Mary Zimmerman

Butyrate suppresses colonic inflammation through HDAC1-dependent Fas upregulation and Fas-mediated apoptosis of T cells

Butyrate, an intestinal microbiota metabolite of dietary fiber, has been shown to exhibit protective effects toward inflammatory diseases such as ulcerative colitis (UC) and inflammation-mediated colorectal cancer. Recent studies have shown that chronic IFN-γ signaling plays an essential role in inflammation-mediated colorectal cancer development in vivo, whereas genome-wide association studies have linked human UC risk loci to IFNG, the gene that encodes IFN-γ. However, the molecular mechanisms underlying the butyrate-IFN-γ-colonic inflammation axis are not well defined. Here we showed that colonic mucosa from patients with UC exhibit increased signal transducer and activator of transcription 1 (STAT1) activation, and this STAT1 hyperactivation is correlated with increased T cell infiltration. Butyrate treatment-induced apoptosis of wild-type T cells but not Fas-deficient (Fas(lpr)) or FasL-deficient (Fas(gld)) T cells, revealing a potential role of Fas-mediated apoptosis of T cells as a mechanism of butyrate function. Histone deacetylase 1 (HDAC1) was found to bind to the Fas promoter in T cells, and butyrate inhibits HDAC1 activity to induce Fas promoter hyperacetylation and Fas upregulation in T cells. Knocking down gpr109a or slc5a8, the genes that encode for receptor and transporter of butyrate, respectively, resulted in altered expression of genes related to multiple inflammatory signaling pathways, including inducible nitric oxide synthase (iNOS), in mouse colonic epithelial cells in vivo. Butyrate effectively inhibited IFN-γ-induced STAT1 activation, resulting in inhibition of iNOS upregulation in human colon epithelial and carcinoma cells in vitro. Our data thus suggest that butyrate delivers a double-hit: induction of T cell apoptosis to eliminate the source of inflammation and suppression of IFN-γ-mediated inflammation in colonic epithelial cells, to suppress colonic inflammation.

Decitabine and Vorinostat Cooperate To Sensitize Colon Carcinoma Cells to Fas Ligand-Induced Apoptosis In Vitro and Tumor Suppression In Vivo

The death receptor Fas and its physiological ligand (FasL) regulate apoptosis of cancerous cells, thereby functioning as a critical component of the host cancer immunosurveillance system. To evade Fas-mediated apoptosis, cancer cells often downregulate Fas to acquire an apoptosis-resistant phenotype, which is a hallmark of metastatic human colorectal cancer. Therefore, targeting Fas resistance is of critical importance in Fas-based cancer therapy and immunotherapy. In this study, we demonstrated that epigenetic inhibitors decitabine and vorinostat cooperate to upregulate Fas expression in metastatic human colon carcinoma cells. Decitabine also upregulates BNIP3 and Bik expression, whereas vorinostat decreased Bcl-xL expression. Altered expression of Fas, BNIP3, Bik, and Bcl-xL resulted in effective sensitization of the metastatic human colon carcinoma cells to FasL-induced apoptosis. Using an experimental metastasis mouse model, we further demonstrated that decitabine and vorinostat cooperate to suppress colon carcinoma metastasis. Analysis of tumor-bearing lung tissues revealed that a large portion of tumor-infiltrating CD8+ T cells are FasL+, and decitabine and vorinostat-mediated tumor-suppression efficacy was significantly decreased in Fasgld mice compared with wild-type mice, suggesting a critical role for FasL in decitabine and vorinostat-mediated tumor suppression in vivo. Consistent with their function in apoptosis sensitization, decitabine and vorinostat significantly increased the efficacy of CTL adoptive transfer immunotherapy in an experimental metastasis mouse model. Thus, our data suggest that combined modalities of chemotherapy to sensitize the tumor cell to Fas-mediated apoptosis and CTL immunotherapy is an effective approach for the suppression of colon cancer metastasis.

TNFα Cooperates with IFN-γ to Repress Bcl-xL Expression to Sensitize Metastatic Colon Carcinoma Cells to TRAIL-mediated Apoptosis

Background TNF-related apoptosis-inducing ligand (TRAIL) is an immune effector molecule that functions as a selective anti-tumor agent. However, tumor cells, especially metastatic tumor cells often exhibit a TRAIL-resistant phenotype, which is currently a major impediment in TRAIL therapy. The aim of this study is to investigate the synergistic effect of TNFα and IFN-γ in sensitizing metastatic colon carcinoma cells to TRAIL-mediated apoptosis. Methodology/Principal Findings The efficacy and underlying molecular mechanism of cooperation between TNFα and IFN-γ in sensitizing metastatic colon carcinoma cells to TRAIL-mediated apoptosis were examined. The functional significance of TNFα- and IFN-γ-producing T lymphocyte immunotherapy in combination with TRAIL therapy in suppression of colon carcinoma metastasis was determined in an experimental metastasis mouse model. We observed that TNFα or IFN-γ alone exhibits minimal sensitization effects, but effectively sensitized metastatic colon carcinoma cells to TRAIL-induced apoptosis when used in combination. TNFα and IFN-γ cooperate to repress Bcl-xL expression, whereas TNFα represses Survivin expression in the metastatic colon carcinoma cells. Silencing Bcl-xL expression significantly increased the metastatic colon carcinoma cell sensitivity to TRAIL-induced apoptosis. Conversely, overexpression of Bcl-xL significantly decreased the tumor cell sensitivity to TRAIL-induced apoptosis. Furthermore, TNFα and IFN-γ also synergistically enhanced TRAIL-induced caspase-8 activation. TNFα and IFN-γ was up-regulated in activated primary and tumor-specific T cells. TRAIL was expressed in tumor-infiltrating immune cells in vivo, and in tumor-specific cytotoxic T lymphocytes (CTL) ex vivo. Consequently, TRAIL therapy in combination with TNFα/IFN-γ-producing CTL adoptive transfer immunotherapy effectively suppressed colon carcinoma metastasis in vivo. Conclusions/Significance TNFα and IFN-γ cooperate to overcome TRAIL resistance at least partially through enhancing caspase 8 activation and repressing Bcl-xL expression. Combined CTL immunotherapy and TRAIL therapy hold great promise for further development for the treatment of metastatic colorectal cancer.

Unphosphorylated STAT1 Promotes Sarcoma Development through Repressing Expression of Fas and Bad and Conferring Apoptotic Resistance

STAT1 exists in phosphorylated (pSTAT1) and unphosphorylated (uSTAT1) forms each regulated by IFN-γ. Although STAT1 is a key mediator of the IFN-γ signaling pathway, an essential component of the host cancer immunosurveillance system, STAT1 is also overexpressed in certain human cancers where the functions of pSTAT1 and uSTAT1 are ill defined. Using a murine model of soft tissue sarcoma (STS), we show that disruption of the IFN effector molecule IRF8 decreases pSTAT1 and increases uSTAT1 in STS cells, thereby increasing their metastatic potential. We determined that the IRF8 gene promoter was hypermethylated frequently in human STS. An analysis of 123 human STS specimens revealed that high uSTAT1 levels in tumor cells was correlated with a reduction in disease-specific survival (DSS), whereas high pSTAT1 levels in tumor cells were correlated with an increase in DSS. In addition, uSTAT1 levels were negatively correlated with pSTAT1 levels in these STS specimens. Mechanistic investigations revealed that IRF8 suppressed STAT1 transcription by binding the STAT1 promoter. RNAi-mediated silencing of STAT1 in STS cells was sufficient to increase expression of the apoptotic mediators Fas and Bad and to elevate the sensitivity of STS cells to Fas-mediated apoptosis. Together, our findings show how the phosphorylation status of pSTAT1 determines its function as a tumor suppressor, with uSTAT1 acting as a tumor promoter that acts by elevating resistance to Fas-mediated apoptosis to promote immune escape.

IFN-γ upregulates survivin and Ifi202 expression to induce survival and proliferation of tumor-specific T cells.

Background A common procedure in human cytotoxic T lymphocyte (CTL) adoptive transfer immunotherapy is to expand tumor-specific CTLs ex vivo using CD3 mAb prior to transfer. One of the major obstacles of CTL adoptive immunotherapy is a lack of CTL persistence in the tumor-bearing host after transfer. The aim of this study is to elucidate the molecular mechanisms underlying the effects of stimulation conditions on proliferation and survival of tumor-specific CTLs. Methodology/Principal Findings Tumor-specific CTLs were stimulated with either CD3 mAb or cognate Ag and analyzed for their proliferation and survival ex vivo and persistence in tumor-bearing mice. Although both Ag and CD3 mAb effectively induced the cytotoxic effecter molecules of the CTLs, we observed that Ag stimulation is essential for sustained CTL proliferation and survival. Further analysis revealed that Ag stimulation leads to greater proliferation rates and less apoptosis than CD3 mAb stimulation. Re-stimulation of the CD3 mAb-stimulated CTLs with Ag resulted in restored CTL proliferative potential, suggesting that CD3 mAb-induced loss of proliferative potential is reversible. Using DNA microarray technology, we identified that survivin and ifi202, two genes with known functions in T cell apoptosis and proliferation, are differentially induced between Ag- and CD3 mAb-stimulated CTLs. Analysis of the IFN-γ signaling pathway activation revealed that Ag stimulation resulted in rapid phosphorylation of STAT1 (pSTAT1), whereas CD3 mAb stimulation failed to activate STAT1. Chromatin immunoprecipitation revealed that pSTAT1 is associated with the promoters of both survivin and ifi202 in T cells and electrophoresis mobility shift assay indicated that pSTAT1 directly binds to the gamma activation sequence element in the survivin and ifi202 promoters. Finally, silencing ifi202 expression significantly decreased T cell proliferation. Conclusions/Significance Our findings delineate a new role of the IFN-γ signaling pathway in regulating T cell proliferation and apoptosis through upregulating survivin and ifi202 expression.

Ceramide targets xIAP and cIAP1 to sensitize metastatic colon and breast cancer cells to apoptosis induction to suppress tumor progression

BackgroundCeramide is a bioeffector that mediates various cellular processes, including apoptosis. However, the mechanism underlying ceramide function in apoptosis is apparently cell type-dependent and is not well-understood. We aimed at identifying molecular targets of ceramide in metastatic human colon and breast cancer cells, and determining the efficacy of ceramide analog in suppression of colon and breast cancer metastasis.MethodsThe activity of and mechanism underlying ceramide as a cytotoxic agent, and as a sensitizer for Fas-mediated apoptosis was analyzed in human cell lines established from primary or metastatic colon and breast cancers. The efficacy of ceramide analog LCL85 in suppression of metastasis was examined in preclinical mouse tumor models.ResultsExposure of human colon carcinoma cells to ceramide analog LCL85 results in apoptosis in a dose-dependent manner. Interestingly, a sublethal dose of LCL85 increased C16 ceramide content and overcame tumor cell resistance to Fas-mediated apoptosis. Subsequently, treatment of tumor cells with exogenous C16 ceramide resulted in increased tumor cell sensitivity to Fas-mediated apoptosis. LCL85 resembles Smac mimetic BV6 in sensitization of colon carcinoma cells to Fas-mediated apoptosis by inducing proteasomal degradation of cIAP1 and xIAP proteins. LCL85 also decreased xIAP1 and cIAP1 protein levels and sensitized metastatic human breast cancer cells to Fas-mediated apoptosis. Silencing xIAP and cIAP1 with specific siRNAs significantly increased the metastatic human colon carcinoma cell sensitivity to Fas-mediated apoptosis, suggesting that IAP proteins mediate apoptosis resistance in metastatic human colon carcinoma cells and ceramide induces IAP protein degradation to sensitize the tumor cells to apoptosis induction. Consistent with its apoptosis sensitization activity, subtoxic doses of LCL85 suppressed colon carcinoma cell metastatic potential in an experimental lung metastasis mouse model, as well as breast cancer growth and spontaneous lung metastasis in an orthotopic breast cancer mouse model.ConclusionWe have identified xIAP and cIAP1 as molecular targets of ceramide and determined that ceramide analog LCL85 is an effective sensitizer in overcoming resistance of human cell lines established from metastatic colon and breast cancers to apoptosis induction to suppress metastasis in vivo.

Cutting Edge: IRF8 Regulates Bax Transcription In Vivo in Primary Myeloid Cells

A prominent phenotype of IRF8 knockout (KO) mice is the uncontrolled expansion of immature myeloid cells. The molecular mechanism underlying this myeloproliferative syndrome is still elusive. In this study, we observed that Bax expression level is low in bone marrow preginitor cells and increases dramatically in primary myeloid cells in wt mice. In contrast, Bax expression level remained at a low level in primarymyeloid cells in IRF8 KO mice. However, in vitro IRF8 KO bone marrow-differentiated myeloid cells expressed Bax at a level as high as that in wild type myeloid cells. Furthermore, we demonstrated that IRF8 specifically binds to the Bax promoter region in primary myeloid cells. Functional analysis indicated that IRF8 deficiency results in increased resistance of the primary myeloid cells to Fas-mediated apoptosis. Our findings show that IRF8 directly regulates Bax transcription in vivo, but not in vitro during myeloid cell lineage differentiation.

Lymphotoxin β receptor mediates caspase-dependent tumor cell apoptosis in vitro and tumor suppression in vivo despite induction of nf-κb activation

Ligation of the lymphotoxin β receptor (LTβR) has been shown to induce both tumor growth inhibition and promotion. The functions of LTβR in these two contrasting cellular processes require further study. We demonstrated here that mice deficient in LTβ R ligands, LTα, LIGHT or both LTβ and LIGHT, exhibit greater susceptibility to methylcholanthrene-induced tumor development. LTα, LTβ and LIGHT were expressed in tumor-infiltrating immune cells, and LTβR was expressed on human colon carcinoma and soft tissue sarcoma (STS) cells. Human LTβR agonist monoclonal antibody (mAb) BS-1 induced both growth inhibition and NF-κB activation in human colon carcinoma, mammary carcinoma and STS cells. Interestingly, BS-1 also significantly inhibited growth of doxorubicin-resistant and radiation-resistant human STS cells in vitro. In the molecular mechanism level, we demonstrated that BS-1 induces caspases 8 and 3 activation and cytochrome c release in tumor cells, suggesting that the LTβR mediates apoptosis at least partially through a caspase-dependent mechanism. Furthermore, mouse LTβR mAb ACH6 suppressed colon carcinoma cell metastatic potential in an experimental metastasis mouse model. Although blocking NF-κB activation did not alter tumor cell growth rate and tumor cell response to LTβR mAb-induced growth inhibition in vitro, surprisingly, blocking NF-κB activation significantly enhanced colon carcinoma cell metastatic potential in vivo, suggesting that the LTβR-mediated apoptosis pathway and NF-κB signaling pathway might cooperate to suppress tumor growth in vivo. In summary, our findings deter mine that LTβR mediates tumor cell apoptosis in colon carcinoma, mammary carcinoma and sarcoma and that LTβR-activated NF-κB potentially functions as a tumor suppressor.

Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into mouse tail veins and colonize in the lungs, thereby, resembling the last steps of tumor cell spontaneous metastasis: survival in the circulation, extravasation and colonization in the distal organs. From a therapeutic point of view, the experimental metastasis model is the simplest and ideal model since the target of therapies is often the end point of metastasis: established metastatic tumor in the distal organ. In this model, tumor cells are injected i.v into mouse tail veins and allowed to colonize and grow in the lungs. Tumor-specific CTLs are then injected i.v into the metastases-bearing mouse. The number and size of the lung metastases can be controlled by the number of tumor cells to be injected and the time of tumor growth. Therefore, various stages of metastasis, from minimal metastasis to extensive metastasis, can be modeled. Lung metastases are analyzed by inflation with ink, thus allowing easier visual observation and quantification.

Abstract PR05: IRF8 regulates GM-CSF expression in T cells and tumor cells to mediate myeloid-derived suppressor cell differentiation

Myeloid cells are a heterogenous and abundant population of haematopoietic cells that are virtually present in all mammalian tissues, where they monitor local microenvironment to maintain homeostasis. All myeloid cells originate from the pluripotent hematopoietic stem cells that undergo progressive restriction in their lineage potential to give rise to mature granulocytes and macrophages. Lineage restriction and differentiation are regulated by timely activation of specific set of lineage-specific transcription factors in concert with down-regulation of other set(s) of transcription factors that are important for alternative cell lineage potential. Altered expression of these lineage-specific transcription factors often leads to deregulation of myelopoiesis and resultant hematopoietic disorders. Therefore, lineage-specific transcription factors are essential for myeloid cell lineage differentiation and maturation. Mice with a null mutation of irf8, the gene that encodes IFN regulatory factor 8 (IRF8), exhibit massive accumulation of CD11b+Gr1+ immature myeloid cells (IMCs). Therefore, IRF8 is a myeloid cell lineage-specific transcription factor that plays an essential function in the regulation of myelopoiesis. Particularly, IRF8 may determine differentiation, lineage commitment, and immune function of monocytes versus granulocytes under physiological conditions. A hallmark of cancer-bearing mice is the accumulation of CD11b+Gr1+ myeloid-derived suppressor cells (MDSCs). Interestingly, IRF8 is silenced in MDSCs from tumor-bearing mice. Therefore, IRF8 is apparently a key transcription factor that mediates MDSC differentiation. However, the molecular mechanism underlying IRF8 regulation of MDSCs is largely unknown. Because MDSCs is induced by inflammation, we therefore hypothesized that IRF8 may repress the expression of proinflammatory factors to mediate differentiation of MDSCs/IMCs under physiological and pathological conditions. To test this hypothesis, we made use of conventional IRF8 KO mice, mice with IRF8 deficiency only in myeloid cells, mice with IRF8 deficiency only in T cells, and tumor-bearing mouse models. Here we report an intriguing finding that although IRF8 conventional mice exhibit deregulated myeloid cell differentiation and resultant accumulation of CD11b+Gr1+ IMCs, surprisingly, mice with IRF8 deficiency only in myeloid cells exhibit normal myeloid cell lineage differentiation. Instead, mice with IRF8 deficiency only in T cells exhibited deregulated myeloid cell differentiation and IMC accumulation. We further demonstrated that IRF8-deficient T cells exhibit elevated GM-CSF expression and secretion. Treatment of mice with GM-CSF increased IMC accumulation, and adoptive transfer of IRF8-deficient T cells, but not GM-CSF-deficient T cells, increased IMC accumulation in the recipient chimera mice. Moreover, overexpression of IRF8 decreased GM-CSF expression in T cells. These data thus determine that IRF8 functions in T cells to repress GM-CSF expression to suppress IMCs. However, in tumor-bearing mice, IRF8 is silenced in MDSCs but not in T cells, suggesting a different mechanism of MDSC regulation by IRF8. We observed that silencing IRF8 using IRF8-specific siRNA dramatically increase GM-CSF expression in tumor cells. Therefore, IRF8 represses GM-CSF expression in tumor cells to mediate MDSC differentiation. In summary, we determine that IRF8 regulates GM-CSF expression in T cells and tumor cells, respectively, to mediate myelopoiesis under physiological and pathological conditions. This abstract is also presented as Poster A84. Citation Format: Amy Paschall, Ruihua Zhang, Kankana Bardhan, Chen-Feng Qi, Liang Peng, Geming Lu, Jianjun Yang, Miriam Merad, Mary Zimmerman, Tracy McGaha, Gang Zhou, Andrew Mellor, Scott I. Abrams, Herbert Morse, Keiko Ozato, Huabao Xiong, Kebin Liu. IRF8 regulates GM-CSF expression in T cells and tumor cells to mediate myeloid-derived suppressor cell differentiation. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr PR05.