnight, followed by anti-rabbit secondary antibody (Vector Laboratories) (60 min), an avidin–biotin complex conjugate (Vector Laboratories) (60 min), diaminobenzidine (5 min) and methyl-green counterstain. Nuclear p-STAT3 expression was recorded as no expression, low-level expression, high-level expression compared to normal colonic epithelial cells. Appropriate positive and negative controls were included in each run of immunohistochemistry. A whole tissue section of colonic carcinoma known to be p-STAT3 positive was used as a positive control. For a negative control, the same tumor was used and the primary antibody was omitted. Each immunohistochemical maker was interpreted by one of the investigators (p-STAT3 by Y.B.; TP53 by S.O.; CTNNB1 by T.M.; JCVT by K.N.) unaware of other data. For agreement studies, a random selection of more than 100 cases was examined for each marker by a second observer (p-STAT3 by T.M.; TP53 by K.N.; CTNNB1 by S.O.; JCVT by Y.B.) unaware of other data. The concordance between the two observers (all p<0.0001) was 0.77 (weighted κ =0.67; N=178) for p-STAT3, 0.87 (κ=0.75; N=108) for TP53, 0.90 for CTNNB1 nuclear expression (κ=0.80; N=292), 0.78 for CTNNB1 cytoplasmic expression (κ=0.54; N=292), 0.86 for CTNNB1 membrane expression (κ=0.72; N=292), and 0.87 (κ=0.74; N=147) for JCVT, indicating good to substantial agreement.
For all statistical analyses, we used SAS program (Version 9.1, SAS Institute, Cary, NC). All p values were two-sided. When we performed multiple hypothesis testing, a p value for significance was adjusted by Bonferroni correction to p=0.0022 (=0.05/23). For categorical data, the chi-square test was performed. The Cicchetti-Allison weight was used for calculating the weighted κ agreement coefficients for p-STAT3 (trichotomized variable). Kaplan-Meier method and log-rank test were used for survival analyses. For analyses of colorectal cancer-specific mortality, patients who died of causes other than colorectal cancer were censored at the time of death. We constructed a multivariate, stage-stratified Cox proportional hazards model to compute mortality hazard ratio (HR) according to p-STAT3 status, initially including sex, age at diagnosis (continuous), year of diagnosis (continuous), body mass index (BMI; <30 vs. ≥30 kg/m2), family history of colorectal cancer in any first degree relative (present vs. absent), tumor location (rectum vs. distal colon vs. proximal), tumor grade (low vs. high), Crohn’s-like reaction (present vs. absent/minimum), peritumoral reaction (present vs. absent/minimum), intratumoral periglandular reaction (present vs. absent/minimum), tumor infiltrating lymphocytes (present vs. absent/minimum), CIMP (high vs. low/0), MSI (high vs. low/MSS), LINE-1 methylation (continuous), BRAF, KRAS, PIK3CA, 18q LOH, TP53, nuclear CTNNB1, and JCVT. Tumor stage (I, IIA, IIB, IIIA, IIIB, IIIC, IV, unknown) was used as a stratifying variable using the “strata” option in the SAS “proc phreg” command to avoid residual confounding and overfitting. A backward elimination with a threshold of p=0.20 was used to select variables in the final model. For cases with missing information in any of categorical variables [tumor location (1.9%), tumor grade (0.4%), Crohn’s-like reaction (1.9%), peritumoral reaction (1.8%), intratumoral periglandular reaction (1.7%), tumor infiltrating lymphocytes (1.8%), MSI (1.9%), CIMP (1.8%), BRAF (1.5%), KRAS (1.1%), PIK3CA (10%), 18q LOH(41%), TP53 (0.8%), CTNNB1 (4.7%), and JCVT (26%)], we included those cases in a majority category of a given covariate to avoid overfitting. We confirmed that excluding cases with missing information in any of the covariates did not substantially alter results (data not shown). The proportionality of hazard assumption was satisfied by evaluating time-dependent variables, which were the cross-product of the STAT3 variable and survival time (p>0.05). An interaction was assessed by the Wald test on the cross product of p-STAT3 variable and another variable of interest (without data-missing cases) in a multivariate Cox model.
Additionally, multivariate logistic regression analysis was performed to assess independent effect of tumor p-STAT3 expression on either peritumoral lymphocytic reaction or intratumoral periglandular reaction (as a binary outcome variable) using the SAS “proc logistic” command. p-STAT3 expression was used as a binary variable (high/low vs. negative) because low-level expression and high-level expression showed similar effects. Odds ratio (OR) was adjusted for age (continuous), sex, BMI (<30 vs. ≥30 kg/m2), family history of colorectal cancer in any first degree relative (present vs. absent), tumor location (proximal vs. distal), tumor grade (high vs. low), CIMP (high vs. low/0), MSI (high vs. low/MSS), LINE-1 methylation (continuous), BRAF, KRAS, PIK3CA, 18q LOH (present vs. absent), TP53, nuclear CTNNB1, and JCVT. A backward elimination with a threshold of p=0.20 was used to select variables in the final model.
Among 724 colorectal cancers, we observed p-STAT3-high expression in 131 tumors (18%) and p-STAT3-low expression in 244 tumors (34%) by immunohistochemistry (Figure 1). Table 1 shows p-STAT3 expression status in relation to various clinical, pathologic and molecular features. p-STAT3 expression status was positively associated with peritumoral lymphocytic reaction (p<0.0001) and intratumoral periglandular reaction (p<0.0001).
To assess independent effect of p-STAT3 expression on peritumoral lymphocytic reaction and intratumoral periglandular reaction, we performed a multivariate logistic regression analysis, adjusting for potential confounders (Table 2). Tumor p-STAT3 expression was independently associated with peritumoral lymphocytic reaction [multivariate OR, 3.23; 95% confidence interval (CI), 1.89–5.53; p<0.0001] and intratumoral periglandular reaction (multivariate OR, 3.01; 95% CI, 1.77–5.12; p<0.0001). Male gender was also independently associated with peritumoral lymphocytic reaction (multivariate OR, 4.32; 95% CI, 2.19–8.53; p<0.0001) and intratumoral periglandular reaction (multivariate OR, 4.30; 95% CI, 2.18–8.47; p<0.0001). Considering multiple hypothesis testing, the apparent relationship of MSI-high with peritumoral or intratumoral periglandular reaction should be interpreted with caution; these relations did not reach the Bonferroni-corrected significance level of p=0.0022.
Among the 724 patients with median follow-up of 129 months for those who were censored, there were 340 deaths, including 210 colorectal cancer-specific deaths. In Kaplan-Meier analysis, p-STAT3 expression was significantly associated with shorter colorectal cancer-specific survival (log rank p=0.0020) and overall survival (log rank p=0.012) (Figure 2). Five-year colorectal cancer-specific survival was 80.3% (95% CI, 75.7–84.1%) among p-STAT3-negative cases, 73.7% (95% CI, 67.6–78.8%) among p-STAT3-low cases, and 63.4% (95% CI, 54.4–71.1%) among p-STAT3-high cases. Five-year overall survival was 77.0% (95% CI, 72.1–81.0%) among p-STAT3-negative cases, 68.4% (95% CI, 62.2–73.8%) among p-STAT3-low cases, and 57.3% (95% CI, 48.3–65.2%) among p-STAT3-high cases.
p-STAT3 expression was significantly associated with shorter cancer-specific survival in univariate Cox regression analysis (Ptrend=0.0005) and in the multivariate Cox model adjusting for clinical, pathologic and molecular features (Ptrend=0.015) (Table 3). In particular, compared to p-STAT3-negative cases, p-STAT3-high cases experienced a significantly higher colorectal cancer-specific mortality [unadjusted HR 1.85; 95% CI, 1.30–2.63: multivariate HR 1.61; 95% CI, 1.11–2.34]. The attenuation in the effect of p-STAT3 in the multivariate analysis was principally the result of adjusting for disease stage. When we simply adjusted for disease stage, p-STAT3-high expression was associated with HR of 1.56 (95% CI, 1.09–2.24). Analyses using overall mortality yielded similar, though attenuated, results (Table 3).
We examined whether the influence of p-STAT3 expression on colorectal cancer-specific survival was modified by any of the other variables including sex, age, BMI, family history of colorectal cancer, tumor location, stage, tumor grade, lymphocytic reactions, CIMP, MSI, BRAF, KRAS, PIK3CA, LINE-1 methylation, 18q LOH, TP53, CTNNB1, and JCVT. We did not observe a significant modifying effect by any of the variables (all Pinteraction>0.10).
We conducted this study to examine the prognostic significance of p-STAT3 expression in a large cohort of colorectal cancers. STAT3 is aberrantly activated in a wide variety of human cancers and plays crucial roles in tumor cell proliferation, survival, invasion and tumor-promoting inflammation (1–8). Given the potential for STAT3 as an anti-cancer therapeutic target (9–15), a better understanding of STAT3 activation in human cancer tissues is important. Nonetheless, prognostic studies on p-STAT3 expression in colorectal cancer have been inconclusive. We have found that p-STAT3 expression is significantly associated with poor prognosis in a database of 724 colorectal cancers, suggesting a potential role of p-STAT3 expression as a prognostic biomarker in colorectal cancer.
Examining biomarkers or prognostic factors is important in cancer research (42–50). Studies examining the relation between p-STAT3 expression and prognosis in colorectal cancer have yielded inconsistent results (16–19) (Table 4); p-STAT3 expression was associated with poor prognosis in two studies [N=90 (16) and N=108 (17)], whereas one study [N=104 (19)] showed good prognosis associated with p-STAT3 expression, and another study [N=126 (18)] showed no prognostic value of p-STAT3. All of these previous studies were limited by small sample sizes and low statistical power (Table 4). Importance of large-scale studies cannot be emphasized enough, because small studies (for example, N<150) with null results have much higher likelihood of being unpublished than small studies with “significant” results, leading to publication bias. In contrast to the prior small studies (16–19), our study examined p-STAT3 expression in a much larger cohort of stage I-IV colorectal cancers. Nonetheless, our finding on the relationship between p-STAT3 expression and poor prognosis in colorectal cancer needs to be confirmed by independent datasets in the future.
Experimental studies using colon cancer cell lines have suggested an oncogenic role of STAT3. Inhibition of STAT3 with small interfering RNA has induced apoptosis and cell cycle arrest in colon cancer cells (22). STAT3 activation triggered through interleukin-6 and through a constitutively active STAT3 mutant has enhanced cell proliferation and tumor growth (21). STAT3 activation has been shown to promote tumor growth through the induction of matrix metalloproteinase (20). A small study (N=90) has shown a positive association between p-STAT3 expression and nuclear accumulation of CTNNB1 (16). In our current study, however, we did not find significant association between p-STAT3 expression and CTNNB1 status. This discrepancy might be caused by differences in immunohistochemical methods, correlative errors, or study samples, or simply due to a chance variation in the small underpowered study. In addition, STAT3 signaling has been implicated in TFF3 and VEGFA-mediated invasion and growth of colon cancer cells (23). Our observational data certainly support a role of STAT3 in colorectal cancer progression.
Interestingly, we have found the relationship of p-STAT3 expression with peritumoral lymphocytic reaction and intratumoral periglandular reaction, independent of the clinical and molecular variables that we examined. STAT3 has been implicated in tumor-promoting inflammation and the regulation of host immune response in the tumor microenvironment (1, 4–8). Our current data certainly support a potential role of STAT3 in the propagation of tumor-promoting inflammation. Because of the relationship between MSI and lymphocytic reactions as well as the fundamental importance of MSI in governing the phenotype of colorectal cancer, we examined the relationship between p-STAT3 and MSI as well as a potential interaction between p-STAT3 and MSI in survival analysis. However, we did not observe a significant association between p-STAT3 and MSI-high, or a significant interaction between p-STAT3 and MSI. Future studies are necessary to elucidate biological mechanisms by which tumor STAT3 activation influences lymphocytic reaction in the tumor microenvironment.
There are limitations in this study. For example, data on cancer treatment were limited. Nonetheless, it is unlikely that chemotherapy use substantially differed according to STAT3 status in tumor, since such data were unavailable for treatment decision making. In addition, our multivariate survival analysis finely adjusted for disease stage (I, IIA, IIB, IIIA, IIIB, IIIC, IV, unknown), on which treatment decision making was mostly based. As another limitation, beyond cause of mortality, data on cancer recurrences were unavailable in these cohort studies. Nonetheless, with a median follow-up of greater than 10 years for censored cases, colorectal cancer-specific mortality was a reasonable colorectal cancer-specific outcome.
There are advantages in utilizing the database of the two prospective cohort studies. Data on family history, cancer staging, and other clinical, pathologic, and tumoral molecular variables were prospectively collected, blinded to patient outcome. Cohort participants who developed cancer were treated at hospitals throughout the U.S., in 48 States except for North Dakota and Alaska, and thus more representative colorectal cancer cases in the general U.S. population than patients in one to a few academic hospitals. In addition, we assessed the effect of p-STAT3 expression independent of other critical molecular events [e.g., BRAF, PIK3CA, LINE-1 hypomethylation, and microsatellite instability (MSI)] or pathologic features (e.g., lymphocytic reaction) that have been documented to be associated with colorectal cancer prognosis (26, 27).
In summary, our large study has shown that individuals with STAT3-activated colorectal cancers experience a poorer prognosis, supporting a tumor-promoting role of STAT3. Considering that drugs targeting the STAT3 pathway are intensively being developed and tested in clinical trials for various human cancers (9–12), p-STAT3 expression in colorectal cancer might serve as a predictive tissue biomarker and can be used for patient selection in clinical trials of these drugs. In this respect, our findings may have substantial clinical implications.
STAT3 is constitutively activated in a variety of human cancers including colorectal cancer and plays crucial roles in cancer cell proliferation, survival and metastasis. Accumulating evidence has implicated STAT3 as a promising target for cancer therapy and chemoprevention. However, the relation between p-STAT3 expression (i.e., STAT3 activation) in colorectal cancer and patient survival has been controversial. We have utilized the database of more than 700 colorectal cancers in two independent, prospective cohort studies, with available clinical information, adequate follow-up, and other important molecular events in colorectal cancers. To our knowledge, this is the first large study to demonstrate influence of p-STAT3 expression on adverse clinical outcome independent of clinical, pathologic and molecular features of colorectal cancer. Thus, our findings are relevant to practice in oncology.
Financial support: This work was supported by U.S. National Institute of Health (NIH) grants P01 CA87969 (to S. Hankinson), P01 CA55075 (to W. Willett), P50 CA127003 (to C.S.F.), K07 CA122826 (to S.O.) and R01 CA151993 (to S.O.), and by grants from the Bennett Family Fund and the Entertainment Industry Foundation through National Colorectal Cancer Research Alliance. T.M. and K.N. were supported by fellowship grants from the Japan Society for Promotion of Science. The content is solely the responsibility of the authors and does not necessarily represent the official views of NCI or NIH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
We deeply thank the Nurses’ Health Study and Health Professionals Follow-up Study cohort participants who have agreed to provide us with biological specimens; hospitals and pathology departments throughout the U.S. for generously providing us with tissue specimens. We thank the following state cancer registries for their help: AL, AZ, AR, CA, CO, CT, DE, FL, GA, ID, IL, IN, IA, KY, LA, ME, MD, MA, MI, NE, NH, NJ, NY, NC, ND, OH, OK, OR, PA, RI, SC, TN, TX, VA, WA, WY.