Signal Transducer And Activator Of Transcription 3 Mutant Research Articles

Targeting PARG induces tumor cell growth inhibition and antitumor immune response by reducing phosphorylated STAT3 in ovarian cancer

BackgroundOvarian cancer is the most lethal gynecological malignancy, with limited treatment options after failure of standard therapies. Despite the potential of poly(ADP-ribose) polymerase inhibitors in treating DNA damage response (DDR)-deficient...

Fucoxanthin induces human melanoma cytotoxicity by thwarting the JAK2/STAT3/BCL-xL signaling axis.

Melanoma is the most lethal skin malignancy. Fucoxanthin is a marine carotenoid with significant anticancer activities. Intriguingly, Fucoxanthin's impact on human melanoma remains elusive. Signal Transducer and Activator of Transcription 3 (STAT3) represents a promising target in cancer therapy due to its persistent activation in various cancers, including melanoma. Herein, we revealed that Fucoxanthin is cytotoxic to human melanoma cell lines A2758 and A375 while showing limited cytotoxicity to normal human melanocytes. Apoptosis is a primary reason for Fucoxanthin's melanoma cytotoxicity, as the pan-caspase inhibitor z-VAD-fmk drastically abrogated Fucoxanthin-elicited clonogenicity blockage. Besides, Fucoxanthin downregulated tyrosine 705-phosphorylated STAT3 (p-STAT3 (Y705)), either inherently present in melanoma cells or inducible by interleukin 6 (IL-6) stimulation. Notably, ectopic expression of STAT3-C, a dominant-active STAT3 mutant, abolished Fucoxanthin-elicited melanoma cell apoptosis and clonogenicity inhibition, supporting the pivotal role of STAT3 blockage in Fucoxanthin's melanoma cytotoxicity. Moreover, Fucoxanthin lowered BCL-xL levels by blocking STAT3 activation, while ectopic BCL-xL expression rescued melanoma cells from Fucoxanthin-induced killing. Lastly, Fucoxanthin was found to diminish the levels of JAK2 with dual phosphorylation at tyrosine residues 1007 and 1008 in melanoma cells, suggesting that Fucoxanthin impairs STAT3 signaling by blocking JAK2 activation. Collectively, we present the first evidence that Fucoxanthin is cytotoxic selectively against human melanoma cells while sparing normal melanocytes. Mechanistically, Fucoxanthin targets the JAK2/STAT3/BCL-xL antiapoptotic axis to provoke melanoma cell death. This discovery implicates the potential application of Fucoxanthin as a chemopreventive or therapeutic strategy for melanoma management.

Methoxyhispolon Methyl Ether, a Hispolon Analog, Thwarts the SRC/STAT3/BCL-2 Axis to Provoke Human Triple-Negative Breast Cancer Cell Apoptosis In Vitro.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with few treatment options. A promising TNBC treatment approach is targeting the oncogenic signaling pathways pivotal to TNBC initiation and progression. Deregulated activation of signal transducer and activator of transcription 3 (STAT3) is fundamental to driving TNBC malignant transformation, highlighting STAT3 as a promising TNBC therapeutic target. Methoxyhispolon Methyl Ether (MHME) is an analog of Hispolon, an anti-cancer polyphenol found in the medicinal mushroom Phellinus linteus. Still, MHME's anti-cancer effects and mechanisms remain unknown. Herein, we present the first report about MHME's anti-TNBC effect and its action mechanism. We first revealed that MHME is proapoptotic and cytotoxic against human TNBC cell lines HS578T, MDA-MB-231, and MDA-MB-463 and displayed a more potent cytotoxicity than Hispolon's. Mechanistically, MHME suppressed both constitutive and interleukin 6 (IL-6)-induced activation of STAT3 represented by the extent of tyrosine 705-phosphorylated STAT3 (p-STAT3). Notably, MHME-evoked apoptosis and clonogenicity impairment were abrogated in TNBC cells overexpressing a dominant-active mutant of STAT3 (STAT3-C); supporting the blockade of STAT3 activation is an integral mechanism of MHME's cytotoxic action on TNBC cells. Moreover, MHME downregulated BCL-2 in a STAT3-dependent manner, and TNBC cells overexpressing BCL-2 were refractory to MHME-induced apoptosis, indicating that BCL-2 downregulation is responsible for MHME's proapoptotic effect on TNBC cells. Finally, MHME suppressed SRC activation, while v-src overexpression rescued p-STAT3 levels and downregulated apoptosis in MHME-treated TNBC cells. Collectively, we conclude that MHME provokes TNBC cell apoptosis through the blockade of the SRC/STAT3/BCL-2 pro-survival axis. Our findings suggest the potential of applying MHME as a TNBC chemotherapy agent.

Blockade of the SRC/STAT3/BCL-2 Signaling Axis Sustains the Cytotoxicity in Human Colorectal Cancer Cell Lines Induced by Dehydroxyhispolon Methyl Ether.

Colorectal cancer (CRC) is the third most prevalent human cancer globally. 5-Fluorouracil (5-FU)-based systemic chemotherapy is the primary strategy for advanced CRC treatment, yet is limited by poor response rate. Deregulated activation of signal transducer and activator of transcription 3 (STAT3) is fundamental to driving CRC malignant transformation and a poor prognostic marker for CRC, underscoring STAT3 as a promising CRC drug target. Dehydroxyhispolon methyl ether (DHME) is an analog of Hispolon, an anticancer polyphenol abundant in the medicinal mushroom Phellinus linteus. Previously, we have established DHME's cytotoxic effect on human CRC cell lines by eliciting apoptosis through the blockade of WNT/β-catenin signaling, a preeminent CRC oncogenic pathway. Herein, we unraveled that compared with 5-FU, DHME is a more potent killer of CRC cells while being much less toxic to normal colon epithelial cells. DHME suppressed both constitutive and interleukin 6 (IL-6)-induced STAT3 activation represented by tyrosine 705 phosphorylation of STAT3 (p-STAT3 (Y705)); notably, DHME-induced CRC apoptosis and clonogenicity limitation were abrogated by ectopic expression of STAT3-C, a dominant-active STAT3 mutant. Additionally, we proved that BCL-2 downregulation caused by DHME-mediated STAT3 blockage is responsible for DHME-induced CRC cell apoptosis. Lastly, DHME inhibited SRC activation, and v-src overexpression restored p-STAT3 (Y705) levels along with lowering the levels of apoptosis in DHME-treated CRC cells. We conclude DHME provokes CRC cell apoptosis by blocking the SRC/STAT3/BCL-2 axis besides thwarting WNT/β-catenin signaling. The notion that DHME targets two fundamental CRC signaling pathways underpins the potential of DHME as a CRC chemotherapy agent.

Sertindole, an Antipsychotic Drug, Curbs the STAT3/BCL-xL Axis to Elicit Human Bladder Cancer Cell Apoptosis In Vitro.

Bladder cancer is the leading urinary tract malignancy. Epidemiological evidence has linked lower cancer incidence in schizophrenia patients to long-term medication, highlighting the anticancer potential of antipsychotics. Sertindole is an atypical antipsychotic agent with reported anticancer action on breast and gastric cancers. Yet, sertindole's effect on bladder cancer remains unaddressed. We herein present the first evidence of sertindole's antiproliferative effect and mechanisms of action on human bladder cancer cells. Sertindole was cytotoxic against bladder cancer cells while less cytotoxic to normal urothelial cells. Apoptosis was a primary cause of sertindole's cytotoxicity, as the pan-caspase inhibitor z-VAD-fmk rescued cells from sertindole-induced killing. Mechanistically, sertindole inhibited the activation of signal transducer and activator of transcription 3 (STAT3), an oncogenic driver of bladder cancer, as sertindole lowered the levels of tyrosine 705-phosphorylated STAT3 along with that of STAT3's target gene BCL-xL. Notably, ectopic expression of the dominant-active STAT3 mutant impaired sertindole-induced apoptosis in addition to restoring BCL-xL expression. Moreover, bladder cancer cells overexpressing BCL-xL were refractory to sertindole's proapoptotic action, arguing that sertindole represses STAT3 to downregulate BCL-xL, culminating in the induction of apoptosis. Overall, the current study indicated sertindole exerts bladder cancer cytotoxicity by provoking apoptosis through targeted inhibition of the antiapoptotic STAT3/BCL-xL signaling axis. These findings implicate the potential to repurpose sertindole as a therapeutic strategy for bladder cancer.

Abstract 13400: STAT3 ASO-Loaded Microbubbles With Pulsed Ultrasound for Enhanced Therapeutic Effect in Head and Neck Tumors

Introduction: Signal transducer and activator of transcription 3 ( STAT3 ) is an oncogenic transcription factor implicated in carcinogenesis, tumor progression, and drug resistance in many cancers, including head and neck squamous cell carcinoma ( HNSCC ). STAT3 antisense oligonucleotide ( ASO ) specifically targeting the nucleotide sequence of STAT3 has therapeutic promise, but effective delivery remains challenging. We developed microbubble ( MB ) delivery vehicles loaded with STAT3 ASO and hypothesized that compared to intravenous ( i.v. ) delivery, ultrasound-targeted microbubble cavitation ( UTMC )-mediated ASO delivery confers superior tumor growth inhibition. Methods: Lipid MBs loaded with STAT3 ASO (S3MB) or mutant ASO (Mut-MB) were prepared. First, murine squamous carcinoma cells (SCC-7) in contact with MBs were exposed to pulsed ultrasound (1 MHz, 0.5 MPa, 100 μs pulse duration, 1 ms pulse interval) for 10 sec to establish efficacy with respect to cytotoxicity, apoptosis, and gene knockdown. Thereafter, the anti-tumor effect of 3 treatments with STAT3 ASO (10 μg) delivered i.v. or via UTMC x 15 min ( f= 1MHz, 0.7MPa, 100 cycles x 5 every 2 sec) was compared in vivo in a murine SCC-7 xenograft model followed up to 17 days. Results: S3MB + UTMC conferred more cytoxocitiy, apoptosis, and downregulation of transcripts for STAT3 and its target genes (Bcl-xL and Cyclin D1) compared to Mut-MB + UTMC in vitro (p<0.05). In tumor bearing mice, compared to i.v. STAT3 ASO (n=8), Mut-MB+UTMC (n=5) or no treatment (n=8), S3MB + UTMC (n=9) significantly attenuated tumor burden, prolonged doubling time, and improved survival (p<0.05). While i.v. STAT3 ASO had no effect on tumor expression of STAT3, Bcl-x, and cyclin D1 at the transcript and protein levels, its delivery via UTMC significantly reduced tumor expression of these genes (p<0.01). Conclusion: At a dose of 10 μg, STAT3 ASO significantly inhibited murine HNSCC tumor growth and downregulated STAT3 and its target genes when delivered via UTMC, compared with intravenous administration alone. Our platform offers a promising imaged-guided, targeted delivery strategy for nucleotide-based therapeutics in cancer applications.

Hemistepsin a Induces Apoptosis of Hepatocellular Carcinoma Cells by Downregulating STAT3.

Hemistepta lyrata (Bunge) Bunge is a biennial medicinal plant possessing beneficial effects including anti-inflammation, and hemistepsin A (HsA) isolated from H. lyrata has been known as a hepatoprotective sesquiterpene lactone. In this report, we explored the cytotoxic effects of H. lyrata on hepatocellular carcinoma (HCC) cells and investigated the associated bioactive compounds and their relevant mechanisms. From the viability results of HCC cells treated with various H. lyrata extracts, HsA was identified as the major compound contributing to the H. lyrata-mediated cytotoxicity. HsA increased expression of cleaved PARP and cells with Sub-G1 phase, Annexin V binding, and TUNEL staining, which imply HsA induces apoptosis. In addition, HsA provoked oxidative stress by decreasing the reduced glutathione/oxidized glutathione ratio and accumulating reactive oxygen species and glutathione-protein adducts. Moreover, HsA inhibited the transactivation of signal transducer and activator of transcription 3 (STAT3) by its dephosphorylation at Y705 and glutathione conjugation. Stable expression of a constitutive active mutant of STAT3 prevented the reduction of cell viability by HsA. Finally, HsA enhanced the sensitivity of sorafenib-mediated cytotoxicity by exaggerating oxidative stress and Y705 dephosphorylation of STAT3. Therefore, HsA will be a promising candidate to induce apoptosis of HCC cells via downregulating STAT3 and sensitizing conventional chemotherapeutic agents.

Interleukin-6–mediated epigenetic control of the VEGFR2 gene induces disorganized angiogenesis in human breast tumors

Disorganized vessels in the tumor vasculature lead to impaired perfusion, resulting in reduced accessibility to immune cells and chemotherapeutic drugs. In the breast tumor-stroma interplay, paracrine factors such as interleukin-6 (IL-6) often facilitate disordered angiogenesis. We show here that epigenetic mechanisms regulate the crosstalk between IL-6 and vascular endothelial growth factor receptor 2 (VEGFR2) signaling pathways in myoepithelial (CD10+) and endothelial (CD31+, CD105+, CD146+, and CD133-) cells isolated from malignant and nonmalignant tissues of clinically characterized human breast tumors. Tumor endothelial (Endo-T) cells in 3D cultures exhibited higher VEGFR2 expression levels, accelerated migration, invasion, and disorganized sprout formation in response to elevated IL-6 levels secreted by tumor myoepithelial (Epi-T) cells. Constitutively, compared with normal endothelial (Endo-N) cells, Endo-T cells differentially expressed DNA methyltransferase isoforms and had increased levels of IL-6 signaling intermediates such as IL-6R and signal transducer and activator of transcription 3 (STAT3). Upon IL-6 treatment, Endo-N and Endo-T cells displayed altered expression of the DNA methyltransferase 1 (DNMT1) isoform. Mechanistic studies revealed that IL-6 induced proteasomal degradation of DNMT1, but not of DNMT3A and DNMT3B and subsequently led to promoter hypomethylation and expression/activation of VEGFR2. IL-6-induced VEGFR2 up-regulation was inhibited by overexpression of DNMT1. Transfection of a dominant-negative STAT3 mutant, but not of STAT1, abrogated VEGFR2 expression. Our results indicate that in the breast tumor microenvironment, IL-6 secreted from myoepithelial cells influences DNMT1 stability, induces the expression of VEGFR2 in endothelial cells via a promoter methylation-dependent mechanism, and leads to disordered angiogenesis.

The JAK2/STAT3 pathway is involved in the anti-melanoma effects of brevilin A

AimsMelanoma is lethal. Constitutively active signal transducer and activator of transcription 3 (STAT3) has been proposed as a pathogenic factor and a therapeutic target of melanoma. Brevilin A, a sesquiterpene lactone isolated from Centipeda minima (L.) A. Br. et Aschers., has been shown to exert antineoplastic effects and inhibit the STAT3 pathway in nasopharyngeal, lung, prostate and breast cancer cells. This study aimed to determine whether brevilin A has anti-melanoma effects, and whether STAT3 signaling is involved in the effects. Main methodsA mouse A375 xenograft model, as well as A375 and A2058 cell models were employed to assess the in vivo and in vitro anti-melanoma effects of brevilin A. A375 cells stably expressing STAT3C, a constitutively active STAT3 mutant, were used to determine the role of STAT3 signaling in brevilin A's anti-melanoma effects. Key findingsIntraperitoneal injection of brevilin A dose-dependently inhibited melanoma growth in mice and suppressed STAT3 phosphorylation in the tumors. In cultured cells, brevilin A reduced cell viability, induced apoptosis, suppressed migration and invasion, decreased protein levels of phospho-JAK2 (Y1007/1008) and phospho-STAT3 (Tyr705), and restrained STAT3 nuclear localization. STAT3 over-activation diminished brevilin A's effects on cell viability and migration. Collectively, brevilin A exerts anti-melanoma effects and these effects are at least in part attributed to the inhibition of the JAK2/STAT3 pathway. SignificanceOur findings provide a pharmacological basis for developing brevilin A as a new phytotherapeutic agent against melanoma.

Signal transducer and activator of transcription-3 drives the high-fat diet-associated prostate cancer growth

Prostate cancer (PCa) is the second leading cause of cancer death in men. PCa progression can be associated with obesity. Signal transducer and activator of transcription-3 (STAT3) plays a crucial role in PCa growth. However, whether STAT3 plays a role in high-fat diet (HFD)-associated PCa growth is unknown. Our data show that HFD feeding increases tumor size, STAT3 phosphorylation, and palmitic acid (PA) level in the xenograft tissues of the PCa-bearing xenograft mouse model. In vitro studies show that PA increases STAT3 expression and phosphorylation (STAT3-Y705) in PCa. Computational modeling suggests strong and stable binding between PA and unphosphorylated STAT3 at R593 and N538. The binding changes STAT3 structure and activity. Functional studies show that both STAT3 mutants (R583A and N538A) and STAT3 dominant negative significantly reduce PA-enhanced STAT3 phosphorylation, PA-increased PCa cell proliferation, migration, and invasion. In the xenograft mouse models, the HFD-increased tumor growth and STAT3 phosphorylation in tumors are reversed by STAT3 inhibition. Our study not only demonstrates the regulatory role of PA/STAT3 axis in HFD-associated PCa growth but also suggests a novel mechanism of how STAT3 is activated by PA. Our data suggest STAT3 as a therapeutic target for the treatment of HFD-associated PCa.

STAT3 associates with vacuolar H+-ATPase and regulates cytosolic and lysosomal pH

Dysregulated intracellular pH is emerging as a hallmark of cancer. In spite of their acidic environment and increased acid production, cancer cells maintain alkaline intracellular pH that promotes cancer progression by inhibiting apoptosis and increasing glycolysis, cell growth, migration, and invasion. Here we identify signal transducer and activator of transcription-3 (STAT3) as a key factor in the preservation of alkaline cytosol. STAT3 associates with the vacuolar H+-ATPase in a coiled-coil domain-dependent manner and increases its activity in living cells and in vitro. Accordingly, STAT3 depletion disrupts intracellular proton equilibrium by decreasing cytosolic pH and increasing lysosomal pH, respectively. This dysregulation can be reverted by reconstitution with wild-type STAT3 or STAT3 mutants unable to activate target genes (Tyr705Phe and DNA-binding mutant) or to regulate mitochondrial respiration (Ser727Ala). Upon cytosolic acidification, STAT3 is transcriptionally inactivated and further recruited to lysosomal membranes to reestablish intracellular proton equilibrium. These data reveal STAT3 as a regulator of intracellular pH and, vice versa, intracellular pH as a regulator of STAT3 localization and activity.

Visualization and quantification of dynamic STAT3 homodimerization in living cells using homoFluoppi

Dimerization in signal transduction is a dynamically regulated process and a key regulatory mechanism. Signal transducer and activator of transcription 3 (STAT3) dimerizes after tyrosine phosphorylation upon cytokine stimulation. Because only the STAT3 dimer possesses the trans-activation activity, dimerization is an indispensable process for cytokine signaling. Here we report the detection of dynamic STAT3 dimerization in living cells using the homoFluoppi system. This method allowed us to validate the presence of an intact Src homology 2 domain and STAT3 Tyr705 phosphorylation, which facilitate puncta formation and homodimerization. Puncta formation was reversible, as determined by a decreased punctate signal after washout of oncostatin M. We analyzed STAT3 mutants, which have been reported in patients with hyper IgE syndrome and inflammatory hepatocellular adenoma (IHCA). Analysis of the IHCA mutants using homoFluoppi revealed constitutive activity independent of cytokine stimulation and novel insight into kinetics of dimer dissociation process. Next, we used homoFluoppi to screen for inhibitors of STAT3 dimerization, and identified 3,4-methylenedioxy-β-nitrostyrene as a novel inhibitor. The results of this study show that homoFluoppi is a useful research tool for the analysis of proteins like STAT3 that dynamically dimerize, and is applicable for the screening of dimerization modulators.

Plumbagin inhibits the proliferation and survival of esophageal cancer cells by blocking STAT3-PLK1-AKT signaling

Esophageal squamous cell carcinoma (ESCC) is one of the deadliest cancers, and it requires novel treatment approaches and effective drugs. In the present study, we found that treatment with plumbagin, a natural compound, reduced proliferation and survival of the KYSE150 and KYSE450 ESCC cell lines in a dose-dependent manner in vitro. The drug also effectively inhibited the viability of primary ESCC cells from fresh biopsy specimens. Furthermore, plumbagin-induced mitotic arrest and massive apoptosis in ESCC cells. Notably, the drug significantly suppressed the colony formation capacity of ESCC cells in vitro and the growth of KYSE150 xenograft tumors in vivo. At the molecular level, we found that exposure to plumbagin decreased both polo-like kinase 1 (PLK1) and phosphorylated protein kinase B (p-AKT) expression in both ESCC cell lines. Enforced PLK1 expression in ESCC cells not only markedly rescued cells from plumbagin-induced apoptosis and proliferation inhibition but also restored the impaired AKT activity. Furthermore, signal transducer and activator of transcription 3 (STAT3), a transcription factor of PLK1, was also inactivated in plumbagin-treated ESCC cells; however, the overexpression of a constitutively activated STAT3 mutant, STAT3C, reinstated the plumbagin-elicited blockade of PLK1-AKT signaling in ESCC cells. Taken together, these findings indicate that plumbagin inhibits proliferation and potentiates apoptosis in human ESCC cells in vitro and in vivo. Plumbagin may exert these antitumor effects by abrogating STAT3-PLK1-AKT signaling, which suggests that plumbagin may be a novel, promising anticancer agent for the treatment of ESCC.

Inhibiting STAT3 signaling is involved in the anti-melanoma effects of a herbal formula comprising Sophorae Flos and Lonicerae Japonicae Flos

A herbal formula (SL) comprising Sophorae Flos and Lonicerae Japonicae Flos was traditionally used to treat melanoma. Constitutively active signal transducer and activator of transcription 3 (STAT3) has been proposed as a therapeutic target in melanoma. Here we investigated whether an ethanolic extract of SL (SLE) exerted anti-melanoma activities by inhibiting STAT3 signaling. B16F10 allograft model, A375 and B16F10 cells were employed to assess the in vivo and in vitro anti-melanoma activities of SLE. A375 cells stably expressing STAT3C, a constitutively active STAT3 mutant, were used to determine the role of STAT3 signaling in SLE’s anti-melanoma effects. Intragastric administration of SLE (1.2 g/kg) potently inhibited melanoma growth in mice and inhibited STAT3 phosphorylation in the tumors. In cultured cells, SLE dramatically reduced cell viability, induced apoptosis, suppressed migration and invasion, and restrained STAT3 activation and nuclear localization. STAT3C overexpression in A375 cells diminished SLE’s effects on cell viability, apoptosis and invasion. Collectively, SLE exerted potent anti-melanoma effects partially by inhibiting STAT3 signaling. This study provides pharmacological justification for the traditional use of this formula in treating melanoma, and suggests that SLE has the potential to be developed as a modern alternative and/or complimentary agent for melanoma treatment and prevention.

Biochanin A enhances RORγ activity through STAT3-mediated recruitment of NCOA1

Interleukin (IL)-17-producing T cells play important roles in autoimmunity, chronic inflammation and host protection against extracellular bacteria and fungi. The retinoic acid receptor-related orphan receptors (ROR) α and γ are key regulators of the IL-17-producing phenotype. We previously showed that the isoflavone biochanin A enhanced ROR-mediated transcriptional activity. Here, we investigated the possible mechanisms underlying this ROR activation. Biochanin A-treated murine thymoma EL4 and primary splenocytes demonstrated enhanced induction of IL-17. Biochanin A also induced tyrosine-phosphorylation of signal transducer and activator of transcription 3 (STAT3) in these cells. Stable knockdown of either RORγ or STAT3 in EL4 cells canceled biochanin A-induced upregulation of IL-17 expression. Importantly, biochanin A enhanced complex formation between RORγ and STAT3 or nuclear-receptor coactivator 1 (NCOA1). Furthermore, the biochanin A-induced RORγ-NCOA1 complex was disrupted by a dominant negative mutant of STAT3 or by the STAT3 specific inhibitor Stattic. These results suggest that biochanin A activates RORγ-dependent IL-17 transcription through the enhancement of STAT3 phosphorylation and STAT3-mediated recruitment of NCOA1 to RORγ.

Altholactone Inhibits NF-κB and STAT3 Activation and Induces Reactive Oxygen Species-Mediated Apoptosis in Prostate Cancer DU145 Cells.

Altholactone, a natural compound isolated from Goniothalamus spp., has demonstrated anti-inflammatory and anticancer activities, but its molecular mechanisms are still not fully defined. Nuclear factor kappa B (NF-κB) and signal transducer and activator of transcription 3 (STAT3) play pivotal roles in the cell survival of many human tumors. The objective of this study was to elucidate the mechanism of action of altholactone against prostate cancer DU145 cells and to evaluate whether its effects are mediated by inhibition of NF-κB and STAT3 activity. Altholactone inhibited proliferation of DU145 cells and induced cell cycle arrest in S phase and triggered apoptosis. Reporter assays revealed that altholactone repressed p65- and TNF-α-enhanced NF-κB transcriptional activity and also inhibited both constitutive and IL-6-induced transcriptional activity of STAT3. Consistent with this, altholactone down-regulated phosphorylation of STAT3 and moreover, decreased constitutively active mutant of STAT3 (STAT3C)-induced transcriptional activity. Altholactone treatment also results in down-regulation of STAT3 target genes such as survivin, and Bcl-2 followed by up regulation of pro-apoptotic Bax protein. However, pre-treatment with the antioxidant N-acetylcysteine (NAC) significantly inhibited the activation of Bax and prevented down-regulation of STAT3 target genes. Collectively, our findings suggest that altholactone induces DU145 cells death through inhibition of NF-κB and STAT3 activity.

Abstract 2521: STAT3 is a central regulator of proliferation, invasion and metabolism in GBM tumor initiating cells

Abstract Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor. Currently, treatment for GBM involves surgical resection, chemotherapy, and radiation, resulting in a median survival of 15 months. This poor outcome highlights the need for improved therapeutic approaches to treat GBM patients. The signal transducer and activator of transcription 3 (STAT3) pathway is abnormally active in GBM, primarily in the mesenchymal subtype of GBM. STAT3 regulates various cellular processes including: proliferation, invasion and resistance to therapy. The Weiss lab has established a large collection of brain tumor initiating cell (BTIC) lines derived from GBM patients. These GBM BTIC lines are used to model the subpopulation of cells that are predicted to be the source of resistance and recurrence following treatment. Here, we are focused on the role of STAT3 as a central regulator of proliferation, invasion and metabolism in GBM BTIC lines. We show that activation of the STAT3 pathway using the ligand oncostatin M (OSM) or by stably expressing a constitutively active STAT3 mutant (STAT3C) in GBM BTIC lines leads to an increase in proliferation. STAT3 is known to regulate the expression of multiple matrix metalloproteinases (MMPs) that promote invasion. Using time-lapse imaging of 3D sphere invasion into matrigel, we observed that JAK2 (an activator of STAT3) and direct STAT3 inhibitors decrease invasion of GBM BTICs. Our analysis of gene expression data available from The Cancer Genome Atlas (TCGA) demonstrates that a glutamine metabolism gene expression signature correlates inversely with a STAT3 gene expression signature in GBM patient samples. Conversely, similar analysis reveals that a high glycolysis gene expression signature correlates with a high STAT3 gene expression signature. We hypothesize that targeting the STAT3 pathway will decrease proliferation and invasion while simultaneously sensitizing GBM BTIC lines to inhibition of glutamine metabolism. Glutaminase (GLS) is the first enzyme to act on glutamine, converting glutamine to glutamate, which is then further processed to α-ketoglutarate (αKG). αKG is important for the production of biosynthetic macromolecules that are essential for cell growth. Chemical inhibition of GLS decreases cell growth in culture with varying levels of sensitivity across multiple GBM BTIC lines. Interestingly, activation of the STAT3 pathway using OSM de-sensitizes GBM BTIC lines to GLS inhibition. Furthermore, chemical inhibition of JAK2 sensitizes a subset of GBM BTIC lines to GLS inhibition resulting in a further decrease in cell growth. Overall, we show that STAT3 is a central hub in GBM BTIC lines that modulates proliferation, invasion, and sensitivity to GLS inhibition. Currently, we are continuing these studies in vivo using GBM BTIC orthotopic xenografts in mice. Citation Format: Ian Restall, H. Artee Luchman, Samuel Weiss. STAT3 is a central regulator of proliferation, invasion and metabolism in GBM tumor initiating cells. [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 2521.

Dissecting functions of the N-terminal domain and GAS-site recognition in STAT3 nuclear trafficking

Signal transducer and activator of transcription 3 (STAT3) is a ubiquitous transcription factor involved in many biological processes, including hematopoiesis, inflammation and cancer progression. Cytokine-induced gene transcription greatly depends on tyrosine phosphorylation of STAT3 on a single tyrosine residue with subsequent nuclear accumulation and specific DNA sequence (GAS) recognition. In this study, we analyzed the roles of the conserved STAT3 N-terminal domain (NTD) and GAS-element binding ability of STAT3 in nucleocytoplasmic trafficking. Our results demonstrate the nonessential role of GAS-element recognition for both cytokine-induced and basal nuclear import of STAT3. Substitution of five key amino acids within the DNA-binding domain rendered STAT3 unable to bind to GAS-elements while still maintaining the ability for nuclear localization. In turn, deletion of the NTD markedly decreased nuclear accumulation upon IL-6 treatment resulting in a prolonged accumulation of phosphorylated dimers in the cytoplasm, at the same time preserving specific DNA recognition ability of the truncation mutant. Observed defect in nuclear localization could not be explained by flawed importin-α binding, since both wild-type and NTD deletion mutant of STAT3 could precipitate both full-length and autoinhibitory domain (∆IBB) deletion mutants of importin-α5, as well as ∆IBB-α3 and ∆IBB-α7 isoforms independently of IL-6 stimulation. Despite its inability to translocate to the nucleus upon IL-6 stimulation, the NTD lacking mutant still showed nuclear accumulation in resting cells similar to wild-type upon inhibition of nuclear export by leptomycin B. At the same time, blocking the nuclear export pathway could not rescue cytoplasmic trapping of phosphorylated STAT3 molecules without NTD. Moreover, STAT3 mutant with dysfunctional SH2 domain (R609Q) also localized in the nucleus of unstimulated cells after nuclear export blocking, while upon cytokine treatment the subcellular localization of this mutant had not changed. Our findings support the concept that basal nucleocytoplasmic shuttling of STAT3 is different from active cytokine-induced nuclear import and does not require conserved N- or SH2-terminal domains, preformed dimer formation and GAS-element-specific DNA recognition.

Enhanced Transcriptional Activity and Mitochondrial Localization of STAT3 Co-induce Axon Regrowth in the Adult Central Nervous System.

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor central to axon regrowth with an enigmatic ability to act in different subcellular regions independently of its transcriptional roles. However, its roles in mature CNS neurons remain unclear. Here, we show that along with nuclear translocation, STAT3 translocates to mitochondria in mature CNS neurons upon cytokine stimulation. Loss- and gain-of-function studies using knockout mice and viral expression of various STAT3 mutants demonstrate that STAT3's transcriptional function is indispensable for CNS axon regrowth, whereas mitochondrial STAT3 enhances bioenergetics and further potentiates regrowth. STAT3's localization, functions, and growth-promoting effects are regulated by mitogen-activated protein kinase kinase (MEK), an effect further enhanced by Pten deletion, leading to extensive axon regrowth in the mouse optic pathway and spinal cord. These results highlight CNS neuronal dependence on STAT3 transcriptional activity, with mitochondrial STAT3 providing ancillary roles, and illustrate a critical contribution for MEK in enhancing diverse STAT3 functions and axon regrowth.

Functional characterization of two new STAT3 mutations associated with hyper-IgE syndrome in a Mexican cohort.

Hyper-IgE syndrome (HIES) is an immunodeficiency disorder that is characterized by distinctive immunologic and non-immunologic manifestations. Although mutations in signal transducer and activator of transcription 3 (STAT3) have been associated with HIES, the exact nature of the relationship is unknown. Here, we characterized the functional activity of STAT3 and its mutations in 11 Mexican patients with autosomal dominant HIES. STAT3 phosphorylation was evaluated by flow cytometry, and in silico analyses were performed to estimate the impact of allelic mutations on the DNA binding and SH2 domains of the STAT3 protein. Electrophoretic mobility shift assays were used to assess whether the STAT3 mutants could bind to the consensus oligonucleotide target in vitro. Two novel mutations [g.58891A>T (Asn395Tyr) and g.59078A>T (Asn425Tyr)] as well as one possible somatic mosaicism were found in several of the patients who bore some remarkable features. However, there were no direct correlations between genotypes and HIES clinical features. STAT3 phosphorylation was found to be lower in the patient cohort than in healthy controls. Moreover, the mutated STAT3 proteins could bind to the Sp1, but not to the STAT3, consensus sequence. From these functional studies, the STAT3 mutations found in our patient cohort were concluded to be deleterious for normal STAT3 function.