Functional interaction between responses to lactic acidosis and hypoxia regulates genomic transcriptional outputs

Abstract

Within solid tumor microenvironments, lactic acidosis, and hypoxia each have powerful effects on cancer pathophysiology. However, the influence that these processes exert on each other is unknown. Here, we report that a significant portion of the transcriptional response to hypoxia elicited in cancer cells is abolished by simultaneous exposure to lactic acidosis. In particular, lactic acidosis abolished stabilization of HIF-1α protein which occurs normally under hypoxic conditions. In contrast, lactic acidosis strongly synergized with hypoxia to activate the unfolded protein response (UPR) and an inflammatory response, displaying a strong similarity to ATF4-driven amino acid deprivation responses (AAR). In certain breast tumors and breast tumor cells examined, an integrative analysis of gene expression and array CGH data revealed DNA copy number alterations at the ATF4 locus, an important activator of the UPR/AAR pathway. In this setting, varying ATF4 levels influenced the survival of cells after exposure to hypoxia and lactic acidosis. Our findings reveal that the condition of lactic acidosis present in solid tumors inhibits canonical hypoxia responses and activates UPR and inflammation responses. Furthermore, these data suggest that ATF4 status may be a critical determinant of the ability of cancer cells to adapt to oxygen and acidity fluctuations in the tumor microenvironment, perhaps linking short-term transcriptional responses to long-term selection for copy number alterations in cancer cells.

Figure 1. The transcriptional response of MCF7 to lactic acidosis (LA), hypoxia (Hyp), and the combined hypoxia and lactic acidosis (LA+Hyp) conditions

(A) The gene expression profiles of MCF7 in response to lactic acidosis (10 mM Lactic acid, pH 6.7), hypoxia (1% O₂) and the combined condition(10 mM Lactic acid, pH 6.7 in 1% O₂) at 24 hrs. 1956 probe sets were selected to have at least two fold change in more than two arrays, and arranged by hierarchical clustering as shown. (B) The gene clusters of hypoxia induced genes which were sensitive (blue vertical bar) and resistant (red vertical bar) to the inhibitory effects of lactic acidosis were shown with selected gene names. (C) The levels of indicated lactic acidosis sensitive hypoxia genes (CA9, PGK1, STC1) in response to individual treatment were measured using RT-qPCR (n=3). (D) The levels of indicated lactic acidosis resistant hypoxia genes (VEGFA, HIG2, Cyr61) in response to individual treatment at 24 hrs were measured using RT-qPCR (n=3). Error bars are mean ± SD, significant p-values are indicated as (* p<0.001; ** p<0.01).

Figure 2. The repression of hypoxia response by lactic acidosis is mediated by inhibition of HIF-1α protein synthesis

(A) The mRNA levels of CA9 and EGLN3 in MCF7 cells measured by RT-qPCR treated with indicated lactic acid concentration (pH 6.7) either in normoxia or in hypoxia condition for 24 hrs (n=3). (B) Immunoblot detection of HIF-1α and EGLN3 protein expressions, and S6K phosphorylation at Thr-398 in MCF7 cells treated as (A). (C) The concentration of VEGFA in media was detected by ELISA assay at indicated days and treatments (10 mM Lactic acid pH 6.7. n=3). (D) The mRNA levels of CA9 and EGLN3 in MCF7 treated with control, DMOG or DMOG plus lactic acidosis (10 mM Lactic acid pH 6.7) in normoxia condition (n=3). (E) Immunoblot detection of HIF-1α and EGLN3 protein expressions, and S6K phosphorylation at Thr-398 in MCF7 cells treated as (C). (F) The HIF-1α protein expression level was determined in MCF7 cells treated with lactic acidosis (10 mM Lactic acid, pH 6.7) in either normoxia or hypoxia condition for 18 hrs, then further treated with the protease inhibitor MG132 (10 μM) for additional 4 hrs. Error bars are mean ± SD, significant p-values are indicated as (* p<0.001; ** p<0.0001; *** p<0.001).

Figure 3. The transcriptional response triggered by combined hypoxia and lactic acidosis

(A) Genes synergistically induced by combined hypoxia and lactic acidosis with the names of selected genes shown. (B, C) The mRNA levels of the inflammation response genes TNF, TNFAIP3 and GADD45B (B) and unfolded protein response genes CHOP, XBP1s and ATF3 (C) in MCF7 cells treated with 10 mM lactic acid (pH 6.7) either in normoxia or in hypoxia condition for 24 hrs. Error bars are mean ± SD, significant p-values are indicated as (* p<0.001, n=3; ** p<0.001, n=3). (D) Immunoblot detection of HIF-1α and ATF4 protein expressions, and eIF2α phosphorylation at Ser-51 in MCF7 treated10 mM lactic acid (pH 6.7) either in normoxia or in hypoxia condition for 24 or 48 hrs. (E) A comparison of gene expression profiles induced by combined hypoxia and lactic acidosis in MCF7 and induced by amino acid deprivation in HepG2. (F) GSEA reveals a significant enrichment of genes induced by combined hypoxia and lactic acidosis among the genes induced by histidine deprivation in HepG2.

Figure 4. DNA copy number alterations of the ATF4 locus among the breast tumors and cancer cell lines

(A, B) Strength of the correlation (−Log₂(p-value), Pearson correlation) between DNA (array CGH) and ATF4 mRNA (microarrays) along all chromosomes among breast tumors (A) and cancer cell lines (B). Each spot represents the strength of correlation for one CGH clone. The clone closest to ATF4 is highlighted in red. (C, D) Scatterplot of ATF4 RNA versus DNA from the CGH clone closest to ATF4 in the genome. Figure (C) shows breast tumors and (D) shows cancer cell lines. Each spot is a single sample. (E) The clustered expression of ATF4 and adjacent chromosomal genes in breast cancer cell lines. (F, G) The DNA (F) and mRNA level (G) of ATF4 and VEGFA genes were determined in the indicated breast cancer cell lines by qPCR or RT-qPCR. Error bars are mean ± SD, significant p-values are indicated as (* p<0.001, n=3; ** p>0. 1, n=3; *** p<0.001, n=3). (H) Immunoblot detection of ATF4 and Histone-3 protein in nuclear extracts of indicated cell lines.

Figure 5. Varying levels of ATF4 impact the survival of breast cancer cells under hypoxia and lactic acidosis

(A) The amount of cell death in MCF7 after 3 days treatment with either hypoxia or combined hypoxia and lactic acidosis condition (empty), and 3 days recovery from stresses by re-plating regular culture medium and maintaining in normoxia condition (hatched). The cell death was assessed by the Propidium Iodide (PI) stained sub-G1 population using FACS. (B) Clonogenic survival of MCF7 treated with indicated stress for 2 days then replated with regular medium every four days for 12 days. Cell colonies were stained with crystal violet. (C) MCF7 transfected with 50 nM either control siRNA or siATF4 were treated with hypoxia or combined hypoxia and lactic acidosis. The levels of cell death were determined at 3 days stress treatment and 2 or 3 days post-stresses. Cell death was measured by the PI stained sub-G1 population (n=4). (D) The levels of cell death of MCF7 cells which have been transfected with either control vector or ATF4 expression constructs were treated, collected and assessed as (C) (n=3). (E) Cell death was measure in HT1080 shNT/shATF4 cells treated as (C) (n=3). (F) The mRNA levels of VEGFA, ASNS and HIG2 in the early passage of MCF7 cells stably infected with empty vector pSM2 and shATF4. (G) The mRNA level of ATF4 gene and cell death were measured at early and late passage of MCF7 cells stably infected with empty vector pSM2 and shATF4 (n=3). Error bars are mean ± SD, significant p-values are indicated as (* p<0.01; ** p<0.05; *** p<0.01; # p< 0.001; ## p<0.01).