Network Pharmacology & Validation of Dihydrotanshinone I's Regulation of HIF-1α via STAT3 in Cardiomyocytes.

Objective: Signal transducer and activator of transcription 3 (STAT3) is as a potential drug target for cancer and inflammation. Strategies to block STAT3 dimerization have dominated STAT3 inhibitor discovery, while inhibition of direct STAT3-DNA binding has not been much explored. This study was to identify novel STAT3 inhibitors that may be developed into probes or therapeutics, and to investigate their putative binding site. Methods: A library of in-house compounds was screened against STAT3-DNA binding using an electrophoretic mobility shift assay (EMSA). Inhibition of DNA and STAT3:STAT3 or STAT3:STAT1 interaction and antiproliferative activity in STAT3 expressing or STAT3 knockout cells were tested. Pharmacophore modeling and 3D-QSAR; and docking with molecular mechanics generalized born surface area (MM-GBSA) refinement were also undertaken at the STAT3 SH2 and STAT3 DNA binding domains. Surface plasmon resonance (SPR) analysis was done to determine STAT3 domain interactions. Organic synthesis was also undertaken. Results: Pyrimidinetriones derivatives were identified as novel STAT3 inhibitors, with activity in low micromolar concentrations. Application of 3D-QSAR and docking analysis with MM-GBSA refinement suggested that the compounds bind at the STAT3-DNA binding interface, and not at the SH2 domain where most current STAT3 inhibitors are thought to bind. The results were confirmed with SPR analysis, pointing to the DNA binding domain (DBD) as the putative binding site of this novel class of STAT3 inhibitors. The analysis guided the synthesis of active novel compounds. Conclusion: Pyrimidinetrione were identified as new STAT3 inhibitors that putatively bind at the STAT3 DBD. This study is an excellent example of the use of QSAR and structure-based design to aid the identification of putative ligand binding sites on proteins. The compounds will provide new tools for studying STAT3 biology; and also serve as potential leads for the development of new therapeutics against STAT3 to fight or study diseases such as cancer and inflammation.

Mitochondrial STAT3 Plays a Critical Role in Survival of AML Cells

Rational drug design of benzothiazole-based derivatives as potent signal transducer and activator of transcription 3 (STAT3) signaling pathway inhibitors.

A Sticky Story for Signal Transducer and Activator of Transcription 3 in Platelets

STAT3-VDAC1 Axis Modulates Mitochondrial Function and Plays a Critical Role in the Survival of Leukemic Stem Cells

Constitutive activation of signal transducers and activators of transcription 3 (STAT3) signaling is frequently detected in human cancer including colon cancer, and has emerged as an attractive molecular target for cancer prevention. Recent experimental evidence suggests that the existence of a small population of tumorigenic stem/progenitor cells may be responsible for tumor initiation, chemotherapy and radiation resistance, invasion, recurrence, and metastasis. An increasing body of evidence suggests that the cancer stem cell concept is also relevant to colorectal cancer. To date, however, whether STAT3 is activated in colon cancer stem cells or cancer-initiating cells and what the role of STAT3 signaling may play in these cancer stem cells is still unknown. If STAT3 is activated in colorectal cancer stem cells, inhibiting STAT3 may offer a promising opportunity to target colorectal cancer stem cells and prevent the recurrence of cancer. We utilized the colon cancer stem cells, which are characterized by an aldehyde dehydrogenase (ALDH)-positive (ALDH+) and CD133-positive (CD133+) subpopulation. We demonstrated that ALDH+/CD133+ cells have capacity than ALDH-/CD133- cells to form more tumorspheres and to exhibit more potent tumor-initiating ability in mice. We then examined the STAT3 activation in these colon cancer cells. Interestingly, we observed that the ALDH+/CD133+ subpopulation of colon cancer cells expressed higher levels of phosphorylated STAT3, an active form of STAT3, compared to the ALDH-/CD133- subpopulation and un-separated colon cancer cells, suggesting that STAT3 is activated in colon cancer stem cells. We demonstrated that dietary agent, curcumin and a new curcumin analog, GO-Y030 can inhibit STAT3 phosphorylation, cell viability, and induced apoptosis in colon cancer stem cells. The inhibition of STAT3 was confirmed using a novel STAT3-selecitve curcumin analog, FLLL32, STAT3 ShRNA, as well as STAT3 inhibitors, LLL12 and Stattic. All these STAT3 inhibitors, inhibited STAT3 phosphorylation, cell viability, and tumorsphere growth in colon cancer stem cells. Furthermore, both GO-Y030 and LLL12 inhibited tumor growth of colon cancer stem cells in NOD/SCID mouse model in vivo. In Summary, this is the first report to demonstrate that persistent STAT3 phosphorylation is expressed in colon cancer stem cells. Our study is also the first attempt to target STAT3 in colon cancer stem cells and we demonstrated for the first time that colon cancer stem cells are indeed sensitive to the inhibition by small molecular STAT3 inhibitors, FLLL32, LLL12, Stattic, STAT3 shRNA, and curcumin analog, GO-Y030. Our results suggest that STAT3 is a novel prevention target in colon cancer stem cells and inhibition of activated STAT3 in cancer stem cells may offer an effective preventive approach for colorectal carcinoma. Citation Information: Cancer Prev Res 2010;3(12 Suppl):A100.

Activation of signal transducer and activator of transcription 3 (STAT3) is implicated in tumor progression and correlates with poor prognosis and reduced survival. In colorectal cancer (CRC), STAT3 activation serves as a key indicator of unfavorable outcomes. However, the scarcity of clinically available STAT3 inhibitors hinders the development of personalized treatment strategies targeting STAT3. Therefore, we aimed to develop a novel STAT3 inhibitor based on the molecular structure of STAT3 and our previously reported STAT3 inhibitor LY17 to inhibit the progression of CRC. The binding of the novel STAT3 inhibitor DB-2B to STAT3 was confirmed by computational docking, surface plasmon resonance, isothermal titration calorimetry, and cellular thermal shift assays. Western blotting and immunofluorescent staining demonstrated that DB-2B specifically inhibited STAT3 activation and nuclear translocation. In vitro studies revealed that DB-2B significantly suppressed proliferation, induced apoptosis, arrested cell cycle progression, and attenuated stemness by inhibiting STAT3 activation and its downstream signaling pathways. In vivo, DB-2B exhibited favorable oral bioavailability and safety, while significantly inhibiting the progression of CRC. Collectively, this study presents DB-2B as a promising small-molecule STAT3 inhibitor for the targeted treatment of CRC.

Pancreatic cancer remains a largely incurable disease, with patients facing the worst 5-year survival rate of any cancer. The challenge is to identify the molecular effectors that regulate the survival of pancreatic ductal adenocarcinoma (PDAC) cells, to devise molecular-targeted strategies that are effective in the metastatic setting, and overcome the protective role of the tumor-associated fibrosis and stroma. Strategies targeting multiple molecular effectors in PDAC are likely going to make a bigger impact. Thus, we are identifying molecular targets or synthetic lethal pairs that regulate critical pro-survival or pro-invasive pathways in PDAC. Constitutively activated Signal Transducer and Activator of Transcription 3 (STAT3) protein has been found to be a key regulator of pancreatic cancer and a target for molecular therapeutic intervention. To better model the tumor and its microenvironment, we utilized ex vivo 3-Dimensional (3D) cultures of patient-derived pancreatic cancer cells in the absence and presence of cancer-associated fibroblasts (CAFs). We can quantitate the inhibitory effect on both the tumor and CAFs as they are labeled with different fluorescent markers. In this co-culture model, inhibition of tumor growth is maintained following STAT3 inhibition in the presence of CAFs. We screened several STAT3 small molecule inhibitors, derived from the SH-4-54 class of STAT3 inhibitors, and found inhibition of pancreatic cancer cell proliferation in the low μM range. Our inhibitors bind the STAT3 protein potently, as shown by SPR, and demonstrate no effect in a kinome screen. In vitro studies demonstrated potent cell killing as well as inhibition of STAT3 activation in the 3D co-culture model. We have previously reported that Ref-1 (redox factor-1) regulates STAT3 activity through its redox function and blocking STAT3 through phosphorylation and redox inhibition synergizes for PDAC cell killing. In our 3D co-culture system, Ref-1 inhibitor, APX3330 decreases tumor area and intensity in a dose-dependent manner. The addition of APX3330 to STAT3 pathway inhibition via Ruxolitinib (Rux, Jak 2 inhibitor) or direct STAT3 inhibitor potentiated the killing effect in the tumor. However, the combination treatment did not appear to sensitize CAF cells, suggesting that targeting of Ref-1 and the STAT3 pathway is more specifically targeting tumor cells. Utilizing APX3330, Rux, and our lead STAT3 inhibitors, we evaluated the effects of Ref-1/STAT3 inhibition in PDAC low passage patient-derived cell lines. The activity of STAT3 and specificity of lead compounds was assessed by immunoblotting for levels of phosphorylated proteins including STAT3 (Y705) and STAT5 (Y694) in 3D culture. These studies establish the rationale for the development of STAT3 dual-targeting strategies for the treatment of pancreatic cancer and suggest that Ref-1 and STAT3 may be a synthetic lethal pair. Citation Format: Melissa L. Fishel, Michelle L. Grimard, Mark R. Kelley, David A. Rosa, Andrew Shouksmith, Gary Tin, Ji Park, Patrick T. Gunning. Development of STAT3 dual-targeting strategies for the treatment of pancreatic cancer. [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 1246.

Hyperactive signal transducer and activator of transcription 3 (STAT3) signaling is frequently detected in human triple-negative breast cancer (TNBC) and gastric cancer, leading to uncontrolled tumor growth, resistance to chemotherapy, and poor prognosis. Thus, inhibition of STAT3 signaling is a promising therapeutic approach for both TNBC and gastric cancer, which have high incidences and mortality and limited effective therapeutic approaches. Here, we report a small molecule, WZ-2-033, capable of inhibiting STAT3 activation and dimerization and STAT3-related malignant transformation. We present in vitro evidence from surface plasmon resonance analysis that WZ-2-033 interacts with the STAT3 protein and from confocal imaging that WZ-2-033 disrupts HA-STAT3 and Flag-STAT3 dimerization in intact cells. WZ-2-033 suppresses STAT3-DNA-binding activity but has no effect on STAT5-DNA binding. WZ-2-033 inhibits the phosphorylation and nuclear accumulation of pY705-STAT3 and consequently suppresses STAT3-dependent transcriptional activity and the expression of STAT3 downstream genes. Moreover, WZ-2-033 significantly inhibited the proliferation, colony survival, migration, and invasion of TNBC cells and gastric cancer cells with aberrant STAT3 activation. Furthermore, administration of WZ-2-033 in vivo induced a significant antitumor response in mouse models of TNBC and gastric cancer that correlated with the inhibition of constitutively active STAT3 and the suppression of known STAT3 downstream genes. Thus, our study provides a novel STAT3 inhibitor with significant antitumor activity in human TNBC and gastric cancer harboring persistently active STAT3.

STAT3 Activation Promotes NK Cell Proliferation, NKG2D Expression, and NK Cell Antitumor Activity

MTE23.02 Biomarker Characterization: Challenges and Perspectives

Breast cancer is the leading type of cancer affecting women. It is estimated that breast cancer accounts for over a quarter of all newly diagnosed cancer cases in women. Despite advances in chemotherapy and hormonal treatment, at the present time, metastatic breast cancer remains an incurable disease. Although a large number of chemotherapeutic agents have been developed that are able to cause the regression of metastatic breast cancers, these tumors almost always recur following chemotherapy treatment. It is possible that breast cancer stem cells or breast cancer initiating cells are responsible for the drug-resistance, metastasis, and recurrence of cancer. Constitutive activation of Signal Transducers and Activators of Transcription 3 (STAT3) signaling is frequently detected in many types of cancers, including breast cancer, and has emerged as an attractive molecular target for cancer treatment. However, whether or not STAT3 is activated in breast cancer stem cells and what role STAT3 signaling plays in cancer stem cells is still unknown. If STAT3 is activated in breast cancer stem cells, targeting STAT3 may offer a promising opportunity to eliminate breast cancer stem cells and prevent the recurrence of cancer. We examined STAT3 activation in breast cancer stem cells, which are characterized by an aldehyde dehydrogenase (ALDH)-positive (ALDH+) and ALDH+/CD44+/CD24− subpopulations. Interestingly, we observed that the ALDH+ and ALDH+/CD44+/CD24− subpopulations of breast cancer cells expressed higher levels of phosphorylated STAT3, an active form of STAT3, as compared to the ALDH− and ALDH−/CD44+/CD24+ subpopulations and un-separated breast cancer cells, suggesting that STAT3 is activated in breast cancer stem cells. We also demonstrated that a novel STAT3 inhibitor, LLL12, inhibited STAT3 phosphorylation, cell viability, STAT3 downstream target gene expression, and induced apoptosis in breast cancer stem cells. In addition, the STAT3 inhibitors Stattic and LLL12 inhibited the tumorsphere forming capacity of breast cancer stem cells. Furthermore, in a mouse tumor model using breast cancer stem cells, we demonstrated that LLL12 can suppress tumor growth in vivo. This is the first report to demonstrate that persistent STAT3 phosphorylation is expressed in breast cancer stem cells and that this subpopulation of breast cancer stem cells is sensitive to STAT3 inhibitors. Our results suggest that STAT3 is a novel therapeutic target in breast cancer stem cells and inhibition of activated STAT3 in cancer stem cells may offer a more effective treatment for breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4296.

The American Cancer Society estimates that over 200,000 new cases of invasive breast cancer and over 40,000 deaths are expected among women in United States each year. Despite advances in chemotherapy and hormonal treatment, at the present time, metastatic breast cancer remains an incurable disease. Although a large number of chemotherapeutic agents have been developed that are able to cause the regression of metastatic breast cancers, these tumors almost always recur following chemotherapy treatment. It is possible that breast cancer stem cells or breast cancer initiating cells are responsible for the drug-resistance, metastasis, and recurrence of cancer. Constitutive activation of Signal Transducers and Activators of Transcription 3 (STAT3) signaling is frequently detected in many types of cancers, including breast cancer, and has emerged as an attractive molecular target for cancer treatment. However, whether or not STAT3 is activated in breast cancer stem cells and what role STAT3 signaling plays in cancer stem cells is still unknown. If STAT3 is activated in breast cancer stem cells, targeting STAT3 may offer a promising opportunity to eliminate breast cancer stem cells and prevent the recurrence of cancer. We examined STAT3 activation in breast cancer stem cells, which are characterized by an aldehyde dehydrogenase (ALDH)-positive subpopulation. Interestingly, we observed that the ALDH-positive subpopulation of breast cancer cells expressed higher levels of phosphorylated STAT3, an active form of STAT3, as compared to the ALDH-negative subpopulation and un-separated breast cancer cells, suggesting that STAT3 is activated in breast cancer stem cells. We also demonstrated that a novel STAT3 inhibitor, LLL12, inhibited STAT3 phosphorylation, cell viability, STAT3 downstream target gene expression, and induced apoptosis in breast cancer stem cells. In addition, the STAT3 inhibitors Stattic and LLL12 reduced ALDH-positive subpopulation and inhibited their tumorsphere forming capacity. Furthermore, in our pilot study in a mouse tumor model using breast cancer stem cells, we demonstrated that LLL12 can suppress tumor growth in vivo. This is the first report to demonstrate that persistent STAT3 phosphorylation is expressed in breast cancer stem cells and that this subpopulation of breast cancer stem cells is sensitive to STAT3 inhibitors. Our results suggest that STAT3 is a novel therapeutic target in breast cancer stem cells and inhibition of activated STAT3 in cancer stem cells may offer a more effective treatment for breast cancer. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A54.

Malignant Peripheral Nerve Sheath Tumor (MPNST) is a rare soft tissue sarcoma that can arise from patients with NF1 (neurofibromatosis type 1). These patients are at a much greater risk of developing MPNST than the general population (10% vs. 0.01%, respectively). Existing chemotherapeutic and targeted agents have thus far not been successful in MPNST treatment, with a 5-year patient survival rate of just 35%-50%. Recent research implicates Signal Transducer and Activator of Transcription-3 (STAT3) and Hypoxia Inducible Factor 1 (HIF1) in driving MPNST. STAT3 is a transcription factor implicated in several cancers, and phosphorylated STAT3 (p-STAT3) expression indicates aggressive disease at disease onset in MPNST. STAT3 is activated upon phosphorylation by Janus Kinase (JAK), but its DNA binding and transcriptional activity is regulated by Apurinic/apyrimidinic endonuclease/redox factor-1 (APE1/Ref-1 or Ref-1) redox function. Ref-1 is a multifunctional protein involved in repairing DNA damage via endonuclease activity and in redox regulation of various transcription factors including STAT3, HIF-1α, and NFκB. High expression levels of Ref-1 indicate decreased survival in several cancers. We characterized Ref-1 and p-STAT3 expression in several MPNST samples including: the IN Pediatric BioBank, and established MPNST cell lines, new patient-derived tumor cells, and genetically engineered mouse models. We observed strong Ref-1 and phospho-STAT3 staining in the majority of these samples. We examined the expression of p-STAT3 and Ref-1 in mice that have conditional ablation in the Schwann cells of Nf1-/-; Arf+/- and Nf1-/-; Arf-/- mice and found a significant increase in positivity of p-STAT3 and Ref-1 expression in the sections containing MPNST. It also appears that there may be differences in staining intensity based on Arf genotype as the Nf1-/-; Arf-/- have stronger staining than the Nf1-/-; Arf+/-. Knocking down Ref-1 or STAT3 impairs MPNST growth in vitro. Inhibiting the redox activity of Ref-1 using the redox-specific APE1 inhibitor, APX3330 (and its next generation analogs), or inhibiting STAT3 activity using Napabucasin also resulted in reduced in vitro growth. Investigation into the apoptotic pathways that are activated following treatment demonstrate activation of caspase 3/7. Several biomarkers downstream of Ref-1 and STAT3 were downregulated following Ref-1 redox or STAT3 inhibition. We also performed RNA-sequencing to identify differentially expressed genes (DEGs) following Ref-1 or STAT3 knockdown and implicate new targets associated with clinical response in MPNST. In vivo experiments performed on mice implanted with ST88-14 cells using either APX2009 (APX3330 analog) or Napabucasin both resulted in significantly reduced tumor growth and additional combination studies are underway. Obtaining these results with clinically-tested Ref-1 and STAT3 inhibitors make the translation of this work highly plausible for pediatric patients with MPNST in the future. We will use both PDX and genetically engineered models of MPNST to test and validate these novel targets. Citation Format: Fenil Shah, Olivia Babb, Chi Zhang, Silpa Gampala, Emily Zhang, Steven D Rhodes, Andrew R. Tee, Brian Calver, Ellie Rad, Verena Staedtke, Karen E Pollok, D. Wade Clapp, Mark R. Kelley, Melissa L. Fishel. Signaling through Ref-1 and STAT3 in soft tissue sarcoma (MPNST) and the effects of perturbing this pathway on tumor cell survival and gene expression [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C017. doi:10.1158/1535-7163.TARG-19-C017

BackgroundSignal transducer and activator of transcription 3 (STAT3) plays a critical role in tumor development by regulating signaling pathways involved in cell proliferation, survival, metastasis and angiogenesis. STAT3 is activated in many cancers, including head and neck squamous cell carcinoma (HNSCC) in people. Feline oral squamous cell carcinoma (OSCC) is similar to advanced or recurrent HNSCC as it is poorly responsive to traditional therapies and carries a poor long-term prognosis. The purpose of this study was to characterize expression and activation of STAT3 in feline OSCC cell lines and tumor samples and to investigate the biologic activity of a novel, allosteric STAT3 inhibitor, LLL12, in feline OSCC cell lines.ResultsWe evaluated 3 feline OSCC cell lines and one of these (SCCF2) exhibited high levels of constitutive STAT3 phosphorylation and high sensitivity to LLL12 treatment. Exposure of SCCF2 cells to LLL12 resulted in decreased expression of pSTAT3 and total STAT3, apoptosis as assessed by caspase 3/7 activation, inhibition of colony formation and reduced expression of the STAT3 transcriptional target survivin. In contrast, the STAT3 transcriptional targets VEGF and MCL-1 increased after LLL12 treatment. This was, in part, likely due to LLL12 mediated upregulation of HIF-1α, which is known to drive VEGF and MCL-1 expression. The OSCC cell lines with low basal STAT3 phosphorylation did not exhibit these effects, suggesting that STAT3 inhibition was responsible for the observed results. Lastly, immunohistochemistry for pSTAT3 was performed using a feline OSCC tissue microarray, demonstrating expression in 48 % of samples tested.ConclusionsThese data demonstrate that LLL12 has biologic activity against a feline OSCC cell line expressing pSTAT3 and that STAT3 represents a target for therapeutic intervention in this disease. However, given the up-regulation of several STAT3 transcriptional targets following treatment, further investigation of STAT3 and its related signaling pathways in OSCC is warranted.Electronic supplementary materialThe online version of this article (doi:10.1186/s12917-015-0505-7) contains supplementary material, which is available to authorized users.