High Signal Transducer And Activator Of Transcription 3 Expression Research Articles

Abstract 265: Cotargeting EGFR, STAT3 and Src-Notch pathways: a promising approach to improve the efficacy of EGFR-TKIs in the treatment of NSCLC patients

Abstract Intrinsic or acquired resistance limits the clinical effectiveness of EGFR tyrosine kinase inhibitors (TKIs) for non-small cell lung cancer (NSCLC) patients (p) with EGFR mutations. One of the signaling mediators downstream of activated EGFR is signal transducer and activator of transcription 3 (STAT3). Not only does gefitinib not inhibit STAT3, but it also augments STAT3 tyrosine phosphorylation. EGFR blockade enriches lung cancer stem cells (CSCs) through NOTCH3-dependent signaling. A co-receptor of IL-6 (gp130) associates with Src and triggers activation of YAP and NOTCH. Our study is designed with three parallel objectives: firstly, to demonstrate that single EGFR TKI treatment cannot abrogate STAT3 and Src in EGFR mutant NSCLC cell lines; secondly, to examine whether the combination of gefitinib with compounds that target STAT3, (TPCA-1) and Src (saracatinib), suppresses the mechanisms of resistance; thirdly, to identify biomarkers in clinical tumor samples that may help us predict the outcome of EGFR TKIs and design effective combination therapies. Cell viability assay (MTT), western blotting, quantitative-real time PCR (qRT-PCR) and aldefluor assay-flow cytometry were used. We found that gefitinib increases pSTAT3 Y705 in PC-9 cells (that harbor the exon 19 deletion) in a time- and dose-dependent manner. Nine days after gefitinib treatment STAT3 mRNA level was significantly elevated. PC-9 cells showed dramatic increase in the fraction of ALDH+ cells upon treatment with gefitinib. TPCA-1 increased sensitivity to gefitinib in the PC-9 cells. Combination of gefitinib with TPCA-1 abrogated pSTAT3 Y705 but neither inhibited pPaxillin Y118 (Src induced) and pYAP S127 nor prevented the increment in the ALDH+ CSCs subpopulation. The triple combination of gefitinib, TPCA-1 and saracatinib was highly synergistic and abrogated pSTAT3 Y705, pPaxillin Y118 and pYAP S127. We performed qRT-PCR at baseline tumor samples of 64 EGFR mutant NSCLC p treated with first line EGFR TKIs and found that high expression of STAT3 and YAP were significantly correlated with shorter median progression-free survival (mPFS). mPFS was 9.6 months (m) (95% CI, 5.9 to 14.1) for p with low STAT3 and 18.4m (95% CI, 8.8 to 30.2) for p with high STAT3 mRNA expression (P<0.001). mPFS was 9.6 months (95% CI, 7.7 to 15.2) for p with low YAP and 23.4 months (95% CI, 13.0 to 28.1) for p with high YAP mRNA expression (P = 0.005). A combined STAT3 and YAP risk group model was constructed since the mRNA expression of the 2 transcripts was weakly correlated (r = .0.15; P = 0.305). mPFS was 25.7 months for p with low STAT3 and YAP mRNA (95% CI, 8.5 to 60.9), 9.4 months for p with high STAT3 and YAP mRNA (95% CI, 2.8 to 15.2), and 14.1 months for others (95% CI, 8.2 to 23.4) (P = 0.004). Single EGFR TKI treatment can no longer be considered adequate for p with EGFR mutant lung cancer and a clinical trial co-targeting STAT3 and Src is warranted. Citation Format: Niki Karachaliou, Imane Chaib, Sara Pilotto, Jordi Codony, Xueting Cai, Xuefei Li, Ana Drozdowskyj, Carles Codony, Andrés Felipe Cardona, Guillermo López-Vivanco, Alain Vergnenègre, José Miguel Sánchez, Mariano Provencio, Filippo de Marinis, Enric Carcereny, Noemí Reguart, Rosario García-Campelo, Silvia Marin, Cristina Teixido, Isabella Sperduti, Sonia Rodríguez, Roger Estrada, Raimon Puig de la Bellacasa, José Luis Ramírez, Miguel Angel Molina-Vila, Caicun Zhou, Peng Cao, Patrick Ma, Trever Bivona, Rafael Rosell. Cotargeting EGFR, STAT3 and Src-Notch pathways: a promising approach to improve the efficacy of EGFR-TKIs in the treatment of NSCLC patients. [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 265.

The sonic hedgehog signaling pathway contributes to the development of salivary gland neoplasms regardless of perineural infiltration.

The pleomorphic adenoma (PA), mucoepidermoid carcinoma (MEC), and adenoid cystic carcinoma (ACC) are common tumors arising from salivary glands whose histopathology is heterogeneous. The sonic hedgehog signaling pathway (Hh) and signal transducer and activator of transcription 3 (STAT3) play important roles in cell proliferation, favoring tumor growth. The aim of this investigation was to study components of the Hh pathway, as well as STAT3 in salivary gland neoplasms in an attempt to add information about the biological characteristics of these neoplasms. We used 9 cases of PA, 17 cases of ACC, and 20 cases of MEC. Using immunohistochemistry, SHH, GLI1, SUFU, HHIP, and STAT3 were investigated. For comparative purposes, MCM3 (cellular proliferation marker) was also included. In PA, there was high expression of cytoplasmic SHH and SUFU and low expression of STAT3 and MCM3. In the ACC, there was high expression of GLI1, HHIP, and STAT3 and low expression of SHH, SUFU, and MCM3. In the MEC, we observed high expression of SHH, GLI1, SUFU, and HHIP and low expression of STAT3 and MCM3. There was a statistically significant difference between SHH (p = 0.0064), STAT3 (p = 0.0003), and MCM3 (p = 0.0257) when all tumors were compared and a higher expression in parenchyma for all tumors when stroma and parenchyma were compared (p < 0.05). These findings suggests a possible role of Hh pathway in the development and maintenance of the cytoarchitectural pattern of PA, ACC, and MEC, as well as the participation of STAT3 in the development of ACC, irrespective perineural infiltration.

A meta-analysis of STAT3 and phospho-STAT3 expression and survival of patients with non-small-cell lung cancer

The prognostic role of signal transducer and activator of transcription 3 (STAT3) and phospho-STAT3 in non-small-cell lung cancer (NSCLC) remains controversial. To clarify its impact on survival, we performed a meta-analysis to quantitatively assess STAT3 and phospho-STAT3 expression on the prognosis of NSCLC. Published studies were identified using a systematic and thorough literature search. To be eligible, a study had to investigate STAT3 or phospho-STAT3 expression rates of NSCLC patients in different characteristics and provide patient survival data. A total of 17 retrospective trials were chosen for meta-analysis, including 1793 patients. The estimated pooled log HR (0.67, 95% CI: 0.57-0.77) of 9 trials (STAT3: log HR 0.71, 95% CI 0.38-1.04; phospho-STAT3: log HR 0.67, 95% CI 0.56-0.77) for NSCLC was statistically significant (P < 0.0001), suggesting that high STAT3 or phospho-STAT3 expression is a strong predictor of poor prognosis among patients with NSCLC. For the risk factors, pooled analysis of patients with STAT3 positivity, demonstrated a statistically significant OR (3.82, 95% CI: 2.37-6.16) between poorly differentiated carcinoma and well-moderately, OR (5.68, 95% CI: 3.16-10.21) between stage III-IV patients and stage I-II patients, and OR (3.41, 95% CI: 2.12-5.49) between patients with lymph node metastasis and patients without lymph node metastasis. However, pooled analysis of patients with phospho-STAT3 positivity only demonstrated a statistically significant OR (4.51, 95% CI: 1.57-12.96) between poorly differentiated carcinoma and well-moderately (P < 0.05). High STAT3 or phospho-STAT3 expression is a strong predictor of poor prognosis among patients with NSCLC. The conclusion should be confirmed by large prospective studies with long-term follow-up.

STAT3 Activates Anti-Apoptotic Genes But When In Access Induces Apoptosis Of CLL Cells

In chronic lymphocytic leukemia (CLL) cells, signal transducer and activator of transcription 3 (STAT3) is constitutively phosphorylated on serine 727 residues, and phosphoserine STAT3 induces the transcription and proliferation of anti-apoptosis genes including c-Myc, BCL2, Bcl-XL, and Mcl-1 (Hazan-Halevy, 2010). Therefore, we hypothesized that the CLL cells of patients with high peripheral blood absolute lymphocyte counts (ALCs) have a high level of STAT3 protein expression and a low rate of spontaneous apoptosis. Using Western blotting and flow cytometry, we quantitated STAT3 levels and apoptosis rates in CLL cells from 64 patients with high (N=32) and low (N=32) ALCs. As expected, the levels of STAT3 expression were significantly higher in cells from patients with high ALCs (145,000 ± 49,738/µL) than in cells from patients with low ALCs (12,800 ± 4,654/µL). However, contrary to our hypothesis, Annexin V/PI staining revealed that the levels of procaspase-3, its activated (cleaved) form caspase-3, and cleaved PARP as well as the apoptosis rates of cells from patients with high ALCs were significantly higher than those in cells from patients with low ALCs. These findings suggest that cells from patients with high ALCs are prone to spontaneous apoptosis. An RNA microarray analysis revealed that the levels of apoptotic pathway genes were upregulated in cells from patients with high ALC (P = 0.002), and Reverse-transcriptaction PCR of Caspase-3, Calpain 9, MAPK 8, KRAS, PLCγ-2, and PKC validated the array data. Intrigued by these findings, we sought to determine whether high levels of intracellular STAT3 induce apoptosis. Indeed, overexpression of STAT3 in interleukin-6–stimulated MM1 cells upregulated caspase-3 and caspase-3 protein levels and induced apoptosis. Similarly, in CLL cells levels of caspase3 and procaspase3 remained stable across a wide range of STAT3 levels, but when STAT3 reached a threshold, level of caspase3 and procaspase3 markedly increased.Because sequence analysis revealed that the caspase-3 promoter harbors γ-interferon activation sequence-like elements, we cloned the caspase-3 promoter in MM1 cells and, using a luciferase assay, identified regions with putative STAT3 binding sites. Chromatin immunoprecipitation (ChIP) and an electrophoretic mobility shift essay (EMSA) confirmed that STAT3 binds to the promoter of caspase-3. However, caspase-3 was activated only in cells with high STAT3 expression levels, suggesting that STAT3 binds to caspase-3 with low affinity. To assess STAT3's binding affinity to the caspase-3 promoter, we prepared serial dilutions of CLL cell DNA and, using ChIP and EMSA, found that STAT3's binding affinities to p21 and c-Myc were 8 and 4 times as high, respectively, as its binding affinity to caspase-3, suggesting that high levels of STAT3 protein are required to activate caspase-3. Taken together, these findings suggest that activated STAT3 has a previously unknown pro-apoptotic function. At high intracellular levels, rather than providing CLL cells with survival advantage, STAT3 induces apoptosis by activating caspase-3. Thus, because CLL cell proliferation is coupled with spontaneous CLL cell apoptosis, the number of circulating CLL cells based on the cells' proliferation rate is often lower than expected. Disclosures:No relevant conflicts of interest to declare.

Significance of STAT3 Activation in Breast Cancer

Signal transducer and activator of transcription 3 (STAT3) is a latent transcription factor whose activation plays immense role in cell proliferation, survival, differentiation and development. Activation of STAT3 relies on phosphorylation at tyrosine residues promoting dimerization and subsequent nuclear translocation and activation of transcription. This study explores the relation between STAT3 and activated STAT3 (p-STAT3) in malignancy of breast. A high expression of STAT3 was found in breast cancer compared to adjacent normal breast tissues. A significant correlation between STAT3 and p-STAT3 was also observed. We also examined possible associations of these biomarkers with various clinicopathological parameters of breast cancer patients. The results revealed that both STAT3 and p-STAT3 are strongly associated with some of the major risk factors of breast cancer. Our findings implicate that targeting STAT3 signalling pathway could be an effective therapeutic approach for breast cancer.

Prognostic significance of STAT3 expression and its correlation with chemoresistance of non-small cell lung cancer cells

Activation of signal transducer and activator of transcription 3 (STAT3) plays important roles in tumorigenesis and tumor development. Previously, we have reported that overexpression of STAT3 potentiates growth, survival and radioresistance of non-small cell lung cancer (NSCLC) cells. The aim of this study was to investigate the prognostic significance of STAT3 expression and its correlation with chemoresistance of NSCLC cells. Semi-quantitative RT-PCR was performed to detect the expression of STAT3 mRNA in 12 NSCLC and corresponding adjacent lung tissues. Immunohistochemistry was performed to detect the expression of STAT3 protein in 76 NSCLC tissue samples. Additionally, the correlation between STAT3 expression and prognosis of NSCLC patients was statistically analyzed. The role of STAT3 in chemoresistance of NSCLC cells was also assessed by the vector-based small interfering RNA. The expression level of STAT3 mRNA in NSCLC tissues was significantly higher than that in corresponding adjacent lung tissues (P<0.05). Positive immunostaining of STAT3 protein was mainly located in the cytoplasm of tumor cells. The expression of STAT3 protein was significantly correlated with tumor differentiation, clinical stage and lymph node metastasis of NSCLC patients. Moreover, the 5-year overall survival rate of patients with high STAT3 expression (42.3%) was significantly lower than that of patients with low STAT3 expression (58.8%; P<0.001). Multivariate analysis using the Cox proportional hazard model showed that high STAT3 protein expression was an independent prognostic factor for NSCLC patients (P=0.021). Furthermore, two stably transfected cell lines (A549/shSTAT3 and SPC-A1/shSTAT3) were successfully established, and RNAi-mediated STAT3 inhibition could significantly increase the sensitivity of NSCLC cells to cisplatin by enhancing caspase-3-dependent apoptosis. Together, the expression of STAT3 might be an independent prognostic marker for NSCLC patients and RNAi-mediated STAT3 inhibition would be a potential strategy for chemosensitization of NSCLC cells.

STAT3 Silencing in Dendritic Cells by siRNA Polyplexes Encapsulated in PLGA Nanoparticles for the Modulation of Anticancer Immune Response

In dendritic cells (DCs), the induction of signal transducer and activator of transcription 3 (STAT3) by tumor-derived factors (TDFs) renders DCs tolerogenic and suppresses their antitumor activity. Therefore, silencing STAT3 in DCs is beneficial for cancer immunotherapy. We have shown that STAT3 knockdown in B16 murine melanoma by siRNA polyplexes of polyethylenimine (PEI) or its stearic acid derivative (PEI-StA) induces B16 cell death in vitro and in vivo. Here, we investigated the physical encapsulation of siRNA/PEI and PEI-StA polyplexes in poly(d,l-lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) for STAT3 knockdown in DCs. PLGA NPs containing siRNA polyplexes of PEI (PLGA-P) and PEI-StA (PLGA-PS) had an average diameter of ~350 to 390 nm and a zeta potential of ∼-13 to -19 mV, respectively. The encapsulation efficiency (E.E.) of siRNA in PLGA-P and PLGA-PS was 26% and 43%, respectively. In both NP types, siRNA release followed a triphasic pattern, but it was faster in PLGA-PS. Our uptake study by fluorescence microscopy confirmed DC uptake and endosomal localization of both NP types. After exposure to B16.F10 conditioned medium, DCs showed high STAT3 and low CD86 expression indicating impaired function. STAT3 silencing by PLGA-P and PLGA-PS of STAT3 siRNA restored DC maturation and functionality as evidenced by the upregulation of CD86 expression, high secretion of TNF-α and significant allogenic T cell proliferation. Moreover, encapsulation in PLGA NPs significantly reduced PEI-associated toxicity on DCs. We propose this formulation as a strategy for targeted siRNA delivery to DCs. The potential of this approach is not limited to STAT3 downregulation in DCs but can be used to target the expression of other proteins as well. Moreover, it can be combined with other means for cancer immunotherapy like cancer vaccine strategies.

Nuclear Localization of Signal Transducer and Activator of Transcription 3 in Head and Neck Squamous Cell Carcinoma Is Associated with a Better Prognosis

A high frequency of head and neck squamous cell cancers (HNSCC) contain constitutively activated signal transducer and activator of transcription 3 (STAT3). To further elucidate the prognostic role of STAT3 in HNSCC, the expression pattern of STAT3 was correlated with outcome in two independent data sets. STAT3 protein expression analysis was done on a test cohort of 102 patients with HNSCC recruited between 1992 and 2005. Automated quantitative analysis was used to assess STAT3 protein expression. We evaluated associations with clinicopathologic parameters and survival prognosis. Associations were validated in a second, independent cohort of 58 patients with confirmed HNSCC enrolled in the Early Detection Research Network-sponsored study who underwent surgical resection with curative intent at the University of Pittsburgh Medical Center between 2000 and 2004. STAT3 displayed mixed nuclear and cytoplasmic staining. Survival analysis showed that high nuclear STAT3 expression (top tertile versus the rest) was associated with longer progression-free survival (n = 70, mean survival of 88.9 versus 46.7 months, P = 0.012 for the first cohort; n = 37, mean survival of 60.3 versus 33.0 months, P = 0.009 for the second cohort). After best model selection in the multivariable analysis context, only STAT3 was significant, revealing a lower risk of progression and death for patients with high nuclear STAT3-expressing tumors (hazard ratio, 0.28; 95% confidence interval, 0.10-0.82; P = 0.019; and hazard ratio, 0.23; 95% confidence interval, 0.07-0.76; P = 0.016, respectively). Our results indicate that high nuclear STAT3 expression levels by automated quantitative analysis are associated with favorable outcome in HNSCC.

Integration of HIV-1 caused STAT3-associated B cell lymphoma in an AIDS patient

Signal transducer and activator of transcription 3 (STAT3) is a DNA-binding transcription factor activated by multiple cytokines and interferons. High expression of STAT3 has also been implicated in cancer and lymphoma. Here, we show a case of B cell lymphoma in which a defective human immunodeficiency virus 1 (HIV-1) integrated upstream of the first STAT3 coding exon. The lymphoma cells with anaplastic large cell morphology formed multiple nodular lesions in the lung of an acquired immunodeficiency syndrome (AIDS) patient with Kaposi's sarcoma. The provirus had a 5′ long terminal repeat (LTR) deletion, but the 3′ LTR had stronger promoter activity than the STAT3 promoter in reporter assays. Immunohistochemistry showed increased expression of STAT3 in the nuclei of lymphoma cells. Transfection of STAT3 resulted in transient cell proliferation in primary B cells in vitro. Although this is a very rare case of HIV-1-integrated lymphoma, these data suggest that up-regulation of STAT3 caused by HIV-1 integration resulted in the development of B cell lymphoma in this special case.

Cyclin D1 Overexpression and Response to Bortezomib Treatment in a Breast Cancer Model

Cyclin D1 is frequently overexpressed in breast cancer, and its overexpression is, surprisingly, associated with improved survival. One potential mechanism for this association involves signal transducer and activator of transcription 3 (STAT3). Cyclin D1 and STAT3 expression were assessed in human tumors using microarray analysis and in breast cancer cell lines HBL100, T47D, MCF7, MDA-MB-453, and BT20 and in HBL100 and T47D cells stably overexpressing cyclin D1 using immunoblot analysis. Cyclin D1 protein was stabilized by treatment with the proteasome inhibitor bortezomib, and the effects on STAT3 expression in vitro was determined by using immunoblotting and on xenograft tumor growth and apoptosis in vivo was determined by using terminal deoxyuridine nick-end labeling assays. All statistical tests were two-sided. Tumors with high cyclin D1 expression (n = 17) had low STAT3 expression (mean = 274 arbitrary units), and those with low cyclin D1 expression (n = 31) had high STAT3 expression (mean = 882 arbitrary units) (P<.001). In HBL100 and T47D parental and cyclin D1-overexpressing cells, cyclin D1 overexpression was also inversely associated with STAT3 expression, and cyclin D1 directly reduced the expression of STAT3. Stabilization of cyclin D1 protein by bortezomib treatment further amplified the cyclin D1-dependent repression of STAT3 in vitro and slowed tumor growth in vivo (week 7: untreated mean = 185.7 mm3 versus treated mean = 136.2 mm3, difference = 49.5 mm3, 95% confidence interval [CI] = 18 to 81 mm3, P = .007; week 8: untreated mean = 240.2 mm3 versus treated mean = 157.3 mm3, difference = 82.9 mm3, 95% CI = 9.1 to 156.7 mm3, P = .0014; and week 9: untreated mean = 256.4 mm3 versus treated mean = 170.2 mm3, difference = 86.2 mm3, 95% CI = 22.8 to 149.6 mm3, P = .006) and increased apoptosis (untreated mean = 19% versus treated mean = 54%, difference = 35%, 95% CI = 24.7% to 45.4%; P = .013) of xenograft tumors. Cyclin D1 repression of STAT3 expression may explain the association between cyclin D1 overexpression and improved outcome in breast cancer. In addition, bortezomib can amplify the proapoptotic function of cyclin D1, raising the possibility that cyclin D1 levels may be a marker for predicting the response to this novel drug.