Tumor Cell-Intrinsic c-Myb Upregulation Stimulates Antitumor Immunity in a Murine Colorectal Cancer Model.

The transcription factor c-Myb is overexpressed in many different types of solid tumors, including colorectal cancer. However, its exact role in tumorigenesis is unclear. In this study, we show that tumor-intrinsic c-Myb expression in mouse models of colon cancer and melanoma suppresses tumor growth. Although no differences in proliferation, apoptosis, and angiogenesis of tumors were evident in tumors with distinct levels of c-Myb expression, we observed changes in intratumoral immune cell infiltrates. MC38 tumors with upregulated c-Myb expression showed increased numbers of CD103+ dendritic cells and eosinophils, but decreased tumor-associated macrophages (TAM). Concomitantly, an increase in the number of activated cytotoxic CD8+ T cells upon c-Myb upregulation was observed, which correlated with a pro-inflammatory tumor microenvironment and increased numbers of M1 polarized TAMs. Mechanistically, c-Myb upregulation in immunogenic MC38 colon cancer cells resulted in enhanced expression of immunomodulatory genes, including those encoding ß2-microglobulin and IFNß, and decreased expression of the gene encoding the chemokine receptor CCR2. The increased numbers of activated cytotoxic CD8+ T cells contributed to tumor growth attenuation. In poorly immunogenic CT26, LLC, and B16-BL6 tumor cells, c-Myb upregulation did not affect the immunomodulatory gene expression. Despite this, c-Myb upregulation led to reduced B16-BL6 tumor growth but it did not affect tumor growth of CT26 and LLC tumors. Altogether, we postulate that c-Myb functions as a tumor suppressor in a tumor cell-type specific manner and modulates antitumor immunity.

Identification of TPM2 and CNN1 as Novel Prognostic Markers in Functionally Characterized Human Colon Cancer-Associated Stromal Cells.

Stromal infiltration is associated with poor prognosis in human colon cancers. However, the high heterogeneity of human tumor-associated stromal cells (TASCs) hampers a clear identification of specific markers of prognostic relevance. To address these issues, we established short-term cultures of TASCs and matched healthy mucosa-associated stromal cells (MASCs) from human primary colon cancers and, upon characterization of their phenotypic and functional profiles in vitro and in vivo, we identified differentially expressed markers by proteomic analysis and evaluated their prognostic significance. TASCs were characterized by higher proliferation and differentiation potential, and enhanced expression of mesenchymal stem cell markers, as compared to MASCs. TASC triggered epithelial-mesenchymal transition (EMT) in tumor cells in vitro and promoted their metastatic spread in vivo, as assessed in an orthotopic mouse model. Proteomic analysis of matched TASCs and MASCs identified a panel of markers preferentially expressed in TASCs. The expression of genes encoding two of them, calponin 1 (CNN1) and tropomyosin beta chain isoform 2 (TPM2), was significantly associated with poor outcome in independent databases and outperformed the prognostic significance of currently proposed TASC markers. The newly identified markers may improve prognostication of primary colon cancers and identification of patients at risk.

Commensal Clostridiales strains mediate effective anti-cancer immune response against solid tumors.

Despite overall success, T cell checkpoint inhibitors for cancer treatment are still only efficient in a minority of patients. Recently, intestinal microbiota was found to critically modulate anti-cancer immunity and therapy response. Here, we identify Clostridiales members of the gut microbiota associated with a lower tumor burden in mouse models of colorectal cancer (CRC). Interestingly, these commensal species are also significantly reduced in CRC patients compared with healthy controls. Oral application of a mix of four Clostridiales strains (CC4) in mice prevented and even successfully treated CRC as stand-alone therapy. This effect depended on intratumoral infiltration and activation of CD8+ T cells. Single application of Roseburia intestinalis or Anaerostipes caccae was even more effective than CC4. In a direct comparison, the CC4 mix supplementation outperformed anti-PD-1 therapy in mouse models of CRC and melanoma. Our findings provide a strong preclinical foundation for exploring gut bacteria as novel stand-alone therapy against solid tumors.

Tumor cell endogenous HIF-1α activity induces aberrant angiogenesis and interacts with TRAF6 pathway required for colorectal cancer development.

Hypoxia and inflammation are key factors for colorectal cancer tumorigenesis. The colonic epithelium belongs to the tissues with the lowest partial pressure of oxygen in the body, and chronic inflammation is associated with an increased chance to develop colon cancer. How the colonic epithelium responds to hypoxia and inflammation during tumorigenesis remains to be elucidated. Here we show, that murine colon adenocarcinoma cells with attenuated response to hypoxia, due to a knock-down (KD) of HIF-1α, produce smaller and less hypoxic tumors in an orthotopic mouse model when compared to tumors induced with control cells. HIF-1α-KD tumors showed more functional perfused vasculature associated with increased levels of vessel-stabilizing factors and reduced levels of proangiogenic factors, including extracellular matrix protein Cyr61/CCN1. Intratumoral injection of Cyr61 in HIF-1α-KD tumors revealed an in increased vessel permeability and tumor hypoxia. Further bioinformatics analysis identified a possible interaction between HIF-1α and TRAF6, an upstream effector of the NF-κB pathway that was confirmed by coimmunoprecipitation in MC-38 and CT26 colon adenocarcinoma cells and in situ by proximity ligation assay. Down-regulation of TRAF6 resulted in virtual abrogation of orthotopic tumor growth. Subcutaneous TRAF6-KD tumors were smaller and contained reduced vessel size and differently polarized macrophages. These data demonstrate that the tumor cell response to increased hypoxia in the colon leads to promotion of nonfunctional angiogenesis, regulated by both hypoxia and TRAF6 pathways.

CCL2 Is a Vascular Permeability Factor Inducing CCR2-Dependent Endothelial Retraction during Lung Metastasis.

Increased levels of the chemokine CCL2 in cancer patients are associated with poor prognosis. Experimental evidence suggests that CCL2 correlates with inflammatory monocyte recruitment and induction of vascular activation, but the functionality remains open. Here, we show that endothelial Ccr2 facilitates pulmonary metastasis using an endothelial-specific Ccr2-deficient mouse model (Ccr2ecKO). Similar levels of circulating monocytes and equal leukocyte recruitment to metastatic lesions of Ccr2ecKO and Ccr2fl/fl littermates were observed. The absence of endothelial Ccr2 strongly reduced pulmonary metastasis, while the primary tumor growth was unaffected. Despite a comparable cytokine milieu in Ccr2ecKO and Ccr2fl/fl littermates the absence of vascular permeability induction was observed only in Ccr2ecKO mice. CCL2 stimulation of pulmonary endothelial cells resulted in increased phosphorylation of MLC2, endothelial cell retraction, and vascular leakiness that was blocked by an addition of a CCR2 inhibitor. These data demonstrate that endothelial CCR2 expression is required for tumor cell extravasation and pulmonary metastasis. IMPLICATIONS: The findings provide mechanistic insight into how CCL2-CCR2 signaling in endothelial cells promotes their activation through myosin light chain phosphorylation, resulting in endothelial retraction and enhanced tumor cell migration and metastasis.

Hypoxia attenuates the proinflammatory response in colon cancer cells by regulating IκB.

Two main features common to all solid tumors are tissue hypoxia and inflammation, both of which cause tumor progression, metastasis, therapy resistance and increased mortality. Chronic inflammation is associated with increased cancer risk, as demonstrated for inflammatory bowel disease patients developing colon cancer. However, the interplay between hypoxia and inflammation on the molecular level remains to be elucidated. We found that MC-38 mouse colon cancer cells contain functional hypoxic (HIF-1α) and inflammatory (p65/RelA) signaling pathways. In contrast to cells of the myeloid lineage, HIF-1α levels remained unaffected in MC-38 cells treated with LPS, and hypoxia failed to induce NF-κB. A similar regulation of canonical HIF and NF-κB target genes confirmed these results. RNA deep sequencing of HIF-1α and p65/RelA knock-down cells revealed that a surprisingly large fraction of HIF target genes required p65/RelA for hypoxic regulation and a number of p65/RelA target genes required HIF-1α for proinflammatory regulation, respectively. Hypoxia attenuated the inflammatory response to LPS by inhibiting nuclear translocation of p65/RelA independently of HIF-1α, which was associated with enhanced IκBα levels and decreased IKKß phosphorylation. These data demonstrate that the interaction between hypoxic and inflammatory signaling pathways needs to be considered when designing cancer therapies targeting HIF or NF-κB.