整合素alpha6beta4刺激葡萄糖代谢和NAD(H)产生以驱动三阴性乳腺癌细胞的侵袭性生长

Integrin alpha6beta4, a laminin receptor preferentially expressed on cells of epithelial origin, is highly expressed in over 80% of TNBC cases and contributes to the aggressive nature of this deadly breast cancer subtype. We recently demonstrated that integrin alpha6beta4 enhances Hif-1alpha nuclear accumulation, suggesting its role in a pseudohypoxia signature. Hif-1alpha reprograms cellular metabolic processes, such as glycolysis, as a hallmark of cancer. However, whether glucose metabolism is part of this signature and how integrin alpha6beta4 impacts TNBC glucose metabolism remain unclear. To examine how integrin alpha6beta4 signaling impacts glycolysis, we first applied a Seahorse XF glycolysis stress test to measure the extracellular acidification rate in BT549 cells that stably express integrin beta4 (BT549-beta4) or an empty vector (BT549-EV). The BT549-beta4 cells had significantly increased glycolysis and glycolytic capacity. Next, we used 13 C 6 glucose stable isotope resolved metabolomics to define how integrin alpha6beta4 impacts glucose metabolic pathways. Integrin alpha6beta4 overexpression increased glucose uptake and shunted it into the pentose phosphate pathway compared to control, resulting in increased production of ribose phosphate. Interestingly, the increased ribose phosphate production in BT549-beta4 cells was used for ATP synthesis, which is a precursor of NAD, both of which are essential coenzymes and co-substrates for TNBC metabolism. We then measured the expression levels of key proteins in the glycolysis pathway and found that glucose transporters (Glut1 and Glut3) and several enzymes, such as hexokinases and LDHA, are upregulated by integrin alpha6beta4. We further validated that integrin alpha6beta4 promotes invasive growth and upregulates hexokinase I and II protein levels using the EMT6 syngeneic model (EV vs beta4). To assess the impact of integrin alpha6beta4-promoted glucose metabolism on cell proliferation and invasive growth, we treated EMT6 cells (EV vs. beta4) with various doses of the glucose analog, 2-DG, and monitored cell viability by MTT assays. The results demonstrated that EMT6-beta4 cells are more sensitive to 2-DG compared to EMT6-EV cells. Similar results were obtained from BT549 cells grown in 3D. Culturing BT549 (EV vs. beta4) and MDA-MB-231 (control vs. beta4 KO) cells in low vs. high glucose media, we found that integrin alpha6beta4-driven cell proliferation depends on the levels of glucose. Using shRNAs and specific inhibitors, we found glucose transporters are regulated through integrin alpha6beta4 signaling-driven Hif-1alpha and KLF5 activation. We further found that integrin alpha6beta4 increased AMPK activation under energy stress and the NAD + /NADH ratio in cells is significantly decreased by integrin alpha6beta4 signaling. In summary, our data highlights a novel function of integrin alpha6beta4 in altering glucose metabolism and the significant impact on the dynamics of NAD + /NADH to promote an invasive phenotype of TNBC cells.