Browsing by Subject "STAT3"
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Item APC loss in breast cancer leads to doxorubicin resistance via STAT3 activation(Impact Journals, 2017-11-01) VanKlompenberg, Monica K.; Leyden, Emily; Arnason, Anne H.; Zhang, Jian-Ting; Stefanski, Casey D.; Prosperi, Jenifer R.; Pharmacology and Toxicology, School of MedicineResistance to chemotherapy is one of the leading causes of death from breast cancer. We recently established that loss of Adenomatous Polyposis Coli (APC) in the Mouse Mammary Tumor Virus – Polyoma middle T (MMTV-PyMT) transgenic mouse model results in resistance to cisplatin or doxorubicin-induced apoptosis. Herein, we aim to establish the mechanism that is responsible for APC-mediated chemotherapeutic resistance. Our data demonstrate that MMTV-PyMT;ApcMin/+ cells have increased signal transducer and activator of transcription 3 (STAT3) activation. STAT3 can be constitutively activated in breast cancer, maintains the tumor initiating cell (TIC) population, and upregulates multidrug resistance protein 1 (MDR1). The activation of STAT3 in the MMTV-PyMT;ApcMin/+ model is independent of interleukin 6 (IL-6); however, enhanced EGFR expression in the MMTV-PyMT;ApcMin/+ cells may be responsible for the increased STAT3 activation. Inhibiting STAT3 with a small molecule inhibitor A69 in combination with doxorubicin, but not cisplatin, restores drug sensitivity. A69 also decreases doxorubicin enhanced MDR1 gene expression and the TIC population enhanced by loss of APC. In summary, these results have revealed the molecular mechanisms of APC loss in breast cancer that can guide future treatment plans to counteract chemotherapeutic resistance.Item APC Loss Prevents Doxorubicin-Induced Cell Death by Increasing Drug Efflux and a Chemoresistant Cell Population in Breast Cancer(MDPI, 2023-04-21) Stefanski, Casey D.; Arnason, Anne; Maloney, Sara; Kotsen, Janna; Powers, Elizabeth; Zhang, Jian-Ting; Prosperi, Jenifer R.; Biochemistry and Molecular Biology, School of MedicineChemoresistance is a major health concern affecting cancer patients. Resistance is multifactorial, with one mechanism being the increased expression of ABC transporters (such as MDR1 and MRP1), which are drug efflux transporters capable of preventing intracellular accumulation of drugs and cell death. Our lab showed that the loss of Adenomatous Polyposis Coli (APC) caused an intrinsic resistance to doxorubicin (DOX), potentially through an enhanced tumor-initiating cell (TIC) population and the increased activation of STAT3 mediating the expression of MDR1 in the absence of WNT being activated. Here, in primary mouse mammary tumor cells, the loss of APC decreased the accumulation of DOX while increasing the protein levels of MDR1 and MRP1. We demonstrated decreased APC mRNA and protein levels in breast cancer patients compared with normal tissue. Using patient samples and a panel of human breast cancer cell lines, we found no significant trend between APC and either MDR1 or MRP1. Since the protein expression patterns did not show a correlation between the ABC transporters and the expression of APC, we evaluated the drug transporter activity. In mouse mammary tumor cells, the pharmacological inhibition or genetic silencing of MDR1 or MRP1, respectively, decreased the TIC population and increased DOX-induced apoptosis, supporting the use of ABC transporter inhibitors as therapeutic targets in APC-deficient tumors.Item APE1/Ref-1 Regulates STAT3 Transcriptional Activity and APE1/Ref-1–STAT3 Dual-Targeting Effectively Inhibits Pancreatic Cancer Cell Survival(2012-10) Cardoso, Angelo A.; Jiang, Yanlin; Luo, Meihua; Reed, April M.; Shahda, Safi; He, Ying; Maitra, Anirban; Kelley, Mark R.; Fishel, Melissa L.Pancreatic cancer is a largely incurable disease, and increasing evidence supports strategies targeting multiple molecular mediators of critical functions of pancreatic ductal adenocarcinoma cells. Intracellular redox state modulates the activity of various signal transduction pathways and biological processes, including cell survival, drug resistance and responsiveness to microenvironmental factors. Recently, it has been shown that the transcription factor STAT3 is under redox control, but the mechanisms involved in its regulation are unknown. Here, we demonstrate for the first time that STAT3 DNA binding and transcriptional activity is directly regulated by the redox function of the APE1/Ref-1 endonuclease, using overexpression and redox-specific mutational strategies, and gene knockdown. Also, pharmacological blockade of APE1/Ref-1 by the redox-selective inhibitor E3330 abrogates STAT3 DNA binding. Since APE1/Ref-1 also exerts redox control on other cancer-associated transcription factors, we assessed the impact of dual-targeting of STAT3 signaling and APE1/Ref-1 redox on pancreatic cancer cell functions. We observed that disruption of APE1/Ref-1 redox activity synergizes with STAT3 blockade to potently inhibit the proliferation and viability of human PDAC cells. Mechanistically, we show that STAT3–APE1/Ref-1 dual targeting promotes marked tumor cell apoptosis, with engagement of caspase-3 signaling, which are significantly increased in comparison to the effects triggered by single target blockade. Also, we show that STAT3–APE1/Ref-1 dual blockade results in significant inhibition of tumor cell migration. Overall, this work demonstrates that the transcriptional activity of STAT3 is directly regulated by the redox function of APE1/Ref-1, and that concurrent blockade of STAT3 and APE1/Ref-1 redox synergize effectively inhibit critical PDAC cell functions.Item Applying Small Molecule Signal Transducer and Activator of Transcription-3 (STAT3) Protein Inhibitors as Pancreatic Cancer Therapeutics(American Association for Cancer Research, 2016-05) Arpin, Carolynn C.; Mac, Stephen; Jiang, Yanlin; Cheng, Huiwen; Grimard, Michelle; Page, Brent D. G.; Kamocka, Malgorzata M.; Haftchenary, Sina; Su, Han; Ball, Daniel; Rosa, David A.; Lai, Ping-Shan; Gómez-Biagi, Rodolfo F.; Ali, Ahmed M.; Rana, Rahul; Hanenberg, Helmut; Kerman, Kagan; McElyea, Kyle C.; Sandusky, George E.; Gunning, Patrick T.; Fishel, Melissa L.; Pediatrics, School of MedicineConstitutively activated STAT3 protein has been found to be a key regulator of pancreatic cancer and a target for molecular therapeutic intervention. In this study, PG-S3-001, a small molecule derived from the SH-4-54 class of STAT3 inhibitors, was found to inhibit patient-derived pancreatic cancer cell proliferation in vitro and in vivo in the low micromolar range. PG-S3-001 binds the STAT3 protein potently, Kd = 324 nmol/L by surface plasmon resonance, and showed no effect in a kinome screen (>100 cancer-relevant kinases). In vitro studies demonstrated potent cell killing as well as inhibition of STAT3 activation in pancreatic cancer cells. To better model the tumor and its microenvironment, we utilized three-dimensional (3D) cultures of patient-derived pancreatic cancer cells in the absence and presence of cancer-associated fibroblasts (CAF). In this coculture model, inhibition of tumor growth is maintained following STAT3 inhibition in the presence of CAFs. Confocal microscopy was used to verify tumor cell death following treatment of 3D cocultures with PG-S3-001. The 3D model was predictive of in vivo efficacy as significant tumor growth inhibition was observed upon administration of PG-S3-001. These studies showed that the inhibition of STAT3 was able to impact the survival of tumor cells in a relevant 3D model, as well as in a xenograft model using patient-derived cells.Item Bone Metabolism: The Role of STAT3 and Reactive Oxygen Species(2013-08-14) Newnum, America Bethanne; Li, Jiliang; Marrs, James; Ji, Julie; Atkinson, SimonSignal Transducers and Activators of Transcription 3 (STAT3), a transcription factor expressed in many cell types, including osteoblasts and osteoclasts, is emerging as a key regulator of bone mass and strength. STAT3 mutations cause a rare human immunodeficiency disease characterized by extremely elevated levels of IgE in serum that have associated craniofacial and skeletal features, such as reduced bone mineral density and recurrent pathological fractures. Our microarray data and immunohistochemical staining using a normal rat model have shown that STAT3 mRNA and protein levels markedly increase in response to mechanical loading. In addition, as indicated by STAT3 phosphorylation in MC3T3-E1 osteoblastic cells, STAT3 activity significantly increases in response to 30 to 90 minutes fluid shear stress. In order to further study the role that STAT3 plays in bone responsiveness to loading, tissue-selective STAT3 knockout (KO) mice, in which inactivation of STAT3 occurs in osteoblasts, were generated by breeding the transgenic mice in which Cre recombinase cDNA was cloned downstream of a 3.6 or 2.3 kb fragment of the rat Col1a1 promoter (Col3.6-Cre and Col2.3-Cre, respectively) with a strain of floxed mice in which the two loxP sites flank exons 18-20 of the STAT3 gene were used. Mice engineered with bone selective inactivation of STAT3 in osteoblasts exhibited significantly lower bone mineral density (7-12%, p<0.05) and reduced ultimate force (21-34%, p<0.01) compared to their age-matched littermate controls. The right ulnae of 16-week-old bone specific STAT3 KO mice and the age-matched control mice were loaded with peak forces of 2.5 N and 2.75 N for female and male mice, respectively, at 2 Hz, 120 cycles/day for 3 consecutive days. Mice with inactivation of STAT3 specific in bone were significantly less responsive to mechanical loading than the control mice as indicated by decreased relative mineralizing surface (rMS/BS, 47-59%, p<0.05) and relative bone formation rate (rBFR/BS, 64-75%, p<0.001). Bone responsiveness was equally decreased in mice in which STAT3 is inactivated either in early osteoblasts (Col3.6-Cre) or in mature osteoblasts (Col2.3-Cre). Accumulating evidence indicates that bone metabolism is significantly affected by activities in mitochondria. For instance, although STAT3 is reported to be involved in bone formation and resorption through regulation of nuclear genes, inactivation of STAT3 is shown to disrupt mitochondrial activities and result in an increased level of reactive oxygen species (ROS). Inactivation of STAT3 suppressed load-driven mitochondrial activity, which led to an elevated level of ROS in cultured primary osteoblasts. Oxidative stress induced by administration of buthionine sulfoximine (BSO) significantly inhibits load-induced bone formation in wild type mice. Taken together, the results support the notion that the loss-of-function mutation of STAT3 in osteoblasts and osteocytes diminishes load-driven bone formation and impairs the regulation of oxidative stress in mitochondria.Item THE DEVELOPMENT AND COMMITMENT OF T HELPER SUBSETS(2011-03-09) Stritesky, Gretta L.; Kaplan, Mark H.; Blum, Janice Sherry, 1957-; Dent, Alexander L.; Harrington, Maureen A.T helper cells play a crucial role in providing protection against a wide variety of pathogens. The differentiation and effector function of T helper cell subsets is dependent on cytokine activation of Signal Transducer and Activator of Transcription (STAT) family members. The development of Th17 cells, which are important for immunity to fungi and extracellular bacteria, relies on STAT3. We show that IL-23 in combination with IL-1β promotes maintenance of the Th17 phenotype following multiple rounds of stimulation. However, IL-23 does not promote commitment of Th17 cells, and when Th17 cells are cultured with IL-12 or IL-4 they switch to a Th1 and Th2 phenotype, respectively. The maintenance of the Th17 phenotype by IL-23 also requires STAT4. STAT4-deficient memory cells cultured with IL-23 have reduced IL-17 production following stimulation with either anti-CD3 or IL-18+IL-23 stimulation compared to wild type memory cells. Furthermore, STAT4-deficient mice have impaired in vivo Th17 development following immunization with ovalbumin. This challenges a one-STAT/one-subset paradigm and suggests that multiple STAT proteins can contribute to a single phenotype. To test this further we examined whether STAT3 is required for the development of Th2 cells, a subset known to depend upon the IL-4-induced activation of STAT6 for immunity to parasites and promoting allergic inflammation. We demonstrate that in the absence of STAT3, the expression of Th2-associated cytokines and transcription factors is dramatically reduced. STAT3 is also required for in vivo development of Th2 cells. Moreover, allergic inflammation is diminished in mice that have T cells lacking expression of STAT3. STAT3 does not affect STAT6 activation, but does impact how STAT6 functions in binding target genes. Thus, multiple STAT proteins can cooperate in promoting the development of specific T helper subsets.Item Dichotomous effects of cellular expression of STAT3 on tumor growth of HNSCC(Elsevier, 2021) Bickett, Thomas E.; Knitz, Michael W.; Piper, Miles; Oweida, Ayman J.; Gadwa, Jacob; Darragh, Laurel B.; Nguyen, Diemmy; Bhatia, Shilpa; Bhuvane, Shiv; Phan, Andy V.; Van Court, Benjamin; Corbo, Sophia; Pham, Tiffany; Dent, Alexander L.; Lenz, Laurel; Karam, Sana D.; Microbiology and Immunology, School of MedicineSTAT3 signaling has been shown to regulate cellular function and cytokine production in the tumor microenvironment (TME). Within the head and neck squamous cell carcinoma (HNSCC) TME, we previously showed that therapeutic targeting of STAT3 in combination with radiation resulted in improved tumor growth delay. However, given the independent regulatory effects STAT3 has on anti-tumor immunity, we aimed to decipher the effects of individually targeting STAT3 in the cancer cell, regulatory T cells (Tregs), and natural killer (NK) cell compartments in driving tumor growth and resistance to therapy in HNSCCs. We utilized a CRISPR knockout system for genetic deletion of STAT3 within the cancer cell as well as two genetic knockout mouse models, FoxP3-Cre/STAT3 fl and NKp46-Cre/STAT3 fl, for Tregs and NK cell targeting, respectively. Our data revealed differences in development of resistance to treatment with STAT3 CRISPR knockout in the cancer cell, driven by differential recruitment of immune cells. Knockout of STAT3 in Tregs overcomes this resistance and results in Treg reprogramming and recruitment and activation of antigen-presenting cells. In contrast, knockout of STAT3 in the NK cell compartment results in NK cell inactivation and acceleration of tumor growth. These data underscore the complex interplay between the cancer cell and the immune TME and carry significant implications for drug targeting and design of combination approaches in HNSCCs.Item Drug Inhibition of Redox Factor-1 Restores Hypoxia-Driven Changes in Tuberous Sclerosis Complex 2 Deficient Cells(MDPI, 2022-12-15) Champion, Jesse D.; Dodd, Kayleigh M.; Lam, Hilaire C.; Alzahrani, Mohammad A. M.; Seifan, Sara; Rad, Ellie; Scourfield, David Oliver; Fishel, Melissa L.; Calver, Brian L.; Ager, Ann; Henske, Elizabeth P.; Davies, David Mark; Kelley, Mark R.; Tee, Andrew R.; Pediatrics, School of MedicineTherapies with the mechanistic target of rapamycin complex 1 (mTORC1) inhibitors are not fully curative for tuberous sclerosis complex (TSC) patients. Here, we propose that some mTORC1-independent disease facets of TSC involve signaling through redox factor-1 (Ref-1). Ref-1 possesses a redox signaling activity that stimulates the transcriptional activity of STAT3, NF-kB, and HIF-1α, which are involved in inflammation, proliferation, angiogenesis, and hypoxia, respectively. Here, we demonstrate that redox signaling through Ref-1 contributes to metabolic transformation and tumor growth in TSC cell model systems. In TSC2-deficient cells, the clinically viable Ref-1 inhibitor APX3330 was effective at blocking the hyperactivity of STAT3, NF-kB, and HIF-1α. While Ref-1 inhibitors do not inhibit mTORC1, they potently block cell invasion and vasculature mimicry. Of interest, we show that cell invasion and vasculature mimicry linked to Ref-1 redox signaling are not blocked by mTORC1 inhibitors. Metabolic profiling revealed that Ref-1 inhibitors alter metabolites associated with the glutathione antioxidant pathway as well as metabolites that are heavily dysregulated in TSC2-deficient cells involved in redox homeostasis. Therefore, this work presents Ref-1 and associated redox-regulated transcription factors such as STAT3, NF-kB, and HIF-1α as potential therapeutic targets to treat TSC, where targeting these components would likely have additional benefits compared to using mTORC1 inhibitors alone.Item Drugging the "undruggable" DNA-binding domain of STAT3(Impact Journals, 2016-10-11) Zhang, Jian-Ting; Liu, Jing-Yuan; Department of Pharmacology and Toxicology, IU School of MedicineItem Endothelial STAT3 Modulates Protective Mechanisms in a Mouse Ischemia-Reperfusion Model of Acute Kidney Injury(hindawi publishing corporation, 2017) Dube, Shataakshi; Matam, Tejasvi; Yen, Jessica; Mang, Henry E.; Dagher, Pierre C.; Hato, Takashi; Sutton, Timothy A.; Medicine, School of MedicineSTAT3 is a transcriptional regulator that plays an important role in coordinating inflammation and immunity. In addition, there is a growing appreciation of the role STAT3 signaling plays in response to organ injury following diverse insults. Acute kidney injury (AKI) from ischemia-reperfusion injury is a common clinical entity with devastating consequences, and the recognition that endothelial alterations contribute to kidney dysfunction in this setting is of growing interest. Consequently, we used a mouse with a genetic deletion of Stat3 restricted to the endothelium to examine the role of STAT3 signaling in the pathophysiology of ischemic AKI. In a mouse model of ischemic AKI, the loss of endothelial STAT3 signaling significantly exacerbated kidney dysfunction, morphologic injury, and proximal tubular oxidative stress. The increased severity of ischemic AKI was associated with more robust endothelial-leukocyte adhesion and increased tissue accumulation of F4/80+ macrophages. Moreover, important proximal tubular adaptive mechanisms to injury were diminished in association with decreased tissue mRNA levels of the epithelial cell survival cytokine IL-22. In aggregate, these findings suggest that the endothelial STAT3 signaling plays an important role in limiting kidney dysfunction in ischemic AKI and that selective pharmacologic activation of endothelial STAT3 signaling could serve as a potential therapeutic target.