Interleukin-6/STAT3 Signaling is Prominent and Associated with Reduced Overall Survival in p16 Negative Oropharyngeal Squamous Cell Carcinoma.
ABSTRACT: This study addresses the hypothesis that IL-6/STAT3 signaling is of clinical relevance in oropharyngeal squamous cell carcinoma (OPSCC). We evaluated relationships between key components of this pathway in tumors from a unique cohort of n = 59 fully annotated, treatment-naïve patients with OPSCC. The multiplex Opal platform was utilized for immunofluorescence (IF) analysis of tissues to detect IL-6 and phosphorylated STAT3 (pSTAT3), taking into consideration its nuclear versus cytoplasmic localization. Abundant staining for both IL-6 and pSTAT3 was evident in tumor-rich regions of each specimen. IL-6 correlated with cytoplasmic pSTAT3 but not nuclear or total pSTAT3 in this cohort of OPSCC tumors, regardless of p16 status (r = 0.682, p < 0.0001). There was a significant association between increased total pSTAT3, nuclear pSTAT3, cytoplasmic pSTAT3 and IL-6 in p16 negative tumors. Our data indicate STAT3 phosphorylation was a key feature in p16-negative OPSCC tumors. When IL-6 data was stratified by median expression in tumors, there was no association with overall survival. In contrast, both total and nuclear pSTAT3 were significant predictors of poor overall and disease free survival. This strong inverse relationship with overall survival was present in p16 negative tumors for both total and nuclear pSTAT3, but not in p16 positive OPSCC tumors. Together these data indicate that activation of the STAT3 signaling pathway is a marker of p16 negative tumors and relevant to OPSCC prognosis and a potential target for treatment of this more aggressive OPSCC sub-population.
Project description:Prior studies established constitutively active AP-1, NF-?B, and STAT3 signaling in oral cancer. Differential expression/activation of specific members of these transcription factors has been documented in HPV-positive oral lesions that respond better to therapy. We performed a comprehensive analysis of differentially expressed, transcriptionally active members of these pivotal signaling mediators to develop specific signatures of HPV-positive and HPV-negative oral lesions by immunohistochemical method that is applicable in low-resource settings. We examined a total of 31 prospective and 30 formalin-fixed, paraffin-embedded tissues from treatment-naïve, histopathologically and clinically confirmed cases diagnosed as oral or oropharyngeal squamous cell carcinoma (OSCC/OPSCC). Following determination of their HPV status by GP5 + /GP6 + PCR, the sequential sections of the tissues were evaluated for expression of JunB, JunD, c-Fos, p50, p65, STAT3, and pSTAT3(Y705), along with two key regulatory proteins pEGFR and p16 by IHC. Independent analysis of JunB and p65 showed direct correlation with HPV positivity, whereas STAT3 and pSTAT3 were inversely correlated. A combined analysis of transcription factors revealed a more restrictive combination, characterized by the presence of AP-1 and NF-?B lacking involvement of STAT3 that strongly correlated with HPV-positive tumors. Presence of STAT3/pSTAT3 with NF-?B irrespective of the presence or absence of AP-1 members was present in HPV-negative lesions. Expression of pSTAT3 strongly correlated with all the AP-1/NF-?B members (except JunD), its upstream activator pEGFR<sup>Y</sup><sup>1092</sup> , and HPV infection-related negative regulator p16. Overall, we show a simple combination of AP-1, NF-?B, and STAT3 members' expression that may serve as molecular signature of HPV-positive lesions or more broadly the tumors that show better prognosis.
Project description:Human papillomavirus (HPV) is a major etiologic factor for oropharyngeal squamous cell carcinoma (OPSCC). However, little is known about HPV-related OPSCC in Japan. During the study, formalin-fixed, paraffin-embedded OPSCC specimens from Japanese patients were analyzed for HPV DNA by the polymerase chain reaction (PCR) and for the surrogate marker p16 by immuno-histochemistry. For HPV DNA-positive, p16-negative specimens, the methylation status of the p16 gene promoter was examined by methylation-specific PCR. Overall survival was calculated in relation to HPV DNA and p16 status and was subjected to multivariate analysis. OPSCC cell lines were examined for sensitivity to radiation or cisplatin in vitro. The study results showed that tumor specimens from 40 (38%) of the 104 study patients contained HPV DNA, with such positivity being associated with tumors of the tonsils, lymph node metastasis, and nonsmoking. Overall survival was better for OPSCC patients with HPV DNA than for those without it (hazard ratio, 0.214; 95% confidence interval, 0.074-0.614; P = 0.002). Multivariate analysis revealed HPV DNA to be an independent prognostic factor for overall survival (P = 0.015). Expression of p16 was associated with HPV DNA positivity. However, 20% of HPV DNA-positive tumors were negative for p16, with most of these tumors manifesting DNA methylation at the p16 gene promoter. Radiation or cisplatin sensitivity did not differ between OPSCC cell lines positive or negative for HPV DNA. Thus, positivity for HPV DNA identifies a distinct clinical subset of OPSCC with a more favorable outcome in Japanese.
Project description:We determined that signal transducer and activator of transcription 3 (Stat3) is tyrosine phosphorylated in 37% of primary breast tumors and 63% of paired metastatic axillary lymph nodes. Examination of the distribution of tyrosine phosphorylated (pStat3) in primary tumors revealed heterogenous expression within the tumor with the highest levels found in cells on the edge of tumors with relatively lower levels in the central portion of tumors. In order to determine Stat3 target genes that may be involved in migration and metastasis, we identified those genes that were differentially expressed in primary breast cancer samples as a function of pStat3 levels. In addition to known Stat3 transcriptional targets (Twist, Snail, Tenascin-C and IL-8), we identified ENPP2 as a novel Stat3 regulated gene, which encodes autotaxin (ATX), a secreted lysophospholipase which mediates mammary tumorigenesis and cancer cell migration. A positive correlation between nuclear pStat3 and ATX was determined by immunohistochemical analysis of primary breast cancer samples and matched axillary lymph nodes and in several breast cancer derived cell lines. Inhibition of pStat3 or reducing Stat3 expression led to a decrease in ATX levels and cell migration. An association between Stat3 and the ATX promoter, which contains a number of putative Stat3 binding sites, was determined by chromatin immunoprecipitation. These observations suggest that activated Stat3 may regulate the migration of breast cancer cells through the regulation of ATX.
Project description:BACKGROUND:There has been a sharp rise in the incidence of human papillomavirus (HPV) associated oropharyngeal squamous cell carcinoma (OPSCC) in many countries. Patients with HPV-positive OPSCC have a more favorable prognosis compared with HPV-negative OPSCC, leading to investigation and adoption of de-escalation treatment protocols. The baseline rate of HPV prevalence in certain populations is of epidemiologic significance. We aimed to evaluate the rate of high-risk HPV in a large cohort of Thai patients, including OPSCC, oral SCC (OSCC) and laryngeal SCC (LSCC). METHODS:In total, 504 patients with HN cancer (110 OPSCC, 260 OSCC and 134 LSCC) who had been treated in Chulalongkorn University between 2010 and 2016 formed the sample set. All histological slides were reviewed to validate the diagnosis and render the histological type as keratinizing (K), non-keratinizing (NK) or non-keratinizing with maturation (NK-M). Immunohistochemistry with p16 was performed in all cases and scored semiquantatively. Positive and equivocal cases were tested by the high-risk HPV DNA in situ hybridization (ISH). Validation with quantitative polymerase-chain reaction (qPCR) was performed in p16-positive OPSCC. RESULTS:The OPSCC were represented by NK (7.3%), NK-M (16.4%) and K (76.4%) types, with an HPV incidence of 100, 22.2 and 4.7%, respectively. The average HPV prevalence in OPSCC was 14.5%. The concordance with p16/ISH was 51.6%, while concordance of the NK morphology with positive HPV ISH was 100%. ISH-qPCR concordance in p16-positive OPSCC was 72.7%. Patients with HPV-positive OPSCC had significantly more tumors with a NK histologic type, tonsillar location, earlier clinical stage, less association with smoking, and, finally, better outcome and longer survival time. In non-OPSCC, p16-positive HPV-associated cancers were found in only 1.5% of OSCC (4/260) and LSCC (2/134). CONCLUSION:A low rate of HPV-related OPSCC was found in Thai patients. The NK morphology was an excellent predictor of high-risk HPV infection in OPSCC. For OPSCC patients, HPV-positive ones had a significantly longer survival time than HPV-negative ones. There was a lack of p16-positive HPV-related OSCC and LSCC. Morphology and p16 status had a poor predictive value for detecting HPV in OSCC and LSCC.
Project description:Diffuse large B-cell lymphoma (DLBCL) with an activated B-cell (ABC) gene-expression profile has been shown to have a poorer prognosis compared with tumors with a germinal center B-cell type. ABC cell lines have constitutive activation of STAT3; however, the mechanisms regulating STAT3 signaling in lymphoma are unknown. In studies of class-I histone deacetylase (HDAC) expression, we found overexpression of HDAC3 in phospho STAT3-positive DLBCL and the HDAC3 was found to be complexed with STAT3. Inhibition of HDAC activity by panobinostat (LBH589) increased p300-mediated STAT3(Lys685) acetylation with increased nuclear export of STAT3 to the cytoplasm. HDAC inhibition abolished STAT3(Tyr705) phosphorylation with minimal effect on STAT3(Ser727) and JAK2 tyrosine activity. pSTAT3(Tyr705)-positive DLBCLs were more sensitive to HDAC inhibition with LBH589 compared with pSTAT3(Tyr705)-negative DLBCLs. This cytotoxicity was associated with downregulation of the direct STAT3 target Mcl-1. HDAC3 knockdown upregulated STAT3(Lys685) acetylation but prevented STAT3(Tyr705) phosphorylation and inhibited survival of pSTAT3-positive DLBCL cells. These studies provide the rationale for targeting STAT3-positive DLBCL tumors with HDAC inhibitors.
Project description:Tyrosine phosphorylated signal transducer and activator of transcription 3 (pStat3) is present in numerous cancers and is required for mediating tumorigenesis. Autocrine and paracrine interleukin (IL)-6 signaling is the principal mechanism by which Stat3 is persistently phosphorylated in epithelial tumors including breast, lung, colon and gastric cancer. The Ras oncogene mediates cellular transformation without evidence of pStat3 in cultured cells. Recently, however non-tyrosine phosphorylated Stat3 was shown to have a transcriptional activating function, a role in mitochondrial function and to mediate cell migration.. Here we examined the role of Stat3 in Ras mediated transformation.Ha-rasV12 transformed mammary epithelial cells (MCF10A) cells were transduced with a Stat3shRNA, IL-6shRNA and/or treated with inhibitors of Janus kinases (JAKs) to examine the role of the IL-6 signaling pathway in Ras mediated invasion and tumorigenesis.Cellular migration, invasion, anchorage independent growth and tumorigenesis were largely abrogated in the Stat3-reduced cells compared to control cells. Analysis of MCF10A-Ras tumors revealed high levels of pStat3 and interleukin-6. Tumors derived from transgenic MMTV-K-Ras mice were also found to express pStat3 and IL-6. MCF10A-Ras cells, when grown in a three-dimensional Matrigel culture system revealed the appearance of the junctional protein E-Cadherin as a consequence of reducing Stat3 levels or inhibiting Stat3 activity. Decreasing IL-6 levels in the MCF10A-Ras cells abrogated tumorigenesis and reduced cell migration. By isolating Ras-expressing primary tumors and serially passaging these cells in two-dimensional culture led to a decrease in IL-6 and pStat3 levels with the reappearance of E-Cadherin.The cellular and environmental context can lead to differential IL-6/pStat3 signaling and a dependency on this cytokine and transcription factor for migration, invasion and tumorigenesis.
Project description:Fibroblast growth factor receptor 3 (FGFR3) is expressed in squamous cell carcinoma of the head and neck (SCCHN) including oropharyngeal squamous cell carcinoma (OPSCC) and is a potential therapeutic target. However, information on its correlation with other relevant cancer related proteins stratified by p16 status and its prognostic significance in OPSCC is limited. We examined FGFR3 expression and its correlation with clinical characteristics, p16 status, and mutant p53 (mp53) among 220 retrospectively collected OPSCC cases and 40 prospectively collected SCCHN cases, including a majority of OPSCC. Correlations of FGFR3 Weighted Index (WI) with p16 status and mp53 WI as well as its association with disease-free survival (DFS) and overall survival (OS) were evaluated. FGFR3 expression was detected in 61% and 70% of cases in cohorts 1 and 2, respectively. FGFR3 level was significantly higher in p16-negative tumors in both cohorts (p<0.001 and 0.006). FGFR3 expression was highly correlated with mp53 expression in both p16 + and p16- OPSCC (p<0.0001 and p = 0.0006, respectively). In cohort 1, univariate analysis showed that FGFR3 was associated with DFS but not OS. Kaplan-Meier analysis showed that higher FGFR3 and mp53 level correlated with worse DFS (p = 0.025) and OS (p = 0.009). As expected, p16 positive status was associated with improved OS and DFS (p<0.001 for both). Our results suggest that high FGFR3 expression is associated with p16 negative status and mp53 expression in OPSCC and correlates with a worse clinical outcome. The biological relationship between FGFR3 and mp53 in OPSCC deserves further investigation.
Project description:STAT3 regulates cell growth by up-regulating downstream targets, such as Myc. The frequency of phosphorylated STAT3 (pSTAT3) and Myc expression and their prognostic relevance is unknown within diffuse large B-cell lymphoma (DLBCL) germinal center B-cell (GCB) and non-GCB subtypes. pSTAT3 and Myc were studied by immunohistochemistry (IHC) on tumors from 40 DLBCL patients uniformly treated on a clinical trial of epratuzumab/rituximab-CHOP. A total of 35% of cases were pSTAT3-positive, and pSTAT3 positivity was more frequent in the non-GCB (P = .06) type but did not correlate with event-free survival (EFS). Myc expression was observed in 50% of cases and was more frequent in non-GCB type (P = .07). Myc-positive cases had inferior EFS in all patients, including the GCB and pSTAT3-positive cases, were more likely to express Myc (P = .06). Myc translocations involving the major breakpoint regions were found in 10% (3 of 29) of cases, and all 3 cases were GCB and had an inferior EFS (P = .09). pSTAT3, but not Myc expression, was correlated with elevated pretreatment serum cytokines, such as IL-10 (P = .05), G-CSF (P = .03), and TNF-? (P = .04). pSTAT3 IHC in DLBCL tumors has the potential to identify patients for STAT3 pathway-directed therapy; Myc IHC is a potential marker for inferior EFS in GCB patients.
Project description:BACKGROUND:There is a paucity of studies reporting long-term survival outcomes for HPV/p16 positive oropharyngeal squamous cell carcinoma (OPSCC). This study aims to compare long-term outcomes of advanced stage p16 positive and negative OPSCCs, treated by surgical and non-surgical modalities. METHODS:OPSCC patients from 1998 to 2012 were identified through a prospectively collected cancer registry. P16 immunohistochemistry was used as a surrogate marker for HPV-OPSCC. Overall survival (OS) and aspiration free survival (AFS) comparisons were made between patients treated with chemoradiation (CRT) versus primary surgery and radiation/chemoradiation (S+RT/CRT) at 5, 10 and 15 years post-treatment. RESULTS:A total of 319 patients were included. P16 positive patients and non-smokers had significantly higher long-term (5, 10 and 15-year) OS. Smokers and p16 negative patients treated with S+RT/CRT had improved long-term OS compared to patients who received CRT. Smokers and p16 negative patients had lower long-term AFS. Multivariate analysis showed improved OS was associated with p16 positivity (HR 0.42, 0.28-0.61) and surgery (HR 0.47, 0.32-0.69), whereas lower OS was associated with ECOG ≥ 2 (HR 2.46, 1.61-3.77), smoking (HR 2.37, 1.41-3.99) and higher stage (HR 1.68, 1.05-2.68). CONCLUSIONS:In smokers and p16-negative OPSCC patients, primary surgery may be associated with improved long-term survival and dysphagia-related outcomes.
Project description:BACKGROUND: Signal transducer and activator of transcription 3 (STAT3) is a key signaling molecule and a central cytoplasmic transcription factor, implicated in the regulation of growth. Its aberrant activation has been demonstrated to correlate with many types of human malignancy. However, whether constitutive STAT3 signaling plays a key role in the survival and growth of soft-tissue tumors is still unclear and hence needs to be elucidated further. In our study we examined the expression levels of STAT3 and pSTAT3 in different grades of soft tissue tumors and correlated with its clinicopathological characteristics. METHODS: Expression levels of STAT3 and pSTAT3 in soft tissue tumors were studied using Immunohistochemistry, Western blotting and Reverse transcriptase- PCR and correlated with its clinicopathological characteristics using Chi squared or Fisher's exact test and by logistic regression analysis. Statistical analysis was done using Intercooled Stata software (Intercooled Stata 8.2 version). RESULTS: Of the 82 soft tissue tumor samples, fifty four (65.8%) showed immunoreactivity for STAT3 and twenty eight (34.1%) for pSTAT3. Expression of STAT3 and pSTAT3 was significantly associated with tumor grade (P < 0.001; P < 0.001), tumor location (P = 0.025; P = 0.027), plane of tumor (P = 0.011; P = 0.006), and tumor necrosis (P = 0.001; P = 0.002). Western blotting and RT-PCR analysis showed increased expression of STAT3 and p-STAT3 as grade of malignancy increased. CONCLUSION: These findings suggest that constitutive activation of STAT3 is an important factor related to carcinogenesis of human soft tissue tumors and is significantly associated with its clinicopathological parameters which may possibly have potential diagnostic implications.