STAT3 Knockdown Research Articles

Serine/Threonine Kinase 35, a Target Gene of STAT3, Regulates the Proliferation and Apoptosis of Osteosarcoma Cells

Background/Aims: Serine/threonine kinase 35 (STK35) may be associated with Parkinson disease and human colorectal cancer, but there have been no reports on the expression levels or roles of STK35 in osteosarcoma. Methods: STK35 mRNA expression was determined in osteosarcoma and bone cyst tissues by real-time PCR. Cell proliferation and apoptosis were assessed by Cell Counting Kit-8 (CCK-8) assay and flow cytometry analysis, respectively. Results: STK35 was up-regulated in osteosarcoma tissues as indicated by analyzing publicly available expression data (GEO dataset E-MEXP-3628) and real-time PCR analysis on our own cohort. We subsequently investigated the effects of STK35 knockdown on two osteosarcoma cell lines, MG63 and U2OS. STK35 knockdown inhibited the growth of osteosarcoma cells in vitro and in xenograft tumors. Meanwhile, STK35 knockdown enhanced apoptosis. Expression of the active forms and the activity of two major executioner caspases, caspase 3 and caspase 7, were also increased in osteosarcoma cells with STK35 silenced. Additionally, Gene Set Enrichment Analysis (GSEA) identified that the JAK/STAT signaling pathway was positively correlated with STK35 expression. The mRNA expression of STK35 was repressed by STAT3 small interfering RNA (siRNA), but not by siRNA of STAT4, STAT5A or STAT6. A luciferase reporter assay further demonstrated that STAT3 transcriptionally regulated STK35 expression. A chromatin immunoprecipitation (ChIP) assay confirmed the direct recruitment of STAT3 to the STK35 promoter. The promotion effects of STAT3 knockdown on cell apoptosis were partially abolished by STK35 overexpression. Furthermore, STK35 mRNA expression was positively correlated with STAT3 mRNA expression in osteosarcoma tissues by Pearson correlation analysis. Conclusions: These results collectively reveal that STAT3 regulates the transcription of STK35 in osteosarcoma. STK35 may exert an oncogenic role in osteosarcoma.

MicroRNA-454 inhibits non‑small cell lung cancer cells growth and metastasis via targeting signal transducer and activator of transcription-3.

Lung cancer is one of the most common type of cancers and the leading cause of cancer‑related mortality worldwide. Non-small cell lung cancer (NSCLC) accounts for >80% of lung cancer cases. Emerging studies have suggested that microRNAs are dysregulated in NSCLC and serve important roles in NSCLC initiation and development. However, to the best of our knowledge, the expression, roles and molecular mechanism of microRNA‑454 (miR‑454) have not been investigated in NSCLC. In the present study, miR‑454 was demonstrated to be significantly downregulated in NSCLC tissues and cell lines, as assessed by western blot analysis and reverse transcription‑quantitative polymerase chain reaction. Reduced miR‑454 expression was significantly correlated with aggressive clinicopathological features in NSCLC. In addition, upregulation of miR‑454 suppressed proliferation, migration and invasion NSCLC cells, as assessed by Cell Counting Kit‑8 and invitro migration and invasion assays, respectively. Furthermore, bioinformatics analysis identified STAT3 as a direct target gene of miR‑454, and STAT3 knockdown was demonstrated to simulate the effects of miR‑454 overexpression in NSCLC. In conclusion, the present study provided convincing evidence that miR‑454 is downregulated in NSCLC, and regulates growth and metastasis by directly targeting STAT3, which suggests that miR‑454 may be an efficient therapeutic target for NSCLC.

Schisandrin A reverses doxorubicin-resistant human breast cancer cell line by the inhibition of P65 and Stat3 phosphorylation.

Multidrug resistance (MDR) in breast cancer therapy occurs frequently. Thus, anti-MDR agents from natural products or synthetic compounds were tested extensively. We have also explored the reverse effect and mechanism of Schisandrin A (Sch A), a natural product, on MCF-7 breast cancer doxorubicin (DOX)-resistant subline MCF-7/DOX. MTT assay was performed to measure the viability of MCF-7 cells to assess the reverse effect of Sch A. Western blot analysis was used to study the protein levels. Laser scanning confocal microscopy was performed to detect the intercellular DOX and Rhodamine 123 accumulation. The qRT-PCR was used to analysis the target gene expression. Dual-luciferase reporter assay was performed to test the transcriptional activity of P-glycoprotein (P-gp). Sch A, at the concentration of 20µM, showed selective reverse effect (better than the positive control, verapamil at 5µM) on MCF-7/DOX cell line but not on BEL-7402/DOX, Hep G2/DOX, and K-562/DOX cells. In addition, Sch A enhanced DOX-induced cleavage of Caspase-9 and PARP levels by increasing intracellular DOX accumulation and inhibiting P-gp function. Furthermore, Sch A selectively suppressed P-gp at gene and protein levels in MCF-7/DOX cells which express high level of MDR1 but not MRP1, MRP3, or BCRP. Besides, Sch A showed inhibitory effect on P-gp transcriptional activity. Sch A significantly reduced p-IκB-α (Ser32) and p-Stat3 (Tyr705) levels which mediate P-gp expression. In addition, Stat3 knockdown enhanced the reverse effect of siP65. The combined effect of siStat3 and siP65 was better than Sch A single treatment in MCF-7/DOX cells. Sch A specifically reverses P-gp-mediated DOX resistance in MCF-7/DOX cells by blocking P-gp, NF-κB, and Stat3 signaling. Inhibition of P65 and Stat3 shows potent anti-MDR effect on MCF-7/DOX cells.

The membrane bile acid receptor TGR5 drives cell growth and migration via activation of the JAK2/STAT3 signaling pathway in non-small cell lung cancer

Mounting evidence suggests that an emerging G protein-coupled receptor, TGR5, plays a crucial role ranging from metabolic diseases to cancers. However, the biological functions of TGR5 in non-small cell lung cancer (NSCLC) remain elusive. We found that TGR5 was aberrantly expressed in NSCLC and positively correlated with an advanced clinical stage in NSCLC patients. We further discovered that TGR5 knockdown prevented JAK2 and STAT3 phosphorylation and repressed the expression of STAT3 target genes, thus inhibiting cell proliferation, migration and invasion in NSCLC. Moreover, the promotive effects of TGR5 were significantly reversed by a JAK2 inhibitor or STAT3 knockdown. Additionally, we demonstrated a positive correlation between TGR5 and p-STAT3 expression in NSCLC tissue samples. Patients with both high TGR5 and p-STAT3 expression had the worst prognosis. In addition, the serum levels of deoxycholic acid (DCA), ursodeoxycholic acid (UDCA) and chenodeoxycholic acid (CDCA) in NSCLC patients were much higher than those in the healthy controls and the patients with higher serum DCA levels had stronger TGR5 expression simultaneously. Moreover, DCA markedly promoted NSCLC cell migration and invasion through a TGR5-dependent way. Taken together, these results indicate that TGR5 drives cell growth and migration through JAK2/STAT3 signaling pathway in NSCLC.

STAT3 mediates resistance to anoikis and promotes invasiveness of nasopharyngeal cancer cells.

Nasopharyngeal carcinoma (NPC), a tumor arising from the epithelial cells of the nasopharynx, is endemic in Southeast Asia and Taiwan. The prognosis of NPC patients with local recurrence and metastasis is poor. Resistance to anoikis is a primary characteristic of tumor cells that metastasize. However, the mechanism through which NPC cells resist anoikis and are able to metastasize has not been fully elucidated. In the present study, the acquisition of anoikis resistance was analyzed in the TW01 and TW06 human NPC cell lines growing under anchorage-independent conditions. A considerable number of TW01 and TW06 cells was found to be resistant to anoikis and exhibited a higher capability of migration and invasion. These anoikis-resistant NPC cells exhibited significantly increased expression of signal transducer and activation of transcription3 (Stat3) compared with adherent cells. Furthermore, blockade of STAT3 expression by STAT3 inhibitors or STAT3 silencing significant increased anoikis in anoikis-resistant NPC cells. Moreover, silencing STAT3 not only reduced the capacity of NPC cells to resist anoikis, but also reversed their invasive properties. The expression of epithelial‑to-mesenchymal transition‑related proteins and CD44 was also significantly decreased following STAT3 knockdown. The results of the present study established that STAT3 mediates anoikis resistance, with enhanced cell migration and invasion of NPC cells, and that activation of STAT3 may increase metastatic capacity, indicating the crucial role of STAT3 in conferring anoikis resistance and enhanced invasive properties to NPC cells.

Relaxin alleviates TGFβ1-induced cardiac fibrosis via inhibition of Stat3-dependent autophagy

Cardiac fibrosis is a pathological feature common to a variety of heart diseases such as myocardial infarction, arrhythmias, cardiomyopathies and heart failure. Emerging data has indicted that autophagy is involved in fibrotic synthesis. Relaxin as a pleiotropic hormone can attenuate cardiac fibrosis and hypertrophy, however the exact molecular mechanism remains largely unknown. In this work, we evaluated whether the antifibrotic effect of relaxin relies on regulating autophagy in primary cardiac fibroblasts (CFs). Our results showed that relaxin significantly attenuated TGFβ1-induced autophagy in parallel with the reduction of fibrosis. Moreover, relaxin inhibited the phosphorylation of Stat3/Smad3 signaling. Then we observed that knockdown of Stat3 synchronously suppressed the fibrogenesis and autophagic flux which was stimulated by TGFβ1 in CFs. More importantly, we simultaneously administrated relaxin and Stat3 knockdown into CFs, which did not cause further downregulation of autophagy process and collagen protein compared with only Stat3 knockdown or relaxin treatment. These data suggested that relaxin ameliorates TGFβ-induced fibrosis dependent on Stat3 signaling-mediated autophagy. This study uncovered a previously unrecognized antifibrotic role of relaxin in cardiac fibrosis, which is achieved through the inhibition of Stat3-dependent autophagy, implying a potential therapeutic target in fibrotic diseases.

IL-10-mediated signals act as a switch for lymphoproliferation in Human T-cell leukemia virus type-1 infection by activating the STAT3 and IRF4 pathways.

Human T-cell leukemia virus type-1 (HTLV-1) causes two distinct diseases, adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Since there are no disease-specific differences among HTLV-1 strains, the etiological mechanisms separating these respective lymphoproliferative and inflammatory diseases are not well understood. In this study, by using IL-2-dependent HTLV-1-infected T-cell lines (ILTs) established from patients with ATL and HAM/TSP, we demonstrate that the anti-inflammatory cytokine IL-10 and its downstream signals potentially act as a switch for proliferation in HTLV-1-infected cells. Among six ILTs used, ILTs derived from all three ATL patients grew much faster than those from three HAM/TSP patients. Although most of the ILTs tested produced IFN-γ and IL-6, the production of IL-10 was preferentially observed in the rapid-growing ILTs. Interestingly, treatment with exogenous IL-10 markedly enhanced proliferation of the slow-growing HAM/TSP-derived ILTs. The IL-10-mediated proliferation of these ILTs was associated with phosphorylation of STAT3 and induction of survivin and IRF4, all of which are characteristics of ATL cells. Knockdown of STAT3 reduced expression of IL-10, implying a positive-feedback regulation between STAT3 and IL-10. STAT3 knockdown also reduced survivin and IRF4 in the IL-10- producing or IL-10- treated ILTs. IRF4 knockdown further suppressed survivin expression and the cell growth in these ILTs. These findings indicate that the IL-10-mediated signals promote cell proliferation in HTLV-1-infected cells through the STAT3 and IRF4 pathways. Our results imply that, although HTLV-1 infection alone may not be sufficient for cell proliferation, IL-10 and its signaling pathways within the infected cell itself and/or its surrounding microenvironment may play a critical role in pushing HTLV-1-infected cells towards proliferation at the early stages of HTLV-1 leukemogenesis. This study provides useful information for understanding of disease mechanisms and disease-prophylactic strategies in HTLV-1 infection.

T cell protein tyrosine phosphatase prevents STAT1 induction of claudin‐2 expression in intestinal epithelial cells

T cell protein tyrosine phosphatase (TCPTP) dephosphorylates a number of substrates, including JAK-STAT (signal transducer and activator of transcription) signaling proteins, which are activated by interferon (IFN)-γ, a major proinflammatory cytokine involved in conditions such as inflammatory bowel disease. A critical function of the intestinal epithelium is formation of a selective barrier to luminal contents. The structural units of the epithelium that regulate barrier function are the tight junctions (TJs), and the protein composition of the TJ determines the tightness of the barrier. Claudin-2 is a TJ protein that increases permeability to cations and reduces transepithelial electrical resistance (TER). We previously showed that transient knockdown (KD) of TCPTP permits increased expression of claudin-2 by IFN-γ. Here, we demonstrate that the decreased TER in TCPTP-deficient epithelial cells is alleviated by STAT1 KD. Moreover, increased claudin-2 in TCPTP-deficient cells requires enhanced STAT1 activation and STAT1 binding to the CLDN2 promoter. We also show that mutation of this STAT-binding site prevents elevated CLDN2 promoter activity in TCPTP-deficient epithelial cells. In summary, we demonstrate that TCPTP protects the intestinal epithelial barrier by restricting STAT-induced claudin-2 expression. This is a potential mechanism by which loss-of-function mutations in the gene encoding TCPTP may contribute to barrier defects in chronic intestinal inflammatory disease.

Target specificity, in vivo pharmacokinetics, and efficacy of the putative STAT3 inhibitor LY5 in osteosarcoma, Ewing's sarcoma, and rhabdomyosarcoma.

BackgroundSTAT3 is a transcription factor involved in cytokine and receptor kinase signal transduction that is aberrantly activated in a variety of sarcomas, promoting metastasis and chemotherapy resistance. The purpose of this work was to develop and test a novel putative STAT3 inhibitor, LY5.Methods and findingsAn in silico fragment-based drug design strategy was used to create LY5, a small molecule inhibitor that blocks the STAT3 SH2 domain phosphotyrosine binding site, inhibiting homodimerization. LY5 was evaluated in vitro demonstrating good biologic activity against rhabdomyosarcoma, osteosarcoma and Ewing’s sarcoma cell lines at high nanomolar/low micromolar concentrations, as well as specific inhibition of STAT3 phosphorylation without effects on other STAT3 family members. LY5 exhibited excellent oral bioavailability in both mice and healthy dogs, and drug absorption was enhanced in the fasted state with tolerable dosing in mice at 40 mg/kg BID. However, RNAi-mediated knockdown of STAT3 did not phenocopy the biologic effects of LY5 in sarcoma cell lines. Moreover, concentrations needed to inhibit ex vivo metastasis growth using the PuMA assay were significantly higher than those needed to inhibit STAT3 phosphorylation in vitro. Lastly, LY5 treatment did not inhibit the growth of sarcoma xenografts or prevent pulmonary metastasis in mice.ConclusionsLY5 is a novel small molecule inhibitor that effectively inhibits STAT3 phosphorylation and cell proliferation at nanomolar concentrations. LY5 demonstrates good oral bioavailability in mice and dogs. However LY5 did not decrease tumor growth in xenograft mouse models and STAT3 knockdown did not induce concordant biologic effects. These data suggest that the anti-cancer effects of LY5 identified in vitro were not mediated through STAT3 inhibition.

Activation of RAGE/STAT3 pathway by methylglyoxal contributes to spinal central sensitization and persistent pain induced by bortezomib

Bortezomib is a first-line chemotherapeutic drug widely used for multiple myeloma and other nonsolid malignancies. Although bortezomib-induced persistent pain is easily diagnosed in clinic, the pathogenic mechanism remains unclear. Here, we studied this issue with use of a rat model of systemic intraperitoneal administration of bortezomib for consecutive 5days. Consisted with our previous study, we found that bortezomib treatment markedly induced mechanical allodynia in rats. Furthermore, we first found that bortezomib treatment significantly induced the upregulation of methylglyoxal in spinal dorsal horn of rats. Spinal local application of methylglyoxal also induced mechanical allodynia and central sensitization in normal rats. Moreover, administration of bortezomib upregulated the expression of receptors for advanced glycation end products (RAGE) and phosphorylated STAT3 (p-STAT3) in dorsal horn. Importantly, intrathecal injection of metformin, a known scavenger of methylglyoxal, significantly attenuated the upregulation of methylglyoxal and RAGE in dorsal horn, central sensitization and mechanical allodynia induced by bortezomib treatment, and blockage of RAGE also prevented the upregulation of p-STAT3, central sensitization and mechanical allodynia induced by bortezomib treatment. In addition, inhibition of STAT3 activity by S3I-201 attenuated bortezomib-induced mechanical allodynia and central sensitization. Local knockdown of STAT3 also ameliorated the mechanical allodynia induced by bortezomib administration. Our results suggest that accumulation of methylglyoxal may activate the RAGE/STAT3 signaling pathway in dorsal horn, and contributes to the spinal central sensitization and persistent pain induced by bortezomib treatment.

Abstract 2983: Tumor induced stromal STAT1 deregulates mammary tissue homeostasis and accelerates breast cancer

Abstract The tumor microenvironment (TME) – the dynamic tissue space in which the tumor exists – plays a significant role in tumor initiation and is a key contributor in cancer progression. Little is known about the tumor-induced changes in the adjacent tissue stroma. Herein, we sought to explore tumor-induced changes in the TME at the morphological and molecular level to further characterize cancer progression. We show that tumor-adjacent mammary glands (TAGs) display altered branch morphology, increased expression of αSMA particularly in myofibroblasts, and an increased capacity to form mammospheres in 2D suspension culture. FACS analysis showed that TAGs contain an increased number of Lin-CD24+/CD49+ enriched mammary gland stem cell (MaSCs) population, suggesting deregulated tissue homeostasis in TAGs. We conducted comparative transcriptomics on TAGs and contralateral control glands. Meta-analysis on differentially expressed genes from our RNA-seq dataset plus two breast cancer stromal patient microarray datasets identified shared upregulation of STAT1, which we verified in tumor-adjacent tissues. Knockdown of STAT1 in caveolin-deficient mouse embryonic fibroblasts (CAFs) cocultured with human breast cancer cells altered cancer cell proliferation, further suggesting the role of STAT1 as a stromal contributor of tumorigenesis. Furthermore, in our proof-of-concept in vivo experiment, co-treatment with fludarabine, a FDA-approved STAT1 activation inhibitor and DNA synthesis inhibitor, in combination with doxorubicin, showed enhanced therapeutic efficacy in treating mouse mammary gland tumors. Our results demonstrate that stromal STAT1 expression could promote tumor progression and is a potential therapeutic target for breast cancer. Citation Format: Victoria R. Zellmer, Patricia M. Schnepp, Sarah L. Fracci, Xuejuan Tan, Erin N. Howe, Siyuan Zhang. Tumor induced stromal STAT1 deregulates mammary tissue homeostasis and accelerates breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2983. doi:10.1158/1538-7445.AM2017-2983

Abstract 363: STAT5 inhibition induces apoptosis in peripheral T cell lymphoma

Abstract Peripheral T cell lymphoma (PTCL) is a heterogeneous malignancy with extremely poor prognosis. Five-year overall survival is <40% and can be as low as 12% for specific subtypes. There is, therefore, an urgent need to identify and develop novel therapeutic targets and approaches. A number of recent sequencing studies, including a whole exome sequencing analysis of primary PTCL patients conducted by our laboratory, have revealed recurrent oncogenic driver mutations in the common gamma chain/JAK/STAT signaling pathway, resulting in the aberrant activation of the transcriptional regulator STAT5. STAT5 has additionally been found to be upregulated or contain activating mutations in 30-40% of PTCL cases of specific subtypes. These findings highlight STAT5 as an oncogenic driver of PTCL and suggest a role for it in novel therapeutic approaches. Based on these data, we aimed to evaluate STAT5 mechanistically as a therapeutic target in PTCL. The drug, pimozide, which is an FDA approved neuroleptic agent, was recently identified by drug screen to be a STAT5 inhibitor. We assessed the efficacy of pimozide in PTCL using resazurin dye assay, Fluorescence Activated Cell Sorting (FACS), and immunoblot analysis. Our data demonstrate a concentration dependent reduction in STAT5 activity and the number of viable cells in PTCL cell lines after culture with pimozide. This is shown to be due to an increase in cell death by apoptosis. To verify that pimozide’s efficacy is due to its inhibition of STAT5 activity, we transduced PTCL cell lines with anti-STAT5 shRNA to knock down STAT5 and assessed PTCL cell survival and proliferation. We show that STAT5 knockdown results in a three-fold reduction in PTCL cell viability, which occurs due to an increase in apoptosis. Furthermore, our data supports a TRAIL dependent mechanism for induction of apoptosis shown by an upregulation of TRAIL by PTCL cells after STAT5 knockdown or culture with pimozide. Mitochondrial membrane potential is also disrupted. These findings have potential clinical implications, as we further demonstrate that pimozide inhibits STAT5 phosphorylation, reduces cell viability, and induces apoptosis in primary PTCL patient samples ex vivo. This research supports further exploration of STAT5 as a therapeutic target in PTCL and the development of STAT5 inhibition in the treatment of PTCL. To facilitate the development of non-chemotherapeutic dependent therapeutic approaches for PTCL, we are now assessing STAT5 inhibition in combination with small molecule inducers of apoptosis including TRAIL pathway and PARP inhibitors. PARP inhibitors have shown efficacy in malignancies with DNA damage response pathway mutations, which have been identified in up to half of cases of certain subtypes of PTCL. We also aim to further investigate the use of JAK inhibitors, kinases responsible for STAT5 activation, in combination in PTCL, as several have demonstrated clinical efficacy in other hematologic malignancies. Citation Format: Haley M. Simpson, Aki Furusawa, Kavitha Sadashivaiah, Arnob Banerjee. STAT5 inhibition induces apoptosis in peripheral T cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 363. doi:10.1158/1538-7445.AM2017-363

Ginsenoside Rb2 inhibits osteoclast differentiation through nuclear factor-kappaB and signal transducer and activator of transcription protein 3 signaling pathway

Ginsenoside-Rb2 (Rb2) is a 20(S)-protopanaxadiol glycoside extracted from ginseng possessing various bioactivities which has drawn considerable interest regarding the area of bone metabolism. However, the effect of Rb2 on osteoclast differentiation remains unknown. In this study, we aimed to investigate the potential role of Rb2 in regulating osteoclast differentiation and the underlying molecular mechanisms. Osteoclast differentiation was induced by receptor activator nuclear factor-kappaB (NF-κB) ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) in mouse RAW 264.7 cells. The results showed that Rb2 dose-dependently inhibited the formation of the tartrate resistant acid phosphatase (TRAP)-positive multinucleated cells and TRAP expression. Furthermore, Rb2 promoted osteoprotegerin expression and bone resorption. The expression of osteoclast marker genes including nuclear factor of activated T cells c1 (NFATc1), c-Fos, OSCAR, and cathepsin K were also markedly inhibited by Rb2 treatment. Moreover, Rb2 significantly inhibited the RANKL-induced NF-κB activation. In addition, Rb2 also markedly suppressed the activation of signal transducer and activator of transcription protein 3 (STAT3) signaling pathway. Interestingly, the knockdown of STAT3 significantly strengthened the inhibitory effect of Rb2 on osteoclast differentiation. Taken together, our study suggests that Rb2 inhibits osteoclast differentiation associated with blocking NF-κB and STAT3 signaling pathways.

Targeting polo-like kinase 1 and TRAIL to enhance apoptosis in non-small cell lung cancer (NSCLC) cells.

e14104 Background: TNF-related apoptosis-ligand (TRAIL) can selectively induce apoptosis in cancer cells without causing damage to normal cells. However, some tumors are resistant to TRAIL monotherapy and clinical studies assessing targeted agents towards the TRAIL receptor have failed to show robust therapeutic activity. A number of studies have reported that the combination of targeted agents against the TRAIL receptor with standard systemic therapy and another targeted therapy considerably increased anti-tumor activity in experimental NSCLC models. Polo like kinase 1 (PLK1) is an emerging anti-mitotic target for NSCLC with high selectivity. Depleting PLK1 has been shown to reduce cell proliferation and induce apoptosis in NSCLC cells but not in normal cells. Both PLK1 and TRAIL-induced apoptosis pathways are important in supporting the malignant phenotype. We postulated that inhibiting PLK1 could enhance TRAIL-induced apoptosis. We therefore combined human recombinant TRAIL (rhTRAIL) and the PLK1 inhibitor RO3280, and studied the effect of combinatorial treatment on NSCLC cells. Methods: Cell growth was assessed by MTS assay and apoptotic activity by FACS and western blot analysis. The effect of combinatorial treatment on MAPK/ERK, PI3K/Akt and JAK/STAT signaling pathways that play an essential role in the survival of NSCLC cells were investigated by western blot analysis. Results: In this study, we demonstrate that the combination of a rhTRAIL with RO3280 synergistically reduces cell growth and strongly increases apoptotic activity in NSCLC cells. In response to RO3280 treatment, STAT3 activity is inhibited, possibly contributing to the sensitization of NSCLC cells to TRAIL-induced apoptosis. Blockade of STAT3 activity with a STAT3 inhibitor (stattic) and siRNA-mediated knockdown of STAT3 significantly enhances TRAIL-induced apoptosis in NSCLC cells. Conclusions: Our results suggest that this synergistic effect might occur through the inhibition of STAT3 activity which in turn may further contribute to the sensitization of the NSCLC cells to TRAIL therapy. Taken together, our results support the further exploration of PLK1 inhibitors in combination with TRAIL therapy in the treatment of NSCLC.

Demethylation of miR-495 inhibits cell proliferation, migration and promotes apoptosis by targeting STAT-3 in breast cancer.

In breast cancer (BC), silencing of miRNA genes due to miRNA gene promoter methylation are the important mechanisms directly contributing to tumorigenesis and tumor progression. miRNA-495 (miR-495) has been reported to be a tumor suppressor gene in various cancers, but its role and regulation in BC remains unclear. In the present study, the level of miR-495 was inversely correlated with the expression of STAT-3 in BC tissues and cell lines. miR-495 can directly target 3'-UTR of STAT-3 mRNA and thereby decrease the expression of STAT-3 in MCF-7 and HCC1973 cells by Targetscan and Dual-luciferase assay. We further analyzed miR-495 promoter methylation by sodium bisulfite sequencing method (BSP), and found DNA methyltransferase inhibitor, 5-AzaC concomitantly upregulated expression of miR-495 and downregulated its target gene STAT-3 and its downstream target VEGF. Furthermore, we further observed that 5-AzaC treatment, miR-495 mimics and STAT-3 knockdown significantly inhibited cell function in breast cancer by Transwell assay, EdU flow cytometry, AnnexinV-FITC/PI combined with flow cytometry and Hoechst staining. Taken together, our data are first to demonstrate that the miR-495 is silenced due to promoter methylation in breast cancer. DNA methyltransferase inhibitor 5-AzaC could reverse miR‑495 (suppressor gene) and STAT-3 (oncogene). The anticancer properties of 5-AzaC were preliminarily confirmed in breast cancer.

TMPRSS4 Upregulates TWIST1 Expression through STAT3 Activation to Induce Prostate Cancer Cell Migration.

Transmembrane protease serine 4 (TMPRSS4), a type-II transmembrane serine protease, is involved in the development and progression of wide range of tumors. However, the biological role of TMPRSS4 in prostate cancer remains obscure. Here, we investigated the effect of TMPRSS4 on proliferation and migration in prostate cancer and potential mechanisms. Our findings demonstrated over-expression of TMPRSS4 promoted the PC3 prostate cancer cells migration, which could be reversed by TMPRSS4 silencing. TMPRSS4 induced TWIST1 expression and followed progression of EMT along with upregulation of N-cadherin and downregulation of E-cadherin via STAT3 phosphorylation. Silencing TWIST1 significantly attenuated TMPRSS4-induced PC3 migration. Moreover, knockdown of STAT3 effectively attenuated TMPRSS4-induced TWIST1 expression and TWIST1 promoter activity. Taken together, we demonstrated a mechanistic cascade of TMPRSS4 up-regulating STAT3 activation and subsequent TWIST1 expression, leading to prostate cancer migration.

Interleukin-33 promotes REG3γ expression in intestinal epithelial cells via activation of mTOR, STAT3 and ERK1/2

Abstract REG3γ, an antimicrobial peptides typically expressed by intestinal epithelial cells (IEC), plays crucial roles in maintaining intestinal mucosal barrier functions against microbiota and potentially pathogenic microorganisms in the intestines. However, the mechanisms by which regulate IEC expression of REG3γ are still not fully understood. IL-33, a member of the IL-1 cytokine family, has been implicated in maintenance of intestinal homeostasis. In this study, we investigated whether and how IL-33 regulates IEC expression of REG3γ, thus contributes to the maintenance of intestinal homeostasis. We report here that the expression of REG3γ in IEC of IL-33−/− mice is significantly lower than that in the wild type (WT) mice at both mRNA and protein levels. Treatment with IL-33 induced IEC expression of REG3γ in mouse MSIE intestinal epithelial cell line. Consistently, IL-33 also induced expression of REG3α, the human ortholog of mouse REG3γ, in human epithelial cells. Mechanistically, rIL-33 activated phosphorylation of STAT3, mTOR and ERK1/2 in MSIE cells. Knockdown of STAT3, mTOR and ERK-activating enzyme MEK by using siRNA or CRISPR abrogated IL-33 induction of REG3γ expression in IEC, indicating that IL-33 promoted IEC expression of REG3γ though activating STAT3, mTOR and ERK1/2 pathways. REG3γ has been shown to be able kill gram-positive gut bacteria. Interestingly, total gut bacteria were significantly lower in IL-33−/− mice compared to WT mice, indicating that IL-33 regulation of gut microbiota, possibly through induction of IEC REG3γ. Put all together, our study demonstrated a novel crosstalk between IL-33 and REG3γ in maintenance of intestinal homeostasis.

Tumor-induced Stromal STAT1 Accelerates Breast Cancer via Deregulating Tissue Homeostasis.

The tumor microenvironment (TME), the dynamic tissue space in which the tumor exists, plays a significant role in tumor initiation, and is a key contributor in cancer progression; however, little is known about tumor-induced changes in the adjacent tissue stroma. Herein, tumor-induced changes in the TME were explored at the morphologic and molecular level to further understand cancer progression. Tumor-adjacent mammary glands (TAG) displayed altered branching morphology, expansion of myofibroblasts, and increased mammosphere formation, broadly suggesting a tumor-induced field effect. FACS analysis of TAGs demonstrated an increased number of Lin-CD24+/CD49+ enriched mammary gland stem cells (MaSC), suggesting deregulated tissue homeostasis in TAGs. Comparative transcriptome analysis of TAGs and contralateral control glands coupled with meta-analysis on differentially expressed genes with two breast cancer stromal patient microarray datasets identified shared upregulation of STAT1. Knockdown of STAT1 in cancer-associated fibroblast (CAF) cocultured with human breast cancer cells altered cancer cell proliferation, indicating a role for STAT1 as a stromal contributor of tumorigenesis. Furthermore, depletion of STAT1 in CAFs significantly reduced periductal reactive fibrosis and delayed early breast cancer progression in vivo Finally, cotreatment with fludarabine, a FDA-approved STAT1 activation inhibitor and DNA synthesis inhibitor, in combination with doxorubicin, showed enhanced therapeutic efficacy in treating mouse mammary gland tumors. Taken together, these results demonstrate that stromal STAT1 expression promotes tumor progression and is a potential therapeutic target for breast cancer.Implications: Tumors induce stromal STAT1-dependent cytokine secretion that promotes tumor cell proliferation and can be targeted using clinically-approved inhibitors of STAT1. Mol Cancer Res; 15(5); 585-97. ©2017 AACR.

Role of STAT3 and FOXO1 in the Divergent Therapeutic Responses of Non-metastatic and Metastatic Bladder Cancer Cells to miR-145.

Although miR-145 is the most frequently downregulated miRNA in bladder cancer, its exact stage association and downstream effector have not been defined. Here, we found that miR-145 was upregulated in human patients with bladder cancer with lymph node metastasis and in metastatic T24T cell line. Forced expression of miR-145 promoted anchorage-independent growth of T24T cells accompanied by the downregulation of forkhead box class O1 (FOXO1). In contrast, in non-metastatic T24 cells, miR-145 overexpression inhibited cell growth with upregulation of FOXO1, and the knockdown of FOXO1 abolished the miR-145-mediated inhibition of cell growth. Mechanistic studies revealed that miR-145 directly bound to and attenuated 3'-untranslated region (UTR) activity of foxo1 mRNA in both T24 and T24T cells. Interestingly, miR-145 suppressed STAT3 phosphorylation at Tyr705 and increased foxo1 promoter transcriptional activity in T24 cells, but not in T24T cells, suggesting a role of STAT3 in the divergent responses to miR-145. Supporting this was our finding that STAT3 knockdown mimicked miR-145-mediated upregulation of FOXO1 in T24T cells and inhibition of anchorage-independent growth. Consistently, ectopic expression of miR-145 promoted tumor formation of xenograft T24T cells, whereas such promoting effect became inhibitory due to specific knockdown of STAT3. Together, our findings demonstrate the stage-specific association and function of miR-145 in bladder cancers and provide novel insights into the therapeutic targeting of miR-145. Mol Cancer Ther; 16(5); 924-35. ©2017 AACR.

Low concentration arsenite activated JAK2/STAT3 signal and increased proliferative factor expressions in SV-HUC-1cells after short and long time treatment.

Epidemiological studies have indicated that ingestion of inorganic arsenic resulted in increased risks of bladder cancer and chronic hyperproliferation could play a direct role in the development of cancer. This study examined the effects of arsenite on JAK2/STAT3 pathway and expressions of proliferation and anti-apoptosis factors. The results showed that long term exposure to low doses arsenite enhanced human uroepithelial cells (SV-HUC-1 cells) proliferation and BrdU positive rate was significant increased. mRNA and protein expressions of proliferation factors, such as cyclin D1, COX-2, and proliferating cell nuclear antigen (PCNA), increased in chronically exposed arsenite SV-HUC-1 cells with exposure time. Furthermore, JAK2/STAT3 signal pathway was activated following exposure to arsenite in SV-HUC-1 cells. Knockdown of STAT3 reduced expressions of cyclin D1, COX-2, PCNA, and BCL2 induced by arsenite. In conclusion, arsenic induced proliferation in human uroepithelial cells after short and long term exposure to arsenite and JAK2/STAT3 signaling pathway might be pivotal in arsenite-induced proliferation by regulating cyclin D1, COX-2, PCNA, and BCL2.