miR-181a Promotes Multiple Protumorigenic Functions by Targeting TGFßR3.

Cutaneous squamous cell carcinoma (cSCC) comprises 15‒20% of all skin cancers and has a well-defined progression sequence from precancerous actinic keratosis to invasive cSCC. To identify targets for chemoprevention, we previously reported a cross-species analysis to identify the transcriptional drivers of cSCC development and identified miR-181a as a potential oncomiR. We show that the upregulation of miR-181a promotes multiple protumorigenic properties by targeting an understudied component of TGFß signaling, TGFßR3. miR-181a and TGFßR3 are upregulated and downregulated, respectively, in cSCC. miR-181a overexpression (OE) and TGFßR3 knockdown (KD) significantly suppresses UV-induced apoptosis in HaCaT cells and in primary normal human epidermal keratinocytes. In addition, OE of miR-181a or KD of TGFßR3 by short hairpin RNA enhances anchorage-independent survival. miR-181a OE or TGFßR3 KD enhances cellular migration and invasion and upregulation of epithelial‒mesenchymal transition markers. Luciferase reporter assays demonstrate that miR-181a directly targets the 3'-untranslated region of TGFßR3. miR-181a upregulates phosphorylated SMAD3 levels after TGFß2 administration and results in elevated SNAIL and SLUG expression. Finally, we confirm in vivo that miR-181a inhibition compromises tumor growth. Importantly, these phenotypes can be reversed with TGFßR3 OE or KD in the context of miR-181a OE or KD, respectively, further highlighting the physiologic relevance of this regulation in cSCC.

Apigenin Increases SHIP-1 Expression, Promotes Tumoricidal Macrophages and Anti-Tumor Immune Responses in Murine Pancreatic Cancer.

Pancreatic cancer (PC) has an extremely poor prognosis due to the expansion of immunosuppressive myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM) in the inflammatory tumor microenvironment (TME), which halts the recruitment of effector immune cells and renders immunotherapy ineffective. Thus, the identification of new molecular targets that can modulate the immunosuppressive TME is warranted for PC intervention. Src Homology-2 (SH2) domain-containing Inositol 5'-Phosphatase-1 (SHIP-1) is a lipid signaling protein and a regulator of myeloid cell development and function. Herein, we used the bioflavonoid apigenin (API) to reduce inflammation in different PC models. Wild type mice harboring heterotopic or orthotopic PC were treated with API, which induced SHIP-1 expression, reduced inflammatory tumor-derived factors (TDF), increased the proportion of tumoricidal macrophages and enhanced anti-tumor immune responses, resulting in a reduction in tumor burden compared to vehicle-treated PC mice. In contrast, SHIP-1-deficient mice exhibited an increased tumor burden and displayed augmented proportions of pro-tumor macrophages. These results provide further support for the importance of SHIP-1 expression in promoting pro-tumor macrophage development in the pancreatic TME. Our findings suggest that agents augmenting SHIP-1 expression may provide novel therapeutic options for the treatment of PC.

JNK2 Is Required for the Tumorigenic Properties of Melanoma Cells.

Overexpression and activation of c-Jun N-terminal kinases (JNKs) have been observed in multiple cancer cell lines and tumor samples. Various JNK isoforms have been reported to promote lung and liver cancer, as well as keratinocyte transformation, suggesting an important role of JNK signaling in promoting tumor development. However, there are three JNK isoforms, and it is unclear how each individual isoform, especially the ubiquitously expressed JNK1 and JNK2, functions in melanoma. Our previous study found that C116S mutations in both JNK1 and JNK2 rendered them insensitive to the covalent pan-JNK inhibitor JNK-IN-8 while retaining kinase activity. To delineate the specific roles of JNK1 and JNK2 in melanoma cell proliferation and invasiveness, we expressed the wild type (WT) and C116S mutants in melanoma cell lines and used JNK-IN-8 to enable chemical-genetic dissection of JNK1 and JNK2 activity. We found that the JNK2C116S allele consistently enhanced colony proliferation and cell invasiveness in the presence of JNK-IN-8. When cells individually expressing WT or C116S JNK1/2 were subcutaneously implanted into immunodeficient mice, we again found that bypass of JNK-IN-8-mediated inhibition of JNK signaling by expression of JNK2C116S specifically resulted in enhanced tumor growth in vivo. In addition, we observed a high level of JNK pathway activation in some human BRAF inhibitor (BRAFi) resistant melanoma cell lines relative to their BRAFi sensitive isogenic counterparts. JNK-IN-8 significantly enhanced the response to dabrafenib in resistant cells overexpressing JNK1WT, JNK2WT, and JNK1C116S but had no effect on cells expressing JNK2C116S, suggesting that JNK2 signaling is also crucial for BRAFi resistance in a subset of melanomas. Collectively, our data show that JNK2 activity is specifically required for melanoma cell proliferation, invasiveness, and BRAFi resistance and that this activity is most important in the context of JNK1 suppression, thus providing a compelling rationale for the development of JNK2 selective inhibitors as a potential therapy for the treatment of melanoma.