Background:Meta-analysis shows that women with diabetes have a 20% increased risk of breast cancer and also an increased risk for distant metastasis and mortality. The molecular mechanisms for distant metastasis and mortality in breast cancer patients with diabetes are not very well understood.Methods:We compared the effect of physiological (5 mM) and diabetic (10 mM) levels of glucose on malignant breast epithelial cell invasion and stemness capabilities. We performed microRNA array to determine the dysregulated microRNAs in hyperglycaemic conditions and performed functional and molecular analysis of the gene targets.Results:Hyperglycaemia leads to hyperactivation of cancer stem cell pool and enhances invasive ability of breast cancer cells. MiR-424 seems to be a key regulator of cancer cell stemness and invasion. Knockdown of miR-424 in cancer cells under euglycaemic conditions leads to enhanced invasion and stem cell activity, whereas ectopic expression of miR-424 in cancer cells under hyperglycaemic conditions results in suppressed invasion and stem cell activity. Cdc42, a target of miR-424, influences cancer stem cell activity by positively regulating prdm14 through activation of pak1 (p-21-activated kinase 1) and stat5.Conclusions:Our findings establish miR-424→cdc42→prdm14 axis as a key molecular signalling cascade that might influence breast cancer progression in diabetic patients through hyperactivation of cancer stem cells.

Accumulation of reactive oxygen species (ROS) has been implicated in induction of apoptosis and regulation of key signaling molecules in cancer cells. Phytochemicals are potent source of anticancer drugs as wells as potential inducers of ROS. Neem (Azadirachta indica) is a medicinal plant used for the treatment of various diseases. The main objective of this study is to investigate the anticancer effect of desacetyl nimbinene (DAN; an active ingredient of neem) against breast cancer. Normal and breast cancer cell lines were used for the study. The effect of DAN on cell proliferation, apoptosis, ROS generation, migration, and invasion was analyzed. Antioxidant enzymes superoxide dismutase (SOD)1 and SOD2 were overexpressed to test the effect of DAN-induced ROS generation on breast cancer growth. Key survival and apoptotic protein markers were analyzed to validate the anticancer effect of DAN. Our data demonstrated that DAN inhibited the growth of breast cancer cells by inducing ROS generation. Further investigations revealed that DAN treatment lead to the loss of mitochondrial membrane potential resulting in mitochondria-dependent apoptotic cell death. Increased phosphorylation of c-Jun-N-terminal kinase (JNK) and reduced phosphorylation of p38 were also observed in response to DAN treatment. Inhibition of ROS production by overexpressing antioxidant enzymes SOD1 and SOD2 reduced the DAN-induced cytotoxicity. Additionally, DAN significantly inhibited migration and invasion of MDA-MB-231 breast cancer cells. Overall, our data suggest that DAN exerts its anticancer effect on breast cancer by induction of mitochondria-mediated apoptosis mediated by ROS accumulation.

Introduction: Meta-analysis shows women with diabetes have a 20% increased risk of breast cancer. Diabetes increases the risk of breast cancer mortality. Additionally, patients with breast cancer and preexisting diabetes have an increased risk for distant metastasis compared with non-diabetic counterparts. The molecular mechanisms for distant metastasis in breast cancer patients with diabetes are not very well understood. Our preliminary data suggested that hyperglycemia enhances stem cell activity in triple negative breast cancer (TNBC) cells. Here, we report miR-424 to be a key regulator of breast cancer stem cell pool dynamics under hyperglycemic environment. Materials and Methods: MDA-MB-231, TNBC cell line was maintained under euglycemic (5mM) and hyperglycemic (10mM) culture conditions. RT-PCR based microRNA array was performed followed by validation of significantly altered microRNA to confirm the effect of hyperglycemia on the global microRNA profile. Flow cytometric analysis for CD44+/CD24- was performed to assess the alterations in breast cancer stem cell population. In addition, sphere-forming assay were also performed. Mir-424 over-expressing or knocked down cell lines were established from parental MDA-MB-231 cells and maintained in hyperglycemic or euglycemic condition respectively. Promoter analysis for PR/SET domain 14 (prdm14) was done. Western blotting and immunofluorescence assays were performed for cdc42 (cell division control 42), pStat5 (phospho signal transducer and activator of transcription factor 5) and prdm14. Results: Loss of miR-424 in TNBC cells under euglycemic conditions led to enhanced stem cell activity; whereas over expression of miR- 424 under hyperglycemic conditions resulted in suppressed stem cell activity. Introduction of cdc42, (a miR-424 target gene) in miR-424 over expressing TNBC cells under hyperglycemic conditions leads to increased stem cell activity demonstrating the significance of miR-424-cdc42 signaling in hyperglycemia. Over-expression of miR-424 in TNBC dramatically reduced its metastatic abilities in vivo. Mechanistically, we found that miR-424-cdc42 signaling in hyperglycemia promotes Prdm14 activation, a stem cell regulator through increased phosphorylation of Stat5. Conclusions: Our findings establish a key molecular signaling cascade (miR-424→cdc42→prdm14) that promotes TNBC stem cell activity under hyperglycemic conditions. Citation Format: Sushmita B. Nandy, Alexis Orozco, Gautham Prabhakar, Viktoria Stewart, Stephanie Jones, Paloma Munoz, Ramadevi Subramani, Diego Pedroza, Rajkumar Lakshmanaswamy. miR-424-cdc42, key signaling axis in hyperglycemic regulation of stemness in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1461. doi:10.1158/1538-7445.AM2017-1461