Low pSTAT3 Research Articles

390 Epigenetic and Post-Translational Modification of STAT3 Regulates Its Spatiotemporal Trafficking and Transcriptional Activity in Mesenchymal Cells of Crohn's Disease and Animal Models of Crohn's Disease

STAT3 activity is governed by phosphorylation and acetylation. Post-translational modifications determine localization to the nucleus for transcriptional activity, or to signaling endosomes for regulation of signaling pathways. We recently identified a pattern of high pSTAT3(S727) and low pSTAT3(Y705) levels due to autocrine IL-6 production in mesenchymal cells of strictures in fibrostenotic Crohn's disease. The opposite is seen in the normal intestine in the same patient. This pattern characterizes B2 from other Crohn's phenotypes. We recently showed that STAT3 regulates transcription of TGFβ1 and collagen I. TheAIM of this study is to determine the post-translational and epigenetic modifications that determine the subcellular trafficking and function of STAT3. METHODS: Subepithelial myofibroblasts (SEMF) and smooth muscle cells (SMC) were isolated from strictured intestine and normal intestine of patients with fibrostenotic Crohn's disease and from intestine of TNBS treated C57Bl/6J mice and RAG(-/-) mice following transfer of CD4+CD45RBhi T-cells both after 8 weeks. Isolated cells were used to prepare RNA and cell lysates. Histologic sections were also prepared for immunofluorescence. Cell lysates and immunofluorescent staining of primary cultured cells were used to determine STAT3 subcellular localization of phosphorylated Y705 and S727 and acetylated K685. RESULTS: In both animal models expression of TGF-β1 and Collagen I increased in mesenchymal cells after 8 weeks similar to Crohn's disease. Similar patterns of S727 and Y705 phosphorylation and K685 acetylation and subcellular localization were observed in SEMF and SMC in B2 Crohn's disease (Figure 1), TNBS colitis and T-cell transfer. Phospho S727 and Acetyl K685 were increased and phospho Y705 was decreased in strictures in mice. Phospho S727 and acetyl K685 were localized to the nucleus, phospho Y705 was localized to endosomes in humans (Figrue 2) and mice. The opposite pattern was seen in normal intestine and control mice. IL-6-induced STAT3 nuclear localization of phospho S727 and acetyl K685 was enhanced by inhibition of histone deacetylation with Trichostatin A (500nM) and blocked by the inhibition of acetylation with the p300/CBP acetylation inhibitor, C646 (2.5 μM). IL-6-induced Erk1/2 activation and mesenchymal cell proliferation was inhibited by transfection of a dominant negative STAT3(Y705F) mutant or by pharmacologic inhibition of STAT3 phosphorylation. CONCLUSIONS: Activity of STAT3 is regulated by post-transcriptional modification of phosphorylation state and by epigenetic modification of acetylation of lysine residues that determine its subcellular localization and activity. Distinct subcellular localization to nucleus or signaling endosome differentially regulate transcription of TGF-β1 and collagen IαI, and proliferation of mesenchymal cells, respectively.

O3-10-5 - Loss of Stat3 Tyrosine Phosphorylation in Tumor Relates Poor Prognosis in Patients with Advanced Pancreatic Cancer

Background: Signal transducer and activator of transcription 3 (STAT3) is a central cytoplasmic transcription factor that is activated by phosphorylation of a conserved tyrosine residue. Activated STAT3 is a key to malignant progression in many cancers including pancreatic cancer (PC), while loss of phosphorylated (tyrosine705)-STAT3 (pSTAT3) relates poor prognosis in breast cancer. This study aimed to elucidate a prognostic impact of pSTAT3 in patients with advanced PC.Methods: Patients who were scheduled to undergo chemotherapy as initial treatment for PC with liver metastasis were enrolled. Immunohistochemical analysis of pSTAT3 was performed on a specimen from liver mets, which was harvested by needle biopsy. The nuclear expression of pSTAT3 in tumor was examined, and labeling index of nuclear pSTAT3 positive-tumor cell (pSTAT3LI) was calculated in each tumor. Patients with pSTAT3LI more than the median were assigned to high pSTAT3 group. Clinical data including the efficacy of chemotherapy were collected prospectively. A prognostic impact of pSTAT3LI was analyzed.Result: Fifty-five patients were evaluated (male: 61.8%, median age: 69 years, ECOG-performance status 0: 56.4%). Systemic chemotherapy was performed in all 55 patients (gemcitabine [GEM]: 32.7%, GEM plus erlotinib: 45.5%). Median overall survival (OS) and progression free survival (PFS) in all patients were 5.6 and 2.3 months, respectively. A median of pSTAT3 LI was 0.073 in all patients. Low pSTAT3 group showed poor prognosis than high pSTAT3 group in OS (low vs. high: 3.3 vs. 6.1 months; P = 0.02). There was no significant differences in PFS between low and high pSTAT3 group (1.9 vs. 2.7 months; P = 0.24), however the rate of 2nd line chemotherapy in low pSTAT3 group (19.2%) was decreased, compared to that in high pSTAT3 group (67.9%, P < 0.01).Conclusion: A decrease of STAT3 activation in liver metastasis of PC related a shortened survival time and low rate of 2nd line chemotherapy.

PTPLAD2 is a tumor suppressor in esophageal squamous cell carcinogenesis

Esophageal squamous cell carcinomas (ESCCs) are highly invasive and have poor prognoses. We investigated the role of PTPLAD2, a protein tyrosine phosphatase-like A domain (PTPLAD) family member, in ESCC carcinogenesis. Survival analysis was performed using patient data. ESCC tissue samples lost PTPLAD2 heterozygosity and had decreased PTPLAD2 expression. Low PTPLAD2 expression and high p-STAT3 correlated with poor prognosis. Overexpression of PTPLAD2 in ESCC cells reduced STAT3 phosphorylation, decreased FoxM1, inhibited proliferation and decreased in mouse xenograft tumor formation. Therefore, PTPLAD2 is a potential tumor suppressor and prognostic indicator that reduces STAT3 phosphorylation. PTPLAD2 is a possible clinical target for ESCC treatment.

Extent of neutrophil (NTP) infiltration in benign lymph nodes (LNs) to predict survival in patients with muscle-invasive bladder cancer (MIBC).

e15522 Background: In preclinical models, NTPs appear to establish a pre-metastatic niche that fosters the invasion of metastases (Kowanetz et al. PNAS 2010). This observation still requires clinical validation in MIBC. Methods: Benign LN tissue was obtained from patients (pts) who had undergone cystectomy and LN dissection for documented MIBC. Immunohistochemical (IHC) staining for CD15 (a NTP marker) was performed. Interleukin-17 (IL-17) and phosphorylated signal transducer and activator of transcription 3 (pSTAT3), putative mediators of NTP recruitment, were assessed through the same method (Laan et al. J Immunol 1999; Fielding et al. J Immunol 2008). Positively staining cells were counted and averaged over 8 high power fields. Pts were stratified by the median cell count for each biomarker. Analyses of overall survival (OS) were performed using the Kaplan-Meier method and log-rank test. Results: Of 55 pts with MIBC, 19 pts received no neoadjuvant chemotherapy (NAC), while 36 pts had received NAC with either GC (n=17) or MVAC (n=19). CD15 and IL-17 expression was significantly lower in pts with prior NAC (P&lt;0.001 for both), while expression of pSTAT3 was similar in both groups. Furthermore, across the whole cohort, a strong association was seen between expression of CD15 and IL-17 (r=0.73, P&lt;0.001). Amongst patients with no prior NAC, median OS was higher in those pts with low CD15 v high CD15 (158.7 mos v 36.9 mos, P=0.02), and low pSTAT3 v high pSTAT3 (NR v 106.4 mos, P=0.04). Median OS was numerically higher in pts with low IL-17 v high IL-17 (114.5 v 36.9 mos; P=0.14). Patients with both low CD15/IL-17 had a particularly favorable outcome. Amongst pts with prior exposure to NAC, no difference in survival was noted based on CD15, pSTAT3 or IL-17. Conclusions: NTP recruitment to benign LNs (potentially mediated by IL-17) may be prognostic of OS in pts with MIBC who have not received NAC. Bolstered by these findings, the prognostic value of NTP recruitment will be examined prospectively in SWOG 1011, a trial comparing limited v extended LN dissection in pts with MIBC.

Phase II study of ruxolitinib in patients with pStat3+ breast cancer.

TPS1134 Background: Multiple lines of evidence implicate the IL-6/JAK2/Stat3 signaling pathway in metastatic progression and therapeutic resistance in breast cancer (Marotta et al, JCI2011; Britschgi et al, Cancer Cell 2012). Ruxolitinib, an oral inhibitor of JAK1 and JAK2, is approved for the treatment of intermediate or high-risk myelofibrosis, but has not been extensively tested in solid tumors. Methods: Pts with triple-negative breast cancer or inflammatory breast cancer of any subtype are eligible for prescreening of archival tumor tissue for pStat3 expression by immunohistochemistry. Pts with high (Cohort A; T-score &gt;5) or low (Cohort B; T-score 3-4) pStat3 expression, measurable disease, adequate organ function, ECOG PS 0-2, and progression through &gt; 1 line of prior therapy may proceed to receive ruxolitinib, 25 mg orally twice daily. Staging studies are performed at baseline (BL) and every 8 weeks (wk). Baseline tumor biopsy is required for pts who have accessible disease, with an optional biopsy at progression. Blood for IL-6, CRP, and circulating tumor cells are collected at BL, Wk 4, and off-treatment. Patient reported outcomes including EORTC QLQ C-30 and the M.D. Anderson Symptom Inventory are collected at BL, Wk 4, Wk 8, and off-treatment. Statistical Considerations: The primary endpoint of this open-label phase 2 trial is objective response by RECIST 1.1. The study is designed to distinguish between a response rate of 5% versus 20% in each cohort, separately. If &gt; 2 responses out of 21 pts are observed in the first stage of Cohort A, a further 20 pts will be entered on that cohort; the agent will be deemed worthy of further study if &gt; 5 of the total 41 pts achieve an objective response (power 0.90, type I error 0.046). Cohort B will open to accrual if Cohort A passes the first stage. If &gt; 2 responses out of 21 pts are observed in the first stage of Cohort B, a further 20 patients will be entered into Cohort B, and the agent will be deemed worthy of further study if &gt; 5 of the total 41 pts achieve an objective response (power 0.90, type I error 0.046). Study Status: A total of 85 patients have consented for prescreening of tumor tissue. As of January 3, 2013, 5 of a planned 41 patients with high pStat3 IHC scores have been treated with ruxolitinib, and accrual is ongoing. Clinical trial information: NCT01562873.

Prediction of survival in patients with muscle-invasive bladder cancer (MIBC) by neutrophil (NTP) infiltration in benign lymph nodes (LNs).

273 Background: In preclinical models, NTPs appear to establish a pre−metastatic niche that fosters the invasion of metastases (Kowanetz et al PNAS 2010). This observation still requires clinical validation in MIBC. Methods: Benign LN tissue was obtained from patients (pts) who had undergone cystectomy and LN dissection for documented MIBC. Immunohistochemical (IHC) staining for CD15, a NTP marker, was performed for the entire cohort. Phosphorylated signal transducer and activator of transcription 3 (pSTAT3), vascular endothelial growth factor receptor−1 (VEGFR1), and CD68 (a macrophage marker) were further assessed in an initial cohort (detailed subsequently) via IHC. Positively staining cells were counted and averaged over 8 high power fields (hpfs). Pts were stratified by the median cell count for each biomarker. Analyses of overall survival (OS) were performed using the Kaplan−Meier method and log−rank test. Results: In an initial cohort of 19 pts who had received no neoadjuvant chemotherapy (NC), a median CD15 count of 284/hpf was noted. Median OS was higher in those pts with low CD15 as compared to high CD15 (158.7 mos v 36.9 mos, P=0.02). Median OS was also improved in those with high pSTAT3 v low pSTAT3 (not reached v 106.4 mos, P=0.04), but no difference was noted in OS in groups stratified by clinical stage, VEGFR1 staining, or CD68 staining. To determine if the prognostic value of CD15 staining was retained in pts with exposure to NC, the cohort was expanded to include an additional 36 pts who had received either preoperative GC (n=17) or MVAC (n=19) chemotherapy. In this group, no significant difference in OS was noted based using the previously applied CD15 cutoff. Conclusions: NTP recruitment to benign LNs may be prognostic of OS in pts with MIBC who have not received NC. pSTAT3, a putative mediator of NTP recruitment, may play a role in this phenomenon. VEGFR1 and CD68, which may mediate pre−metastatic niche through a different mechanism, do not predict OS in our dataset (Kaplan et al Nature 2005). Bolstered by these findings, the prognostic value of NTP recruitment will be examined prospectively in SWOG 1011, a trial comparing limited v extended LN dissection in pts with MIBC.

Prognostic Significance of B-Cells and pSTAT3 in Patients with Ovarian Cancer

BackgroundSeveral previous studies have identified a strong association between T-cell infiltration and clinical outcome in ovarian cancer. The role of B-cells remains controversial, however.MethodsForty-nine paraffin-embedded omental specimens derived from patients with high grade epithelial ovarian cancer were assessed. Immunohistochemical analyses were performed to characterize expression of CD19+ B-cells and pSTAT3 as high (>50% positively staining cells [PSCs]) or low (<50% PSCs). The Kaplan-Meier method with log-rank test was used to determine the association between clinicopathologic parameters and overall survival (OS). A multi-variate Cox proportional hazards regression analysis including nature of debulking (primary vs secondary), histology, tumor grade, receipt of prior chemotherapy, B-cell infiltration and pSTAT3 expression was performed.ResultsMedian OS was 160.6 months in those patients with low B-cell expression vs 47.3 months in those with high B-cell expression (P = 0.0015). Similarly, median OS was improved in those patients with low pSTAT3 expression (160.6 vs 47.9 months, P = 0.02). In a multivariate model to predict survival, only the degree of B-cell infiltration and clinical stage were retained. pSTAT3 expression did not enter the final model, possibly be due to a high positive correlation with B-cell infiltration (r = 0.82, P<0.0001).ConclusionsIncreased B-cell infiltration and pSTAT3 expression in omental tissue are associated with poorer survival.

Loss of SOCS3 expression is associated with an increased risk of recurrent disease in breast carcinoma

Constitutive activation of JAK/STAT pathway is observed in various solid tumors and hematological malignancies. SOCS3 acts as a key negative regulator of JAK/STAT pathway and represents one of the candidate tumor suppressor genes. In the current study, we aimed to evaluate SOCS3 expression in breast carcinoma and to explore the prognostic significance of SOCS3. The expression of SOCS3 was measured by Western blot and immunohistochemistry in breast carcinoma cells and a large cohort of tissue microarray, respectively. Among 367 human primary breast tumors, SOCS3 protein was detected in 103 patients. Deficient SOCS3 expression correlated significantly with lymph node metastasis (P = 0.003), blood vessel invasion (P = 0.029), VEGF (P = 0.001) and Ki-67 (P = 0.027). Univariate and multivariate analyses revealed that SOCS3 expression was an independent prognostic factor for disease-free survival (P < 0.0001). A positive SOCS3 protein expression correlated significantly with a low pSTAT3 protein expression in breast carcinoma (P = 0.015). The patients with a SOCS3 (+)/pSTAT3 (-) phenotype had a better prognosis than any other combination (DFI: P < 0.0001, BCSS: P = 0.013). Deficient expression of SOCS3 is associated with an aggressive phenotype and portends a poor clinical outcome in breast carcinoma.

Tissue micro array analysis of ganglioside N-glycolyl GM3 expression and signal transducer and activator of transcription (STAT)-3 activation in relation to dendritic cell infiltration and microvessel density in non-small cell lung cancer

BackgroundTumor immune escape and angiogenesis contribute to tumor progression, and gangliosides and activation of signal transducer and activator of transcription (STAT)-3 are implicated in these processes. As both are considered as novel therapeutic targets, we assessed the possible association of ganglioside GM3 expression and STAT3 activation with suppression of dendritic cell (DC) activation and angiogenesis in non-small cell lung cancer (NSCLC).MethodsImmunohistochemistry was performed on a tissue array to determine N-glycolyl GM3 (GM3) and phosphorylated STAT3 (pSTAT3) expression in 176 primary NSCLC resections. Median values of GM3 and pSTAT3 expression were used as cut off. Microvessel density (MVD) was determined by CD34 staining and morphology. CD1a and CD83 were used to determine infiltrating immature and mature dendritic cells, respectively.Results94% and 71% of the NSCLC samples expressed GM3 and nuclear pSTAT3, respectively. Median overall survival was 40.0 months. Both low GM3 expression and high pSTAT3 expression were associated with a worse survival, which reached near significance for GM3 (P = 0.08). Microvessel density (MVD), determined by CD34 staining and morphology, was lower in NSCLC samples with high GM3 expression. CD1a+ cells (immature DCs) were more frequent in NSCLC tissues as compared to peritumoral lung tissue, while CD83+ cells (mature DCs) were more frequent in peritumoral lung tissue. CD83+ DCs were less frequent in NSCLC tissues with high GM3 expression.ConclusionGM3 and pSTAT3 are widely expressed in NSCLC. Based on CD83 expression, GM3, but not pSTAT3, appeared to be involved in tumor-induced DC suppression. pSTAT3 expression was not associated with MVD, while GM3 might play an anti-angiogenic role.

Association of Serum and Follicular Fluid Leptin Concentrations with Granulosa Cell Phosphorylated Signal Transducer and Activator of Transcription 3 Expression in Fertile Patients with Polycystic Ovarian Syndrome

Our objective was to evaluate whether polycystic ovarian syndrome (PCOS)-associated infertility is related to alterations of leptin, leptin receptor (Ob-R), and the phosphorylated signal transducer and activator of transcription 3 (p-STAT3)/suppressor of cytokine signal 3 (SOCS3) system in the ovary. A case-control study was conducted in a university hospital. Thirty-one infertile PCOS women with oligoovulation plus polycystic ovarian morphology and 79 infertile women with tubal blockage (control) participated in the study. The subjects were stratified according to in vitro fertilization outcomes: successful and failed subgroups. Serum and follicular fluid (FF) leptin levels were measured with ELISA. RT-PCR and Western blotting were performed to assess expression of mRNA encoding leptin and Ob-R and proteins of p-STAT3 and SOCS3 in granulosa cells (GCs). Leptin levels in serum and FF of PCOS women were significantly higher than those of control (P < 0.01). There were no significant differences in expression of leptin mRNA and short and long Ob-Rs between PCOS and control (P > 0.05). The p-STAT3 level was decreased in PCOS compared with control (P < 0.01), whereas SOCS3 remained significantly unchanged (P > 0.05). Further analysis showed that serum and FF leptin levels were significantly higher, whereas p-STAT3 in GCs was lower in the failed subgroup of PCOS than those in the successful subgroup of PCOS (P < 0.05). Hyperleptinemia and high FF leptin are important pathologies of PCOS with infertility. Lower levels of p-STAT3 in GCs may be related to ovarian leptin resistance and fecundity in PCOS women. Relatively high serum and FF leptin and low p-STAT3 in GCs may account for decreased fertilization, implantation, and pregnancy rates of in vitro fertilization in PCOS women.

Altered p-STAT3 (tyr705) expression is associated with histological grading and intratumour microvessel density in hepatocellular carcinoma

Background: Constitutive activation of signal transducer and activator of transcription 3 at tyrosine residue 705 (p-STAT3 (tyr705)) has been associated with many types of human cancers. However, its potential roles...