SUMMARY STING is an ER-associated membrane protein that is critical for innate-immune sensing of pathogens. STING-mediated activation of the IFN-I pathway through the TBK1/IRF3 signaling axis involves both cyclic-dinucleotide binding, and its translocation from the ER to vesicles. However, how these events are coordinated, and the exact mechanism of STING activation, remain poorly understood. Here, we found that the Shigella effector protein IpaJ potently inhibits STING signaling by blocking its translocation from the ER to ERGIC, even in the context of dinucleotide binding. Reconstitution using purified components revealed STING translocation as the rate-limiting event in maximal signal transduction. Furthermore, STING mutations associated with autoimmunity in humans were found to cause constitutive ER exit, and to activate STING independent of cGAMP binding. Together, these data provide compelling evidence for an ER-retention and ERGIC/Golgi-trafficking mechanism of STING regulation that is subverted by bacterial pathogens and is deregulated in human genetic disease.

Gliomas are the most common and devastating central nervous system neoplasms. A gender bias exists in their development: females are at lower risk than males, implicating estrogen-mediated protective effects. Estrogen functions are mediated by two ER subtypes: ERα, that functions as tumor promoter and ERβ that function as tumor suppressor. We examined the potential use of ERβ agonists as a novel therapeutic to curb the growth of gliomas. Western analysis of six glioma model cells showed detectable expression of ERβ with little or no ERα. Treatment of glioma cells with ERβ agonists resulted in significant decrease in proliferation. IHC analysis of tumor tissues revealed that ERβ expression is down regulated in high-grade gliomas. We found that ERβ agonists promote both expression and tumor suppressive functions of ERβ in glioma cells. Liquiritigenin, a plant-derived ERβ agonist significantly reduced in vivo tumor growth in a xenograft model. Compared to control mice, animals treated with liquiritigenin had greater than 50% reduction in tumor volume and size. IHC analysis of tumors revealed a significant increase in the nuclear ERβ expression with a concomitant decrease in cell proliferation in the liquiritigenin-treated group. Our results suggest that ERβ signaling has a tumor suppressive function in gliomas. Since ERβ agonists are currently in clinical trials and are well tolerated with fewer side effects, identification of an ERβ agonist as a therapeutic agent can be readily extended to clinical use with current chemotherapies, providing an additional tool for enhancing survival in glioma patients.

Tumor metastasis is the leading cause of death among breast cancer patients. PELP1 is a nuclear receptor coregulator that is upregulated during breast cancer progression to metastasis and is an independent prognostic predictor of shorter survival of breast cancer patients. Here, we show that PELP1 modulates expression of metastasis-influencing microRNAs (miRs) to promote cancer metastasis. Whole genome miR array analysis using PELP1 over expressing and under expressing model cells revealed that miR-200a and miR-141 levels inversely correlated with PELP1 expression. Consistent with this, PELP1 knockdown resulted in lower expression of miR-200a target genes ZEB1 and ZEB2. PELP1 knockdown significantly reduced tumor growth and metastasis compared with parental cells in an orthotopic xenograft tumor model. Furthermore, re-introduction of miR-200a and miR-141 mimetics into PELP1 overexpressing cells reversed PELP1 target gene expression, decreased PELP1 driven migration/invasion in vitro, and significantly reduced in vivo metastatic potential in a preclinical model of experimental metastasis. Our results demonstrated that PELP1 binds to miR-200a and miR-141 promoters and regulates their expression by recruiting chromatin modifier HDAC2 as revealed by ChIP, siRNA and HDAC inhibitor assays. Taken together, our results suggest that PELP1 regulates tumor metastasis by controlling the expression and functions of the tumor metastasis suppressors miR-200a and miR-141.

Trex1−/− mice suffer from systemic inflammation caused largely by chronic activation of the cGAS-STING-TBK1-IRF3 signaling pathway. We showed previously that Trex1-deficient cells have reduced mTORC1 activity, although the underlying mechanism is unclear. Here, we performed detailed metabolic analysis in Trex1−/− mice and cells that revealed defects in mitochondrial respiration, reduced adiposity, increased energy expenditure and glycolysis. We also found that Trex1−/− mice and cells exhibit chronically suppressed mTORC1 activity. Furthermore, we provide genetic evidence corroborated with cellular and biochemical validation to show that metabolic dysregulation in Trex1−/− mice is caused by STING-dependent activation of TBK1, but not the downstream IRF3-mediated signaling and inflammation, and that TBK1 binds to the mTORC1 complex and inhibits its activity in Trex1−/− cells. Our data thus establish chronic STING-dependent activation of TBK1 as a novel regulatory axis of mTORC1 and cellular metabolism.

Purpose: Gliomas are the most common type of primary brain tumors with bleak prognosis. Gliomas exhibit gender bias with females at lower risk than males, implicating estrogen mediated protective effects. Estrogen functions are mediated by two ER subtypes: ERbeta, that functions as tumor promoter and ERbeta that function as tumor suppressor. In this study, we examined the status of ERbeta and tested whether natural ERbeta agonists have therapeutic potential on gliomas. Experimental Design: Tumor microarrays (TMAs) were utilized to determine the levels of ERbeta in gliomas. Therapeutic effect of three ERbeta agonists was determined using proliferation, foci formation and cell cycle progression assays. Functionality of ERbeta signaling in gliomas was determined by reporter assays and localization by confocal microscopy. The therapeutic potential of liquiritigenin was determined by systemic administration in a glioma xenograft model. Results: TMA analysis revealed that ERα expression is down regulated in high-grade gliomas. Glioma model cells exhibited detectable ERα with functional ERα signaling. Treatment of glioma cells with ERα agonists resulted in significant decreases in proliferation. Further, ERbeta agonist treatment induced the expression of ERα. Compared to control mice, animals treated with liquiritigenin had 50% reduction in tumor volume and size. IHC analysis of tumors revealed a significant increase in the nuclear ERα expression with a concomitant decrease in cell proliferation in the liquiritigenin-treated group. Conclusion: We found that ERα agonists promote both expression and tumor suppressive functions of ERα. Liquiritigenin, a plant-derived ERα agonist significantly reduced in vivo tumor growth in a xenograft model. Our results suggest that ERα signaling has a tumor suppressive function in gliomas and thus ERα agonists may represent a novel class of drugs for curbing glioma progression. Since ERα agonists are currently in clinical trials and are well tolerated with fewer side effects, identification of an ERα agonist as a therapeutic agent can be readily extended to clinical use with current chemotherapies, providing an additional tool for enhancing survival in glioma patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 953. doi:1538-7445.AM2012-953

Hormonal therapy resistance is a major clinical problem. Emerging evidence suggests that estrogen (ER) participates in extra-nuclear signaling events in the cytoplasm / membrane and such actions may play a role in therapy resistance. Recent studies suggested that mTOR pathway play a critical role in ER-mediated cell proliferation and growth factor signaling crosstalk. Proline, Glutamic-acid and Leucine-rich Protein 1 (PELP1) participates in ER extra-nuclear actions. PELP1 is a prognostic indicator of shorter breast cancer specific survival and PELP1 expression is predominantly in the cytoplasm in a subset of breast tumors. The objective of this application is to test whether cross talk occurs between mTOR-PELP1 signaling axis and whether mTOR targeting drugs can be used to target PELP1 oncogenic functions leading to therapy resistance. We have tested this hypothesis using ER-positive breast cancer cells that over express PELP1 (MCF7-PELP1, ZR75-PELP1) and models cells lacking PELP1 (MCF7-PELP1 shRNA, ZR-75-PELP1 shRNA). Vector transfected ZR75 and MCF7 cells were used as controls. Rapamycin and AZD8055 were used as pharmacological inhibitors to block mTOR pathway. Over expression of PELP1 enhanced the estrogen and Heregulin mediated cell proliferation in breast cancer cells. Rapamycin (10−7M) or AZD8055 (10−8M) treatment significantly reduced PELP1 driven growth in both MCF7 and ZR75 cells that are treated with estrogen. Similarly, Rapamycin and AZD8055 treatment of MCF7 and ZR75 cells significantly reduced PELP1 mediated increase in cell growth of Heregulin stimulated cells. Mechanistic studies using yeast two hybrid screen and reciprocal immunoprecipitations demonstrated that PELP1 directly interacts with mTOR and PP2A. PELP1cyto model cells that uniquely express PELP1 in the cytoplasm, exhibit excessive activation of mTOR signaling pathway upon estrogen and growth factor stimulation. Over expression of PELP1 in the breast cancer model cells increased phosphorylation of mTOR axis components (phos-mTOR, -S6K, -4EBP1,-AKT). Further, Knock down of PELP1 with siRNA significantly reduced the activation of mTOR signaling components upon estrogen and Heregulin stimulation. Immunohistochenistry studies using xenograft tumor tissues with PELP1 overexpression and PELP1cyto driven tumors with PELP1 deregulation showed correlation of PELP1 expression with the activation of mTOR signaling components and that PELP1cyto driven tumors have excessive activation of mTOR signaling components. Combination therapy of Tamoxifen and Rapamycin or AZD8055, sensitized PELP1 deregulated cells to hormonal therapy. Our results suggest that PELP1 driven oncogenic functions involve PELP1 modulation of mTOR signaling and blockade of mTOR signaling render PELP1 driven tumors highly sensitive to therapeutic inhibition. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 958. doi:1538-7445.AM2012-958