STAT3 Activation In Cancer Cells Research Articles

Role of STAT3 in pancreatic cancer.

Pancreatic cancer remains a serious and deadly disease, impacting people globally. There remain prominent gaps in the current understanding of the disease, specifically regarding the role of the signal transducer and activator of transcription (STAT) family of proteins in pancreatic tumors. STAT proteins, particularly STAT3, play important roles in pancreatic cancer, especially pancreatic ductal adenocarcinoma (PDAC), which is the most prevalent histotype. The role of STAT3 across a continuum of molecular processes, such as PDAC tumorigenesis and progression, immune escape, drug resistance and stemness, and modulation of the tumor microenvironment (TME), are only a tip of the iceberg. In some ways, the role of STAT3 in PDAC may hold greater importance than that of oncogenic Kirsten rat sarcoma virus (KRAS). This makes STAT3 a highly attractive target for developing targeted therapies for the treatment of pancreatic cancer. In this review, the current knowledge of STAT3 in pancreatic cancer has been summarized, particularly relating to STAT3 activation in cancer cells, cells of the TME, and the state of targeting STAT3 in pre-clinical and clinical trials of PDAC.

Enhanced anti-tumor effects of the PD-1 blockade combined with a highly absorptive form of curcumin targeting STAT3.

PD‐1/PD‐L1 immune checkpoint inhibitors are promising cancer immunotherapies however responses are still limited and the development of more effective combination immunotherapy is needed. We previously reported that STAT3 activation in cancer cells and immune cells was involved in immune‐resistant mechanisms. In this study, we evaluated the effect of highly absorptive forms of curcumin extracts and synthetic curcumin on anti‐tumor T cell responses. The curcumin po administration resulted in the significant augmentation of in vivo induction of tumor antigen‐specific T cells through restoration of dendritic cells (DCs) by inhibiting directly STAT3 in DCs and indirectly via reduced IL‐6 production from STAT3 activated cancer cells in 2 syngeneic MC38 and CT26 murine tumor models. Curcumin also showed direct DC enhancing activity and enhanced T cell induction for the immunized antigens in non‐tumor‐bearing mice and human hosts. Curcumin restored DC functions in xenogeneic nude mouse model implanted with high IL‐6‐producing human clear cell ovarian cancer cells. The combination of curcumin and PD‐1/PD‐L1 Abs demonstrated a synergistic anti‐tumor activity in MC38 murine tumor models. These results indicated that curcumin augments the induction of tumor antigen‐specific T cells by restoring the T cell stimulatory activity of DCs targeting activated STAT3 in both cancer cells and immune cells. Combination immunotherapy with curcumin and PD‐1/PD‐L1 Ab is an attractive strategy in the development of effective immunotherapy against various cancers.

Progranulin sustains STAT3 hyper-activation and oncogenic function in colorectal cancer cells.

Persistent activation of Signal Transducer and Activator of Transcription (STAT)3 occurs in a high percentage of tumors, including colorectal cancer (CRC), thereby contributing to malignant cell proliferation and survival. Although STAT3 is recognized as an attractive therapeutic target in CRC, conventional approaches aimed at inhibiting its functions have met with several limitations. Moreover, the factors that sustain hyper‐activation of STAT3 in CRC are not yet fully understood. The identification of tumor‐specific STAT3 cofactors may facilitate the development of compounds that interfere exclusively with STAT3 activity in cancer cells. Here, we show that progranulin, a STAT3 cofactor, is upregulated in human CRC as compared to nontumor tissue/cells and its expression correlates with STAT3 activation. Progranulin physically interacts with STAT3 in CRC cells, and its knockdown with a specific antisense oligonucleotide (ASO) inhibits STAT3 activation and restrains the expression of STAT3‐related oncogenic proteins, thus causing cell cycle arrest and apoptosis. Moreover, progranulin knockdown reduces STAT3 phosphorylation and cell proliferation induced by tumor‐infiltrating leukocyte (TIL)‐derived supernatants in CRC cell lines and human CRC explants. These findings indicate that CRC exhibits overexpression of progranulin, and suggest a role for this protein in amplifying the STAT3 pathway in CRC.

Abstract 2968: FTY720 inhibits mutant Kras-induced lung cancer via disrupting Stat3-S1PR1 vicious cycle and downregulating tumor PD-L1 expression

Abstract Aberrant activation of signal transducer and activator of transcription 3 (Stat3) occurs in many cancers and plays a critical role in tumor progression. The system that Stat3-induced Sphingosine-1-phosphate receptor 1 (S1PR1) expression and the S1P-S1PR1 pathway reciprocally regulate Stat3 activity was considered a major positive feedback loop for persistent Stat3 activation in cancer cells and the cells of tumor microenvironment. Tumor cells expressing the ligand for the receptor programmed death-1 (PD-1), PD-L1, have been shown to increase apoptosis of antigen-specific human T-cell to evade the immune system. In this study, we determined the influence of disrupting Stat3-S1PR1 vicious cycle on tumor formation and PD-L1 expression in lung cancer. We have established mutant Kras-induced lung cancer mice model and found that Stat3 phosphorylation was elevated in tumor tissues of lung cancer. Treatment of lung cancer cells with AG490 (JAK2 inhibitor) inhibited Stat3 activation and AG490 administration to the mice decreased the numbers of lung cancer nodules, indicating that blockage of Stat3 activation could inhibit mutant Kras-induced lung cancer in mice. Similar effects were also detected by using Stat3 inhibitor, S3I-201. We found that lung cancer cells expressed more pStat3 showed higher PD-L1 expression levels than that of lower pStat3 expressing cells. Inhibition of Stat3 activation by AG490 decreased PD-L1 expression in vitro and in vivo, indicating that PD-L1 expression could be regulated by Stat3. Moreover, FTY720 (S1P antagonist) could inhibit S1PR1 expression, down-regulate Stat3 activity, pJAK1 expression and inhibit IL-6 secretion in lung cancer cell lines and causes inhibition of the mutant Kras-induced lung cancer in mice. Furthermore, FTY720 administration dose-dependently suppressed tumor PD-L1 expression in mice lung cancer tissues. Taken together, our study indicated that FTY720 could suppress mutant Kras induced-lung cancer formation via inhibiting persistent Stat3 activation and down-regulating tumor PD-L1 expression. Citation Format: Hsuan-Heng Yeh, Tsung-I Hsu, Jan-Jong Hung, Wen-Pin Su, Wu-Chou Su. FTY720 inhibits mutant Kras-induced lung cancer via disrupting Stat3-S1PR1 vicious cycle and downregulating tumor PD-L1 expression. [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 2968. doi:10.1158/1538-7445.AM2014-2968

Abstract B33: Target STAT3 signaling for the prevention of ER-negative breast cancer

Abstract Estrogen receptor (ER)-negative breast cancer counts for approximately 30-40% of breast cancer cases. Although a large portion of ER-positive breast cancer can be prevented with ER modulators and aromatase inhibitors, these drugs fail to prevent any ER-negative breast cancers or the rest, resistant ER-positive breast cancers. Particularly, there is no targeted therapy for preventing ER-negative breast cancer, due to its lack of ER expression and resulting non-responsive nature to ER modulators. Thus, prevention of ER-negative breast cancer is an unmet need and challenge, and it is urgent to identify novel molecular targets and develop effective chemopreventive agents for the prevention of all types of breast cancers. The STAT3 transcription factor is a major contributor in the development of multiple cancers since STAT3 signaling is required for the growth of stem cell-like cancer cells. In our preliminary studies, we found that STAT3 is activated in human mammary epithelial cells that overexpress tert and ras oncogenes, in the premalignant mammary glands of MMTV-erbB2 mice before mammary tumors develop, and in human breast premalignant hyperplasia lesion and ductal carcinoma in situ (DCIS). Thus, STAT3 activation is an early molecular event in breast carcinogenesis and therefore, STAT3 represents a promising, new molecular target for the prevention of breast cancer. We hypothesized that STAT3 is a driving factor in breast cancer development, and blockade of STAT3 activity will prevent ER-negative breast cancer formation. However, STAT3 is traditionally termed as “un-druggable” target. Previous strategies of inhibiting STAT3 activity did not sufficiently block STAT3 activation in cancer cells due to their limited potency, poor solubility and bioavailability. We therefore developed orally active STAT3 inhibitors for cancer prevention use. Our newly developed STAT3 inhibitors suppress STAT3 activation and ER-negative breast cancer cell proliferation at low μM to nM potency, induce apoptosis, and suppress xenograft tumor growth in vivo. The inhibitors also show low toxicity and superior aqueous solubility as well as oral bioavailability. We treated the MMTV-erbB2 transgenic mice that develop spontaneous ER-negative mammary tumors, at the age of 3 months when mammary glands were fully developed. The STAT3 inhibitor, HJC0152, was given via oral gavage for 5 days per week at a dosage of 25 mg/kg for 2-8 months. While our experiments are still ongoing, we have started to observe significant chemopreventive effect of HJC0152 on reducing oncogene-induced ER-negative mammary tumor formation in MMTV-Neu model. These studies suggest that inhibition of STAT3 activation is a promising strategy for the prevention of ER-negative breast cancer. This work was supported by Grants P50 CA097007, P30DA028821, and R21MH093844 (J.Z.) from NIH, R. A. Welch Foundation Chemistry and Biology Collaborative Grant from Gulf Coast Consortia (GCC), John Sealy Memorial Endowment Fund (J.Z.), the DFI Seed Funding Program from MD Anderson Cancer Center (Q.S.). Citation Format: Zhengduo Yang, Haijun Chen, Chunyong Ding, Christopher Wild, Jia Zhou, Qiang Shen. Target STAT3 signaling for the prevention of ER-negative breast cancer. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr B33.

Metformin Enhances Cisplatin Cytotoxicity by Suppressing Signal Transducer and Activator of Transcription–3 Activity Independently of the Liver Kinase B1–AMP-Activated Protein Kinase Pathway

Metformin has been used as first-line treatment in patients with type 2 diabetes, and is reported to reduce cancer risk and progression by activating the liver kinase B1 (LKB1)-AMP-activated protein kinase (AMPK) pathway. Cisplatin remains the main drug for treating advanced non-small-cell lung cancer. However, drug resistance often develops through several mechanisms during the treatment course, including one mechanism mediated by the activation of the IL-6/signal transducer and activator of transcription (STAT)-3 pathway, related to the generation of reactive oxygen species (ROS). This study demonstrated a correlation between STAT3 phosphorylation and cisplatin cytotoxicity, using AS2 (PC14PE6/AS2)-derived cell lines (AS2/S3C) that contained constitutively active STAT3 plasmids as a model. A STAT3 inhibitor (JSI-124) enhanced the cisplatin sensitivity in AS2 cells, whereas metformin inhibited STAT3 phosphorylation and enhanced cisplatin cytotoxicity. By contrast, another AMPK activator (5-aminoimidazole-4-carboxamide-riboside) failed to produce these effects. LKB1-AMPK silencing by small, interfering RNA or mammalian target of rapamycin (mTOR) inhibition by rapamycin or pp242 did not alter the effect of metformin on STAT3 activity suppression, suggesting that metformin can modulate the STAT3 pathway through an LKB1-AMPK-independent and probably mTOR-independent mechanism. Metformin also inhibited cisplatin-induced ROS production and autocrine IL-6 secretion in AS2 cells. Both mechanisms contributed to the ability of metformin to suppress STAT3 activation in cancer cells, which resulted in the decreased secretion of vascular endothelial growth factor by cancer cells. The growth of subcutaneous tumor xenografts was significantly delayed by a combination of cisplatin and metformin. This is the first study to demonstrate that metformin suppresses STAT3 activation via LKB1-AMPK-mTOR-independent but ROS-related and autocrine IL-6 production-related pathways. Thus, metformin helps to overcome tumor drug resistance by targeting STAT3.

Abstract 872: Novel potent and orally active STAT3 inhibitors for cancer therapies.

Abstract Signal transducers and activators of transcription (STATs) are proteins which have dual roles as cytoplasmic signaling proteins and transcription factors that activate sets of genes including those contribute to tumorigenesis. Among the STATs family members, STAT3 has been shown to play an important role in breast carcinogenesis and is often overexpressed in primary breast tumors. Increased STAT3 transcriptional activity is reported to correlate with ER-negative phenotype in breast cancer cell lines and in primary human invasive ductal breast carcinomas. Therefore, STAT3 is a promising target for breast cancer therapies. Current strategies of inhibiting STAT3 activity did not sufficiently block STAT3 activation in cancer cells due to their limited potency, poor solubility or bioavailability. Despite several STAT3 inhibitors have been advanced into early clinical trials, there is an urgent need to develop orally active, potent STAT3 inhibitors as cancer therapeutics. We have designed, synthesized and screened a series of novel STAT3 inhibitors utilizing fragment-based drug design strategy. A number of small molecules with novel scaffolds have been discovered with low-micromolar to nanomolar potency in inhibiting the proliferation of various human breast cancer and pancreatic cancer cells. The identified compounds HJC0152, HJC0123, HJC0125, and HJC0416 significantly suppress STAT3 promoter activity and STAT3 phosphorylation (pY705), reduce total STAT3 protein expression, induce cell growth arrest, and early apoptosis in ER-negative breast cancer cell line MDA-MB-231. These novel compounds have demonstrated a better efficacy in suppressing the growth of MDA-MB-231 breast xenograft tumors via intraperitoneal and oral administrative routes, with remarkably improved aqueous solubility and less toxicity than the lead compounds. In conclusion, we have developed a new class of orally active STAT3 inhibitors with superior drug-like properties, for further evaluation in cancer therapeutic and preventive settings. This work was supported by The University of Texas MD Anderson Cancer Center DFI Funding Program. Citation Format: Zhengduo Yang, Haijun Chen, Lili Chu, Yusong Zhang, Chunyong Ding, Huiling Liu, Jia Zhou, Qiang Shen. Novel potent and orally active STAT3 inhibitors for cancer therapies. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 872. doi:10.1158/1538-7445.AM2013-872

Abstract B63: Developing Novel STAT3 Inhibitors for the Treatment and Prevention of Cancer

Abstract Signal transducers and activators of transcription (STATs) are a family of transcription factors involved in the regulation of early embryonic development, immune response, cell proliferation, differentiation, and apoptosis. Particularly, STAT3 regulates lobuloalveolar apoptosis during involution in mammary gland development, and its activation is required for the growth of CD44+CD24- stem cell-like breast cancer cells and the development of multiple cancers including breast cancer. Increased STAT3 transcriptional activity was found to correlate with ER-negative phenotype in breast cancer cell lines and in primary human invasive ductal breast carcinomas. Thus, STAT3 represents a promising target for therapeutic and preventive intervention of breast cancer. Yet current strategies of inhibiting STAT3 activity did not sufficiently block STAT3 activation in cancer cells due to their limited potency, poor solubility or bioavailability. Despite several STAT3 inhibitors have been advanced into early clinical trials, there is an urgent need to develop a more successful strategy targeting STAT3 for cancer therapies, and preventive use in particular. We attempted to identify potent and orally active STAT3 inhibitors for such purposes and have designed, synthesized and screened a series of novel STAT3 inhibitors utilizing fragment-based drug design strategy. A number of small molecules with new scaffolds have been discovered with low-micromolar to nanomolar potency in inhibiting the proliferation of various human breast cancer and pancreatic cancer cells. The identified compound HJC0152 significantly suppresses basal and IL-6-stimulated STAT3 promoter activity and STAT3 phosphorylation, reduces total STAT3 protein expression, induces G2/M cell cycle arrest, and induces early apoptosis. HJC0152 has demonstrated a better potency in suppressing the growth of MDA-MB-231 breast xenograft tumors via intraperitoneal and oral administrative routes, with remarkably improved aqueous solubility (3,300 fold improvement) and less toxicity than the lead compounds. In conclusion, we have developed a new class of orally active STAT3 inhibitors with superior drug-like properties for further preclinical therapeutic and preventive evaluation. Citation Format: Zhengduo Yang, Haijun Chen, Lili Chu, Yusong Zhang, Chunyong Ding, Kristin Terry, Jia Zhou, Qiang Shen. Developing novel STAT3 inhibitors for the treatment and prevention of cancer. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr B63.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

CK2 Inhibitors Enhance the Radiosensitivity of Human Non-Small Cell Lung Cancer Cells Through Inhibition of Stat3 Activation

CK2 interacts and phosphorylates >300 proteins, including Stat3, and is linked to a number of human cancers. Constitutively activated Stat3 has been reported in 50% of human lung cancers. Inhibition of CK2 activity can induce apoptosis and suppression of Stat3 activation in cancer cells. This study examined the effects of CK2 inhibitors on growth inhibition of lung cancer cells and the therapeutic potential on lung cancer. The CK2 inhibitor and radiation both suppressed cancer cell growth in a dose-dependent manner. Besides, the cytotoxic effect of irradiation could be augmented by CK2 inhibitors (p<0.05, two-way analysis of variance and Tukey's Honestly Significant Difference). Moreover, the growth inhibition of CK2 inhibitor and irradiation was both associated with suppression of Stat3 activation. Taken together, inhibition of CK2 activity appears to be a promising treatment strategy for non-small cell lung cancer and CK2 inhibition results in reduced Stat3 activation. Our data warrant further effort to develop CK2-targeted radiosensitizer for lung cancer treatment.

Macrophage infiltration and its prognostic relevance in clear cell renal cell carcinoma

Most malignant tumors evidence infiltration of many macrophages. In this study, we investigated an anti-inflammatory macrophage phenotype (M2) in clear cell renal cell carcinoma (RCC) using CD163 and CD204 as markers. Immunostaining showed a correlation between the number of CD163(+) cells and age, sex, nuclear grade, and TNM classification. High infiltration of CD163(+) cells was significantly associated with poor clinical prognosis in univariate analysis but not in multivariate analysis. We also carried out in vitro studies to examine cell-cell interactions between macrophages and cancer cells. Culture supernatants from RCC cell lines induced polarization of macrophages toward the M2 phenotype. Coculture of macrophages with cancer cells significantly induced activation of signal transducers and activators of transcription-3 (Stat3) in the cancer cells. Direct coculture of RCC cells with macrophages led to stronger activation of Stat3 in the cancer cells than did indirect coculture using Transwell chamber dishes. Because RCC cells expressed membrane-type macrophage colony-stimulating factor (mM-CSF) on the cell surface, we suggested that this mM-CSF plays an important role in direct cell-cell interactions. Stat3 activation in cancer cells that was induced by coculture with macrophages was suppressed by downregulation of the M-CSF receptor (M-CSFR) in macrophages and by an inhibitor of M-CSFR. In conclusion, investigation of CD163(+) tumor-associated macrophages would be useful for assessment of the clinical prognosis of patients with ccRCC. Cell-cell interactions mediated by mM-CSF and M-CSFR binding could contribute to cancer cell activation.

3-D Pharmacophore Identification of Second Generation Small Molecule Inhibitors of Stat3 SH2-Phosphotyrosine Binding That Induce Apoptosis in Acute Myeloid Leukemia Cells.

Abstract 1727Poster Board I-753Signal Transducer and Activator of Transcription 3 (Stat3) is a critical signaling intermediate in hematopoietic cells that is activated by recruitment to tyrosine-phosphorylated receptor complexes, including c-kit, IL-6 receptor, and G-CSF receptor. Stat3 proteins interact via their Src homology 2 (SH2) domain with specific phosphotyrosine (pY) motifs on activated receptors, leading to phosphorylation of Stat3 on tyrosine 705 (pY705). Reciprocal interactions between the SH2 domains and pY705 residues within Stat3 proteins promote tail-to-tail dimerization, which results in nuclear accumulation. Nuclear Stat3 dimers bind to specific DNA sequences and regulate the expression of genes involved in cell survival, proliferation, and angiogenesis. Stat3 is aberrantly activated in many types of cancers, including acute myeloid leukemia (AML), and this finding is associated with poor prognosis. Dominant negative, antisense, and RNAi strategies have repeatedly shown that inhibition of Stat3 activity in cancer cells induces apoptosis and impairs tumor growth, but these approaches are clinically problematic. Candidates for small molecule Stat3 inhibitors have been described, but none has yet entered into clinical use. We used a combination of virtual ligand screening, surface plasmon resonance (SPR) screening, and functional assays to identify 3 novel lead compounds that competitively inhibited Stat3 binding to its pY-peptide ligand, thereby blocking recruitment / phosphorylation and dimerization of Stat3 proteins. Each was selective for Stat3 v. Stat1, inhibited Stat3 nuclear accumulation and induced apoptosis preferentially in breast cancer cell lines with constitutively activated Stat3. The most active of these, Compound 188 (C188), however, was only weakly active in inducing apoptosis of AML cell lines. Two-dimensional fingerprint screening for similarity using the scaffold of C188 and a LifeChemicals compound library identified 202 compounds (C188-1 to C188-202). Each was overlayed onto C188 using a 3-D pharmacophore program (LigandScout) and ranked. The top ranking 39 compounds were tested by SPR for inhibition of Stat3 binding to pY-peptide ligand; 33 of 39 had measurable IC50s with C188-9 having one of the lowest at 2.5 μM. AML cell lines were pre-treated for 1 hour with increasing concentrations of C188-9, then stimulated with G-CSF (100 ng/ml), and pY-Stat3 was quantified by FACS. The IC50s for 4 out of 5 cell lines tested were approximately 4 μM. Immunoblot analyses confirmed that pre-treatment with C188-9 inhibited Stat3 phosphorylation without affecting the level of total Stat3. The IC50s for inhibition of IFN-γ-induced Stat1 phosphorylation were greater than 7 μM in 4 out of 5 cell lines, indicating that C188-9 was relatively selective for Stat3. We also tested apoptosis induction by C188-9 in 7 AML cell lines representing a variety of FAB and cytogenetic subtypes, including GDM-1, HL-60, Kasumi-1, KG-1, K562, NB-4, and THP-1. Cells were exposed to increasing concentrations of C188-9 for 24 hours and then labeled with annexin V-FITC. Apoptotic cells were quantified by FACS. C188-9 induced apoptosis in all cell lines tested, with EC50s ranging from 7.4 μM (GDM-1) to 44.4 μM (K562). The range of EC50s was similar to that found with JSI-124, a commercially available small molecule reported to inhibit Stat3 activity by an unknown mechanism. Similar to C188-9, JSI-124 induced apoptosis in these AML cell lines with EC50s ranging from 1.2 μM (Kasumi-1 and THP-1) to 44.9 μM (K562). Thus, scaffold similarity and 3-D pharmacophore analysis identified second generation chemical probes that target Stat3 at two steps in its activation. One of the compounds, C188-9, induced apoptosis in AML cells at low micromolar concentrations, and therefore merits further study as a promising new targeted therapy for AML and other malignancies. DisclosuresNo relevant conflicts of interest to declare.