PDF file - 431K, Histological analysis of in-vivo LC3 expression in HS766T subcutaneous tumors buffered with sodium bicarbonate. (A) Whole cell lysates from HS766T cells cultured at neutral pH 7.4 or pH 6.7 for 48 hours were analyzed for LC3-II expression. (B) Positive pixel analysis was completed for LC3 staining on whole tissue cross sections from HS766T tumors treated with tap or NaHCO3. An overall significant decrease in total positive and strong positive LC3 pixels was observed in NaHCO3 treated samples. The data are plotted as the mean � standard deviation of three whole tumor cross sections from each treatment group. (C) A 5x magnification of representative tissue regions from HS766T tumors stained for LC3
ARTICLE ABSTRACT
Tumor cell survival relies upon adaptation to the acidic conditions of the tumor microenvironment. To investigate potential acidosis survival mechanisms, we examined the effect of low pH (6.7) on human breast carcinoma cells. Acute low pH exposure reduced proliferation rate, induced a G1 cell cycle arrest, and increased cytoplasmic vacuolization. Gene expression analysis revealed elevated levels of ATG5 and BNIP3 in acid-conditioned cells, suggesting cells exposed to low pH may utilize autophagy as a survival mechanism. In support of this hypothesis, we found that acute low pH stimulated autophagy as defined by an increase in LC3-positive punctate vesicles, double-membrane vacuoles, and decreased phosphorylation of AKT and ribosomal protein S6. Notably, cells exposed to low pH for approximately 3 months restored their proliferative capacity while maintaining the cytoplasmic vacuolated phenotype. Although autophagy is typically transient, elevated autophagy markers were maintained chronically in low pH conditioned cells as visualized by increased protein expression of LC3-II and double-membrane vacuoles. Furthermore, these cells exhibited elevated sensitivity to PI3K-class III inhibition by 3-methyladenine. In mouse tumors, LC3 expression was reduced by systemic treatment with sodium bicarbonate, which raises intratumoral pH. Taken together, these results argue that acidic conditions in the tumor microenvironment promote autophagy, and that chronic autophagy occurs as a survival adaptation in this setting. Cancer Res; 72(16); 3938–47. ©2012 AACR.
