Loss of tumor suppressors promotes inflammatory tumor microenvironment and enhances LAG3+T cell mediated immune suppression.

Low response rate, treatment relapse, and resistance remain key challenges for cancer treatment with immune checkpoint blockade (ICB). Here we report that loss of specific tumor suppressors (TS) induces an inflammatory response and promotes an immune suppressive tumor microenvironment. Importantly, low expression of these TSs is associated with a higher expression of immune checkpoint inhibitory mediators. Here we identify, by using in vivo CRISPR/Cas9 based loss-of-function screening, that NF1, TSC1, and TGF-ß RII as TSs regulating immune composition. Loss of each of these three TSs leads to alterations in chromatin accessibility and enhances IL6-JAK3-STAT3/6 inflammatory pathways. This results in an immune suppressive landscape, characterized by increased numbers of LAG3+ CD8 and CD4 T cells. ICB targeting LAG3 and PD-L1 simultaneously inhibits metastatic progression in preclinical triple negative breast cancer (TNBC) mouse models of NF1-, TSC1- or TGF-ß RII- deficient tumors. Our study thus reveals a role of TSs in regulating metastasis via non-cell-autonomous modulation of the immune compartment and provides proof-of-principle for ICB targeting LAG3 for patients with NF1-, TSC1- or TGF-ß RII-inactivated cancers.

CCL9 Induced by TGFß Signaling in Myeloid Cells Enhances Tumor Cell Survival in the Premetastatic Organ.

Tumor cell survival in the hostile distant organ is a rate-limiting step in cancer metastasis. Bone marrow-derived myeloid cells can form a premetastatic niche and provide a tumor-promoting microenvironment. However, it is unclear whether these myeloid cells in the premetastatic site have any direct effect on tumor cell survival. Here, we report that chemokine CCL9 was highly induced in Gr-1(+)CD11b(+) immature myeloid cells and in premetastatic lung in tumor-bearing mice. Knockdown of CCL9 in myeloid cells decreased tumor cell survival and metastasis. Importantly, CCL9 overexpression in myeloid cells lacking TGFß signaling rescued the tumor metastasis defect observed in mice with myeloid-specific Tgfbr2 deletion. The expression level of CCL23, the human orthologue for CCL9, in peripheral blood mononuclear cells correlated with progression and survival of cancer patients. Our study demonstrates that CCL9 could serve as a good candidate for anti-metastasis treatment by targeting the rate-limiting step of cancer cell survival. In addition, targeting CCL9 may avoid the adverse effects of TGFß-targeted therapy.

Immunosuppressive and Prometastatic Functions of Myeloid-Derived Suppressive Cells Rely upon Education from Tumor-Associated B Cells.

Myeloid-derived suppressive cells (MDSC) have been reported to promote metastasis, but the loss of cancer-induced B cells/B regulatory cells (tBreg) can block metastasis despite MDSC expansion in cancer. Here, using multiple murine tumor models and human MDSC, we show that MDSC populations that expand in cancer have only partially primed regulatory function and limited prometastatic activity unless they are fully educated by tBregs. Cancer-induced tBregs directly activate the regulatory function of both the monocyte and granulocyte subpopulations of MDSC, relying, in part, on TgfßR1/TgfßR2 signaling. MDSC fully educated in this manner exhibit an increased production of reactive oxygen species and NO and more efficiently suppress CD4(+) and CD8(+) T cells, thereby promoting tumor growth and metastasis. Thus, loss of tBregs or TgfßR deficiency in MDSC is sufficient to disable their suppressive function and to block metastasis. Overall, our data indicate that cancer-induced B cells/B regulatory cells are important regulators of the immunosuppressive and prometastatic functions of MDSC.

G1/S arrest induced by histone deacetylase inhibitor sodium butyrate in E1A + Ras-transformed cells is mediated through down-regulation of E2F activity and stabilization of beta-catenin.

Tumor cells are often characterized by a high and growth factor-independent proliferation rate. We have previously shown that REF cells transformed with oncogenes E1A and c-Ha-Ras do not undergo G(1)/S arrest of the cell cycle after treatment with genotoxic factors. In this work, we used sodium butyrate, a histone deacetylase inhibitor, to show that E1A + Ras transformants were able to stop proliferation and undergo G(1)/S arrest. Apart from inducing G(1)/S arrest, sodium butyrate was shown to change expression of a number of cell cycle regulatory genes. It down-regulated cyclins D1, E, and A as well as c-myc and cdc25A and up-regulated the cyclin-kinase inhibitor p21(waf1). Accordingly, activities of cyclin E-Cdk2 and cyclin A-Cdk2 complexes in sodium butyrate-treated cells were decreased substantially. Strikingly, E2F1 expression was also down-modulated at the levels of gene transcription, the protein content, and the E2F transactivating capability. To further study the role of p21(waf1) in the sodium butyrate-induced G(1)/S arrest and the E2F1 down-modulation, we established E1A + Ras transformants from mouse embryo fibroblast cells with deletion of the cdkn1a (p21(waf1)) gene. Despite the absence of p21(waf1), sodium butyrate-treated mERas transformants reveal a slightly delayed G(1)/S arrest as well as down-modulation of E2F1 activity, implying that the observed effects are mediated through an alternative p21(waf1)-independent signaling pathway. Subsequent analysis showed that sodium butyrate induced accumulation of beta-catenin, a downstream component of the Wnt signaling. The results obtained indicate that the antiproliferative effect of histone deacetylase inhibitors on E1A + Ras-transformed cells can be mediated, alongside other mechanisms, through down-regulation of E2F activity and stabilization of beta-catenin.