Oncogenic STAT3 Signaling Research Articles

NRN1 interacts with Notch to increase oncogenic STAT3 signaling in melanoma

BackgroundMelanoma is a highly heterogeneous cancer, in which frequent changes in activation of signaling pathways lead to a high adaptability to ever changing tumor microenvironments. The elucidation of cancer specific signaling pathways is of great importance, as demonstrated by the inhibitor of the common BrafV600E mutation PLX4032 in melanoma treatment. We therefore investigated signaling pathways that were influenced by neurotrophin NRN1, which has been shown to be upregulated in melanoma.MethodsUsing a cell culture model system with an NRN1 overexpression, we investigated the influence of NRN1 on melanoma cells’ functionality and signaling. We employed real time cell analysis and spheroid formation assays, while for investigation of molecular mechanisms we used a kinase phosphorylation kit as well as promotor activity analysis followed by mRNA and protein analysis.ResultsWe revealed that NRN1 interacts directly with the cleaved intracellular domain (NICD) of Notch1 and Notch3, causing a potential retention of NICD in the cytoplasm and thereby reducing the expression of its direct downstream target Hes1. This leads to decreased sequestration of JAK and STAT3 in a Hes1-driven phosphorylation complex. Consequently, our data shows less phosphorylation of STAT3 while presenting an accumulation of total protein levels of STAT3 in association with NRN1 overexpression. The potential of the STAT3 signaling pathway to act in both a tumor suppressive and oncogenic manner led us to investigate specific downstream targets – namely Vegf A, Mdr1, cMet - which were found to be upregulated under oncogenic levels of NRN1.ConclusionsIn summary, we were able to show that NRN1 links oncogenic signaling events between Notch and STAT3 in melanoma. We also suggest that in future research more attention should be payed to cellular regulation of signaling molecules outside of the classically known phosphorylation events.Graphical

PDSS1-Mediated Activation of CAMK2A-STAT3 Signaling Promotes Metastasis in Triple-Negative Breast Cancer.

Genomic alterations are crucial for the development and progression of human cancers. Copy-number gains found in genes encoding metabolic enzymes may induce triple-negative breast cancer (TNBC) adaptation. However, little is known about how metabolic enzymes regulate TNBC metastasis. Using our previously constructed multiomic profiling of a TNBC cohort, we identified decaprenyl diphosphate synthase subunit 1 (PDSS1) as an essential gene for TNBC metastasis. PDSS1 expression was significantly upregulated in TNBC tissues compared with adjacent normal tissues and was positively associated with poor survival among patients with TNBC. PDSS1 knockdown inhibited TNBC cell migration, invasion, and distant metastasis. Mechanistically, PDSS1, but not a catalytically inactive mutant, positively regulated the cellular level of coenzyme Q10 (CoQ10) and intracellular calcium levels, thereby inducing CAMK2A phosphorylation, which is essential for STAT3 phosphorylation in the cytoplasm. Phosphorylated STAT3 entered the nucleus, promoting oncogenic STAT3 signaling and TNBC metastasis. STAT3 phosphorylation inhibitors (e.g., Stattic) effectively blocked PDSS1-induced cell migration and invasion in vitro and tumor metastasis in vivo. Taken together, our study highlights the importance of targeting the previously uncharacterized PDSS1/CAMK2A/STAT3 oncogenic signaling axis, expanding the repertoire of precision medicine in TNBC. SIGNIFICANCE: A novel metabolic gene PDSS1 is highly expressed in triple-negative breast cancer tissues and contributes to metastasis, serving as a potential therapeutic target for combating metastatic disease.

Pharmacological Inhibition of Oncogenic STAT3 and STAT5 Signaling in Hematopoietic Cancers.

Signal Transducer and Activator of Transcription (STAT) 3 and 5 are important effectors of cellular transformation, and aberrant STAT3 and STAT5 signaling have been demonstrated in hematopoietic cancers. STAT3 and STAT5 are common targets for different tyrosine kinase oncogenes (TKOs). In addition, STAT3 and STAT5 proteins were shown to contain activating mutations in some rare but aggressive leukemias/lymphomas. Both proteins also contribute to drug resistance in hematopoietic malignancies and are now well recognized as major targets in cancer treatment. The development of inhibitors targeting STAT3 and STAT5 has been the subject of intense investigations during the last decade. This review summarizes the current knowledge of oncogenic STAT3 and STAT5 functions in hematopoietic cancers as well as advances in preclinical and clinical development of pharmacological inhibitors.

EGFR blockade in GBM brain tumor stem cells synergizes with JAK2/STAT3 pathway inhibition to abrogate compensatory mechanisms in vitro and in vivo.

BackgroundThe EGFR pathway is frequently mutated in glioblastoma (GBM). However, to date, EGFR therapies have not demonstrated efficacy in clinical trials. Poor brain penetration of conventional inhibitors, lack of patient stratification for EGFR status, and mechanisms of resistance are likely responsible for the failure of EGFR-targeted therapy. We aimed to address these elements in a large panel of molecularly diverse patient-derived GBM brain tumor stem cells (BTSCs).MethodsIn vitro growth inhibition and on-target efficacy of afatinib, pacritinib, or a combination were assessed by cell viability, neurosphere formation, cytotoxicity, limiting dilution assays, and western blotting. In vivo efficacy was assessed with mass spectrometry, immunohistochemistry, magnetic resonance imaging, and intracranial xenograft models.ResultsWe show that afatinib and pacritinib decreased BTSC growth and sphere-forming capacity in vitro. Combinations of the 2 drugs were synergistic and abrogated the activation of STAT3 signaling observed upon EGFR inhibition in vitro and in vivo. We further demonstrate that the brain-penetrant EGFR inhibitor, afatinib, improved survival in EGFRvIII mt orthotopic xenograft models. However, upregulation of the oncogenic STAT3 signaling pathway was observed following afatinib treatment. Combined inhibition with 2 clinically relevant drugs, afatinib and pacritinib, synergistically decreased BTSC viability and abrogated this compensatory mechanism of resistance to EGFR inhibition. A significant decrease in tumor burden in vivo was observed with the combinatorial treatment.ConclusionsThese data demonstrate that brain-penetrant combinatorial therapies targeting the EGFR and STAT3 signaling pathways hold therapeutic promise for GBM.

RERG involvement in the RAS pathway and ER-dependent transcription in breast cancer.

e14638 Background: Breast cancer is the highest incidence malignancies in women worldwide. Luminal breast cancers are typically estrogen receptor–positive with better prognosis. However, the rapid disease progression and the high relapse rate of this subtype of breast cancer have become a puzzle in breast cancer treatment. It gave us the motivation to figure out the anomalous molecular mechanism which devoted in. It has been well-documented that the RAS pathway is overloadly activated in more than half of human breast tumors. RAS genes encode a superfamily of small GTPases that contribute to cell growth signals. Methods: Gene expression level were measured by qPCR and Western Blot.Biological effects of each condition were evaluated in cell viability and migration. Results: In this study, we investigated one member of the RAS family, RERG, which was related to the ER pathway and contributed to inhibit Ras activated pathway. Our results showed that knockdown of RERG concomitantly promoted two major oncogenic pathways, Ras and Stat3 signaling pathways, in luminal type breast cancer cell lines. Moreover, ectopic RERG expression significantly inhibited Ras expression. It implicated that RERG mediated in RAS-driven biological effects. Our findings indicated that knockdown of RERG enhanced mobility of the breast cancer cells and made cells more intractable under SERM treatment. Conclusions: We elucidated the tumor-suppressor role of RERG in breast cancer cells though inhibition of the Ras and Stat3 signaling pathways. Therefore, this study might shed light on the important mechanistic insight into the tumorigenesis of ER-positive luminal type cancer and provided the prognostic and therapeutic improved roles of RERG.

Differential Methylation and Acetylation as the Epigenetic Basis of Resveratrol's Anticancer Activity.

Numerous studies support the potent anticancer activity of resveratrol and its regulation of key oncogenic signaling pathways. Additionally, the activation of sirtuin 1, a deacetylase, by resveratrol has been known for many years, making resveratrol perhaps one of the earliest nutraceuticals with associated epigenetic activity. Such epigenetic regulation by resveratrol, and the mechanism thereof, has attracted much attention in the past decade. Focusing on methylation and acetylation, the two classical epigenetic regulations, we showcase the potential of resveratrol as an effective anticancer agent by virtue of its ability to induce differential epigenetic changes. We discuss the de-repression of tumor suppressors such as BRCA-1, nuclear factor erythroid 2-related factor 2 (NRF2) and Ras Associated Domain family-1α (RASSF-1α) by methylation, PAX1 by acetylation and the phosphatase and tensin homologue (PTEN) by both methylation and acetylation, in addition to the epigenetic regulation of oncogenic NF-κB and STAT3 signaling by resveratrol. Further, we evaluate the literature supporting the potentiation of HDAC inhibitors and the inhibition of DNMTs by resveratrol in different human cancers. This discussion underlines a robust epigenetic activity of resveratrol that warrants further evaluation, particularly in clinical settings.

Abstract 5457: The relationship between ADAR1 and STAT3 and its relevance in multiple myeloma

Abstract Multiple myeloma (MM), the second most common hematological cancer, is characterized by the accumulation of malignant plasma cells in the bone marrow (BM). Survival of MM cells is highly dependent on the bone marrow microenvironment, which is enriched with an array of growth factors secreted by the BM stromal cells. Amongst these growth factors, interleukin-6 (IL6) has been reported to be a major driver for MM disease progression. In line with the mechanism of action of IL6, MM cells are associated with constitutive activation of the oncogenic STAT3 signaling. ADAR1, an RNA editing enzyme, exists in two distinct isoforms, namely the P110 and P150. Aberrant ADAR1 expression and activity have been reported in various cancer types, however, this remains elusive in MM. Herein, we analyzed the publicly available myeloma patient datasets and identified that ADAR1 is overexpressed in MM and its expression level is closely associated with patients' overall survival. In vitro and in vivo assays revealed that ADAR1 can drive the growth and proliferation of MM cells, underlining its potential oncogenic effects in MM. Till date, much studies have reported the association of aberrant ADAR1 expression and its canonical functions of RNA editing, however, the knowledge on the upstream regulators of ADAR1 is still largely limited. To elucidate the cause of ADAR1 overexpression in MM, human myeloma cell lines were stimulated with IL6 at different time points. Besides potently activating the STAT3 signaling, IL6 stimulation also renders an increased expression of ADAR1 P150 isoform, at both the protein and mRNA level, with minimal effects seen on the P110 isoform, suggesting a transcriptional regulation of P150 by STAT3. Downstream validations with ChiP-qPCR and Luciferase Reporter Assay revealed that ADAR1 P150 is a direct target of STAT3 transcription factor. Specific STAT3 inhibition caused a downregulation of P150 and its oncogenic properties. Likewise, ectopic introduction of P150 rendered a more proliferative profile in the cells. Interestingly, we also observed that IL6-induced-ADAR1 expression could in turn mediate the activity of STAT3 signaling to enhance the oncogenic signaling within MM. Cells with high ADAR1 demonstrated a more rapid IL6-induced-STAT3 activation. In summary, we identified that ADAR1 overexpression is clinically relevant in MM and is driven by the hyperactivity of IL6-STAT3 signaling. Our data suggests that the P150 isoform, instead of the P110, is the more critical driver of MM disease aggressiveness in the patients with constitutively activated STAT3 pathway. The potential interplay between ADAR1 and STAT3 may represent a novel mechanism by which IL6 confers oncogenicity in MM. Citation Format: Phaik Ju Teoh, Tae Hoon Chung, Henry Yang, Polly Chen, Wee Joo Chng. The relationship between ADAR1 and STAT3 and its relevance in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5457.

Epigenomic characterization of a p53-regulated 3p22.2 tumor suppressor that inhibits STAT3 phosphorylation via protein docking and is frequently methylated in esophageal and other carcinomas.

Rationale: Oncogenic STAT3 signaling activation and 3p22-21.3 locus alteration are common in multiple tumors, especially carcinomas of the nasopharynx, esophagus and lung. Whether these two events are linked remains unclear. Our CpG methylome analysis identified a 3p22.2 gene, DLEC1, as a methylated target in esophageal squamous cell (ESCC), nasopharyngeal (NPC) and lung carcinomas. Thus, we further characterized its epigenetic abnormalities and functions.Methods: CpG methylomes were established by methylated DNA immunoprecipitation. Promoter methylation was analyzed by methylation-specific PCR and bisulfite genomic sequencing. DLEC1 expression and clinical significance were analyzed using TCGA database. DLEC1 functions were analyzed by transfections followed by various cell biology assays. Protein-protein interaction was assessed by docking, Western blot and immunoprecipitation analyses.Results: We defined the DLEC1 promoter within a CpG island and p53-regulated. DLEC1 was frequently downregulated in ESCC, lung and NPC cell lines and primary tumors, but was readily expressed in normal tissues and immortalized normal epithelial cells, with mutations rarely detected. DLEC1 methylation was frequently detected in ESCC tumors and correlated with lymph node metastasis, tumor recurrence and progression, with DLEC1 as the most frequently methylated among the established 3p22.2 tumor suppressors (RASSF1A, PLCD1 and ZMYND10/BLU). DLEC1 inhibits carcinoma cell growth through inducing cell cycle arrest and apoptosis, and also suppresses cell metastasis by reversing epithelial-mesenchymal transition (EMT) and cell stemness. Moreover, DLEC1 represses oncogenic signaling including JAK/STAT3, MAPK/ERK, Wnt/β-catenin and AKT pathways in multiple carcinoma types. Particularly, DLEC1 inhibits IL-6-induced STAT3 phosphorylation in a dose-dependent manner. DLEC1 contains three YXXQ motifs and forms a protein complex with STAT3 via protein docking, which blocks STAT3-JAK2 interaction and STAT3 phosphorylation. IL-6 stimulation enhances the binding of DLEC1 with STAT3, which diminishes their interaction with JAK2 and further leads to decreased STAT3 phosphorylation. The YXXQ motifs of DLEC1 are crucial for its inhibition of STAT3 phosphorylation, and disruption of these motifs restores STAT3 phosphorylation through abolishing DLEC1 binding to STAT3.Conclusions: Our study demonstrates, for the first time, predominant epigenetic silencing of DLEC1 in ESCC, and a novel mechanistic link of epigenetic DLEC1 disruption with oncogenic STAT3 signaling in multiple carcinomas.

Correction: A novel small molecule agent displays potent anti-myeloma activity by inhibiting the JAK2-STAT3 signaling pathway.

The oncogenic STAT3 signaling pathway is emerging as a promising target for the treatment of multiple myeloma (MM). In the present study, we identified a novel STAT3 inhibitor SC99 in a target-based high throughput screen. SC99 inhibited JAK2-STAT3 activation but had no effects on other transcription factors such as NF-κB, and kinases such as AKT, ERK, and c-Src that are in association with STAT3 signaling pathway. Furthermore, SC99 downregulated the expression of STAT3-modulated genes, including Bcl-2, Bcl-xL, VEGF, cyclin D2, and E2F-1. By inhibiting the STAT3 signaling, SC99 induced MM cell apoptosis which could be partly abolished by the ectopic expression of STAT3. Furthermore, SC99 displayed potent anti-MM activity in two independent MM xenograft models in nude mice. Oral administration of SC99 led to marked decrease of tumor growth within 10 days at a daily dosage of 30 mg/kg, but did not raise toxic effects. Taken together, this study identified a novel oral JAK2/STAT3 inhibitor that could be developed as an anti-myeloma agent.

Abstract 1261: Nintedanib induces antitumor effects in triple-negative breast cancer cells by targeting SHP-1/p-STAT3-mediated signaling pathway

Abstract Background: Triple-negative breast cancer (TNBC) is a heterogeneous and aggressive subtype that currently lack of specific targeted therapy. Previous studies have suggested that disturbing the oncogenic STAT3 signaling by activating a protein tyrosine phosphatase SHP-1 as a possible anti-cancer strategy. Our previous study also found that nintedanib (BIBF1120), a multi-kinase inhibitor that blocks mainly anti-angiogenic pathways and has been approved for idiopathic pulmonary fibrosis and for non-small cell lung cancer (in Europe), inhibits hepatocellular carcinoma cell growth independent of its anti-angiokinase activity (J Hepatology 2015). In this study, we tested nintedanib in TNBC cells and examined the underlying molecular mechanism by which nintedanib shows anti-TNBC effects. Methods: TNBC cell lines (MDA-MB-468, MDA-MB-231, HCC-1395) were used for in vitro studies. Cell viability was examined by MTT assay. Apoptosis was examined by flow cytometry and western blot. Signal transduction pathways in cells were assessed by western blot. Purified SHP-1 proteins or cells expressing deletion N-SH2 domain or D61A point mutants (SHP-1 mutants with constant open form conformations) were used to investigate the potential effects of nintedanib on SHP-1. In vivo efficacy of nintedanib was tested in nude mice bearing orthotopic breast cancer cells xenografts. Results: MTT assays revealed nintedanib induced anti-proliferation in the 3 TNBC cell lines. Sub-G1 flow cytometric and western blot analysis showed that nintedanib induced apoptosis in association with downregulation of p-STAT3 and its downstream proteins such as cyclin D1 in both dose and time-dependent manners. Overexpression of STAT3 suppressed nintedanib-mediated apoptosis in MDA-MB-231 cells. Nintedanib further increased SHP-1 activity in purified SHP-1 proteins, suggesting that nintedanib may directly affects SHP-1 activity for p-STAT3 inhibition. Moreover, either specific SHP-1 activity inhibitor or SHP-1 siRNA reduced the apoptotic effects of nintedanib on MDA-MB-231 and MDA-MB-468 cells, validating the role of SHP-1 activation in nintedanib-induced apoptosis. Furthermore, nintedanib-induced apoptosis was attenuated in TNBC cells expressing SHP-1 mutants with constant open form conformations, including deletion mutants of N-SH2 domain or D61A mutants, suggesting that the auto-inhibition structure of SHP-1 mediated the effect of nintedanib. Importantly, intraperitoneal injection of nintedanib showed antitumor activity, increased SHP-1 activity and p-STAT3 downregulation in MDA-MB-231 xenograft tumors. Conclusions Our results suggest that nintedanib induced anti-tumor effect by SHP-1 dependent STAT3 inhibition in human TNBC cells. These findings may provide new signaling pathway insight into the inhibition of TNBC growth by nintedanib. Citation Format: Chun-Yu Liu, Chia-Han Lee, Chun-Teng Huang, Pei-Yi Chu, Wan-Lun Wang, Ling-Ming Tseng, Chung-Wai Shiau, Kuen-Feng Chen. Nintedanib induces antitumor effects in triple-negative breast cancer cells by targeting SHP-1/p-STAT3-mediated signaling pathway. [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 1261.

A novel small molecule agent displays potent anti-myeloma activity by inhibiting the JAK2-STAT3 signaling pathway.

The oncogenic STAT3 signaling pathway is emerging as a promising target for the treatment of multiple myeloma (MM). In the present study, we identified a novel STAT3 inhibitor SC99 in a target-based high throughput screen. SC99 inhibited JAK2-STAT3 activation but had no effects on other transcription factors such as NF-κB, and kinases such as AKT, ERK, and c-Src that are in association with STAT3 signaling pathway. Furthermore, SC99 downregulated the expression of STAT3-modulated genes, including Bcl-2, Bcl-xL, VEGF, cyclin D2, and E2F-1. By inhibiting the STAT3 signaling, SC99 induced MM cell apoptosis which could be partly abolished by the ectopic expression of STAT3. Furthermore, SC99 displayed potent anti-MM activity in two independent MM xenograft models in nude mice. Oral administration of SC99 led to marked decrease of tumor growth within 10 days at a daily dosage of 30 mg/kg, but did not raise toxic effects. Taken together, this study identified a novel oral JAK2/STAT3 inhibitor that could be developed as an anti-myeloma agent.

Abstract 4465: Sequential combination of docetaxel with a novel SHP-1 agonist enhanced anti-tumor effect in triple negative breast cancer cells

Abstract Background: Interfering the oncogenic STAT3 signaling is considered as promising anti-cancer strategy. STAT3 signaling can be negatively regulated by the protein tyrosine phosphatase SHP-1 through direct dephosphorylation of p-STAT3 (Tyr705). Our previous studies have confirmed SC-43 as a novel SHP-1 enhancer which down-regulate p-STAT3 and STAT3-related downstream effectors (Breast Cancer Research 2013). Here, we tested the efficacy of SC-43 in combination with docetaxel in triple negative breast cancer cells. Methods: TNBC cell lines (HCC-1937, MDA-MB-468, MDA-MB-231) were used for in vitro studies. Cell viability was examined by MTT assay. Combination index was determined using Calcusyn anaysis. Apoptosis was examined by flow cytometry and western blot. Signal transduction pathways in cells were assessed by western blot. In vivo efficacy of SC-43 in combination with docetaxel was tested in nude mice with breast cancer xenografts. Results: Sequential combination of docetaxel with SC-43 showed enhanced anti-proliferative activity in all tested TNBC cells. Synergistic effect was demonstrated when docetaxel combined with SC-43 at a dose ratio of 1 to 10, showing combination index below 1.0. Sub-G1 and western blot analysis showed that SC-43 enhanced docetaxel-induced apoptosis. In addition, combining SC-43 with docetaxel efficiently suppressed p-STAT3 and downstream effectors. Ectopic expression of STAT3 reduced the enhanced cytotoxicity by the combination therapy. Previous studies has shown SC-43 enhanced SHP-1 activity, here we showed that SC-43 in combination with docetaxel significantly enhanced SHP-1 activity. More importantly, intraperitoneal injection of SC-43 with tri-weekly docetaxel showed enhanced anti-tumor growth in xenografted tumor mice. Conclusion: Our results suggest that the novel SHP-1 agonist SC-43 enhanced docetaxel-induced cytotoxic killing by SHP-1 dependent STAT3 inhibition in human triple negative breast cancer cells. (Supported by Yen Tjing-Ling Medical Foundation CI-104-07; NSC-102-2325-B-075-006 and MOST 103-2325-B-075-002; and MOHW103-TD-B-111-02) Citation Format: Ling-Min Tseng, Chun-Yu Liu, Shiu-Ping Yang, Wan-Lun Wang, Jung-Chen Su, Chia-Yun Wu, Chung-Wai Shiau, Kuen-Feng Chen. Sequential combination of docetaxel with a novel SHP-1 agonist enhanced anti-tumor effect in triple negative breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4465. doi:10.1158/1538-7445.AM2015-4465

Abstract 3803: Obatoclax analogue SC-2001 inhibits STAT3 phosphorylation by enhancing protein tyrosine phosphatase SHP-1 expression and induces apoptosis in human breast cancer cells

Abstract Background: Interfering the oncogenic STAT3 signaling is considered as promising anti-cancer strategy. Previously, we have reported an obatoclax analogue, SC-2001, as a novel STAT3 inhibitor in hepatocellular carcinoma cells (Cancer Letter 2012). Here, we examined the efficacy and drug mechanism of SC-2001 in breast cancer cells. Methods: Human breast cancer cell lines were used for in vitro studies. Cell viability was examined by MTT assay. Apoptosis was examined by both flow cytometry and western blot. Signal transduction pathways in cells were assessed by western blot. Small interference RNA was used to knockdown SHP-1. Quantitative RT-PCR was used for assessing gene transcription. In vivo efficacy of SC-2001 was tested in xenografted nude mice. Results: SC-2001 inhibited cell growth and induced apoptosis in breast cancer cell lines (MDA-MB-468, MDA-MB-231, MDA-MB-453, MCF-7 and HCC 1937). SC-2001 downregulated the phosphorylation of STAT3 (Tyr 705) and subsequently inhibited its transcriptional activities in a dose-dependent manner. STAT3-regulated proteins, including Mcl-1, survivin and cyclin D1, were also repressed by SC-2001. Over-expression of STAT3 in MDA-MB-468 cells protected cells from SC-2001-induced apoptosis. Moreover, SC-2001 enhanced the expression of SHP-1, a negative regulator of STAT3, in a time-dependent manner. The enhanced SHP-1 expression, in conjunction with increased SHP-1 phosphatase activity, was mediated by upregulated transcription. Furthermore, co-immunoprecipitation experiment showed that SC-2001 upregulated SHP-1 expression through enhanced transcription by RFX-1 transcription factor. Importantly, SC-2001 showed efficacious antitumor activity and p-STAT3 downregulation in MDA-MB-468 xenograft tumors. Conclusion: Our results suggest that SC-2001 induced apoptosis in breast cancer cells, and that this effect is mediated through RFX-1 upregulated SHP-1 expression and SHP-1-dependent STAT3 inactivation. (Supported by Yen Tjing Ling Medical Foundation; NSC 101-2325-B-075-006, NSC-102-2325-B-075-006 and NSC 100-2325-B-010-007; and V100-D-005-4) Citation Format: Chun-Yu Liu, Jung-Chen Su, Ling-Ming Tseng, Pei-Yi Chu, Wei-Tien Tai, Chung-Wai Shiau, Kuen-Feng Chen. Obatoclax analogue SC-2001 inhibits STAT3 phosphorylation by enhancing protein tyrosine phosphatase SHP-1 expression and induces apoptosis in human breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3803. doi:10.1158/1538-7445.AM2014-3803

Obatoclax analog SC-2001 inhibits STAT3 phosphorylation through enhancing SHP-1 expression and induces apoptosis in human breast cancer cells.

Interfering oncogenic STAT3 signaling is a promising anti-cancer strategy. We examined the efficacy and drug mechanism of an obatoclax analog SC-2001, a novel STAT3 inhibitor, in human breast cancer cells. Human breast cancer cell lines were used for in vitro studies. Apoptosis was examined by both flow cytometry and western blot. Signaling pathways were assessed by western blot. In vivo efficacy of SC-2001 was tested in xenograft nude mice. SC-2001 inhibited cell growth and induced apoptosis in association with downregulation of p-STAT3 (Tyr 705) in breast cancer cells. STAT3-regulated proteins, including Mcl-1, survivin, and cyclin D1, were repressed by SC-2001. Over-expression of STAT3 in MDA-MB-468 cells protected cells from SC-2001-induced apoptosis. Moreover, SC-2001 enhanced the expression of protein tyrosine phosphatase SHP-1, a negative regulator of STAT3. Furthermore, the enhanced SHP-1 expression, in conjunction with increased SHP-1 phosphatase activity, was mediated by upregulated transcription by RFX-1. Chromatin immunoprecipitation assay revealed that SC-2001 increased the binding capacity of RFX-1 to the SHP-1 promoter. Knockdown of either RFX-1 or SHP-1 reduced SC-2001-induced apoptosis, whereas ectopic expression of RFX-1 increased SHP-1 expression and enhanced the apoptotic effect of SC-2001. Importantly, SC-2001 suppressed tumor growth in association with enhanced RFX-1 and SHP-1 expression and p-STAT3 downregulation in MDA-MB-468 xenograft tumors. SC-2001 induced apoptosis in breast cancer cells, an effect that was mediated by RFX-1 upregulated SHP-1 expression and SHP-1-dependent STAT3 inactivation. Our study indicates targeting STAT3 signaling pathway may be a useful approach for the development of targeted agents for anti-breast cancer.

Targeting oncogenic STAT3 and STAT5 signaling with on 146040, a small molecule inhibitor of PI3K α/δ and BCR-ABL.

e13562 Background: The signal transducer and activator of transcription (STAT) family of proteins are important intracellular mediators. Two of these family members, STAT3 and STAT5, have been foun...

Erlotinib, dasatinib, erlotinib-dasatinib versus placebo: A randomized, double-blind window study in operable head and neck squamous cell carcinoma (HNSCC).

6033 Background: The epidermal growth factor receptor (EGFR) and Src family kinases are upregulated in HNSCC. EGFR interacts with cSrc to activate oncogenic STAT3 signaling; dual targeting is syner...

Highlights of This Issue

The importance of the Hedgehog (Hh) pathway has been shown recently in several malignancies and in pancreatic cancer models. Signaling is thought to involve paracrine interactions with the tumor microenvironment. Hwang and colleagues found that the Smoothened (SMO) receptor is highly expressed in pancreatic stellate cells (PSC) but not in pancreatic cancer cells, whereas the converse pattern was seen for Hh ligands. Stimulation of the Hh pathway increased PSC activity, which was reversed by a novel SMO inhibitor (AZD8542). Using a coinjection mouse model with PSCs, the authors show that not only did PSCs increase pancreatic tumor growth but also that inhibition by AZD8542 was highly dependent on the presence of PSCs expressing SMO. Furthermore, AZD8542 treatment increased tumor vascularity. These results emphasize the crucial contribution of PSCs in the tumor microenvironment and can help guide future clinical trials involving Hh inhibitors for pancreatic cancer.Based on an earlier observation that increased inducible nitric oxide (NO) synthase in estrogen receptor–negative breast cancer is associated with epidermal growth factor receptor (EGFR) phosphorylation and a stem cell–like phenotype, Switzer and colleagues examined the mechanism of NO-induced EGFR activation. They discovered that NO activates EGFR and Src by a mechanism that includes S-nitrosylation, leading to oncogenic c-Myc, PI3K/Akt, STAT3, and β-catenin signaling; loss of PP2A tumor suppressor activity; and a stem cell–like phenotype. NO signaling also increased chemoresistance to doxorubicin and paclitaxel. Thus, NO signaling may induce therapy resistance, and its inhibition may lead to improved outcomes in breast cancer.Munoz and colleagues describe a novel mechanism of growth promotion in cancer cells in which the mitogen hepatocyte growth factor (HGF) can enhance alternative splicing of the tumor suppressor gene, Kruppel-like factor 6 (KLF6), into a growth-promoting splice isoform, SV1. Although it is known that SV1 can promote metastasis in human cancer, the extracellular signals that drive its generation remain obscure. In cultured cells, HGF promotes Akt phosphorylation, which in turn downregulates 2 splice regulators, SRSF3 and SRSF1, to enhance KLF6 splicing. These findings link a well-known mitogen to abrogation of a tumor suppressor pathway, uncovering new potential targets for growth-suppressive therapies in tumors in which KLF6 splicing activity is high.There is a need for a clearer understanding of the consequences of KRAS activation in pancreatic ductal adenocarcinoma (PDAC) initiation and progression. In this study, Appleman and colleagues determined the signaling pathways activated by KRASG12D and BRAFV600E in primary pancreatic ductal epithelial cells (PDEC) and found that KRASG12D- and BRAFV600E - expressing PDECs depend on a novel MEK/IGF1R/PI3K signaling axis for cell survival after apoptotic challenge and tumor formation in an orthotopic model. Furthermore, they show that combined inhibition of MEK and IGF1R impairs pancreatic cancer cell survival. These findings identify key signaling mechanisms and suggest novel therapeutic strategies that might be applied to PDAC.

Abstract B5: CpG-siRNA conjugates: Overcoming cancer immunoresistance

Abstract Small interfering RNA (siRNA) have been proved to be an effective tool for treating diseases caused by gene expression abnormalities. However, clinical relevance of siRNA technology is compromised by multiple obstacles, such as lack of cellular specificity, low efficiency of cellular uptake and/or poor cytoplasmic release. We have previously demonstrated that natural capability of certain immune cells to recognize non-methylated CpG motifs in DNA oligonucleotides can be harnessed for cell-specific siRNA delivery. The siRNA molecules conjugated to CpG oligonucleotides (CpG-siRNA) are efficiently internalized and bind to the intracellular Toll-like receptor 9 (TLR9) in human and mouse dendritic cells as well as in certain TLR9-positive tumor cells. We designed a CpG-siRNA targeting Stat3 (signal transducer and activator of transcription-3), a transcription factor, which is constitutively activated in majority of mouse and human cancers. Oncogenic Stat3 signaling regulates cancer cell proliferation, survival and promotes tumor immunoresistance. We have previously shown that CpG-Stat3 siRNA can eliminate Stat3 signaling in tumor-infiltrating myeloid cells, resulting in potent antitumor immunity. In the current study, we investigated the intracellular mechanism of action of CpG-siRNA conjugates. We have demonstrated that internalized CpG-siRNAs are recruited into endosomes by dynamin-dependent mechanism and bind to endosomal TLR9 receptor. Further events include uncoupling of siRNA from the CpG moiety by Dicer endonuclease, and rapid translocation of siRNA to endoplasmic reticulum for recruitment into functional RISC (RNA interference silencing complex). We confirmed the target gene silencing both in vitro and in vivo, and proved that it occurs through RNA interference mechanism. Our current results indicate that downstream effectors of TLR9 pathway may be involved in RISC assembly and/or activation. Overall, the CpG-siRNA strategy provides a novel opportunity for overcoming cancer immunoresistance with translational potential for therapy of various malignancies.

Small Molecule Inhibitors of STAT3 for Cancer Therapy

Aberrant activation of the signal transducer and activator of transcription (STAT) 3 occurs in many human tumors. Constitutive STAT3 activity induces specific target genes that stimulate cell proliferation, prevent apoptosis, promote angiogenesis and facilitate tumor immune evasion. Thus, STAT3 is an attractive molecular target for the development of novel cancer therapeutics. Targeting and disruption of oncogenic STAT3 signaling may theoretically be accomplished through various approaches, involving direct and indirect strategies. Small molecular weight compounds have been used for this purpose. This review is intended to be full coverage of the small molecular inhibitors to develop direct STAT3 as a target for cancer therapy and will provide a discussion on the inhibitory modalities developed to date. At present, we retrieved related small molecular inhibitors experimental research papers about STAT3 as a cancer therapy target, the rationale to pursue the protein for the discovery and development of novel anticancer strategies and agents.

Abstract PR-02: STAT2 contributes to promotion of colorectal and skin carcinogenesis

Abstract STAT2 is an essential transcription factor in the type I interferon (IFN-α/β) signal transduction pathway and well described for its role in mediating antiviral immunity and cell growth inhibition. Unlike other members of the STAT family, STAT2 is unique as IFNs are the only cytokines known to date to induce its activation. Given the inflammatory and antiproliferative dual nature of IFNs, our initial hypothesis was that STAT2 prevents inflammation-induced colorectal and skin carcinogenesis by altering the inflammatory microenvironment. In contradiction to our hypothesis, deletion of STAT2 inhibited rather than promoted AOM/DSS-induced colorectal carcinogenesis as measured by prolonged survival, lower adenoma incidence, smaller polyps, and less chronic inflammation. This protective effect was not restricted to the colon as deletion of STAT2 also inhibited DMBA/TPA-induced skin carcinogenesis as indicated by reduced papilloma multiplicity. A potential mechanism by which STAT2 promotes carcinogenesis is through activation of proinflammatory mediators. Deletion of STAT2 decreased AOM/DSS-induced expression and release of proinflammatory mediators such as interleukin 6 and CCL2 and decreased interleukin-6 release from skin carcinoma cells, which in turn decreased STAT3 activation. Therefore, our findings identify STAT2 as a novel contributor to colorectal and skin carcinogenesis that may act to increase the gene expression and secretion of proinflammatory mediators, which then activate the oncogenic STAT3 signaling pathway. This talk is also presented as Poster A27. Citation Information: Cancer Prev Res 2010;3(12 Suppl):PR-02.