Volume 18, Issue 9, Pages 2228-2242 (February 2017) Extracellular Acidic pH Activates the Sterol Regulatory Element-Binding Protein 2 to Promote Tumor Progression  Ayano Kondo, Shogo Yamamoto, Ryo Nakaki, Teppei Shimamura, Takao Hamakubo, Juro Sakai, Tatsuhiko Kodama, Tetsuo Yoshida, Hiroyuki Aburatani, Tsuyoshi Osawa  Cell Reports  Volume 18, Issue 9, Pages 2228-2242 (February 2017) DOI: 10.1016/j.celrep.2017.02.006 Copyright © 2017 The Author(s) Terms and Conditions

Cell Reports 2017 18, 2228-2242DOI: (10.1016/j.celrep.2017.02.006) Copyright © 2017 The Author(s) Terms and Conditions

Figure 1 Low pH Culture Conditions Triggered Differential Transcriptional Regulation Compared with Hypoxia and Nutrient Starvation Culture Conditions (A) Experimental schematic illustrating control (pH 7.4) and low pH (pH 6.8) culture conditions. Cancer cells were cultured in DMEM supplemented with 10% FBS, and the pH was adjusted with NaHCO3. (B) Time course analysis of cell proliferation under low pH culture conditions in PANC-1 and AsPC-1 cells. Cell proliferation was measured using sulforhodamine B (SRB) cell proliferation assays at 24, 48, and 72 hr after cell seeding. (C) Cell adhesion was decreased under extracellular acidic pH. The number of attached cells was measured by SRB cell adhesion assays in PANC-1 cells and AsPC-1 cells at 1 and 3 hr after cell seeding. (D) Cell proliferation under low pH, hypoxia (H), and nutrient starvation (NS) culture conditions in PANC-1 and AsPC-1 cells. Cancer cells were cultured for 72 hr, and cell proliferation was measured by SRB cell proliferation assays. (E) Venn diagram representation of genes upregulated or downregulated under low pH, hypoxia, and nutrient starvation culture conditions, with 24-hr exposure to each condition. (F) Expression of PDK4 and IDI1 mRNAs in response to 24 hr of low pH in PANC-1 and AsPC-1 cells. (G) Expression of PDK1 and LDHA mRNAs in response to 24 hr of H in PANC-1 and AsPC-1 cells. (H) Expression of SREBP1 and LCN2 mRNAs in response to 24 hr of NS in PANC-1 and AsPC-1 cells. (I) Expression of PDK4 and IDI1 mRNAs in response to both NaHCO3 (pH) and lactic acidosis (Lactate) for adjustment of the pH to 6.8 in PANC-1 and AsPC-1 cells, as determined by real-time qPCR analysis after 24 hr. Data are presented as the mean ± SEM of at least three independent experiments. The expression of each transcript is reported relative to that of β-actin and was determined by real-time qPCR analysis in response to exposure to the culture conditions for 24 hr. Student’s t tests were performed for the indicated comparisons (∗∗∗p < 0.005). See also Figure S1. Cell Reports 2017 18, 2228-2242DOI: (10.1016/j.celrep.2017.02.006) Copyright © 2017 The Author(s) Terms and Conditions

Figure 2 Integrated Analysis of RNA-seq- and FAIRE-seq-Predicted SREBP1 and SREBP2 as Key Regulators of Acidic Extracellular pH (A) Heat map representation of the 524 differentially expressed genes under acidic pH. 2-fold upregulated or downregulated genes commonly found in PANC-1 and AsPC-1 cells after 24-hr exposure to control (C), acidic pH 6.8 (pH), hypoxia (H), and nutrient starvation (NS) conditions, respectively. (B) SREBP1, SREBP2, HNF4A, and XBP1 were predicted as upstream transcription factors under acidic pH by ingenuity pathway analysis (IPA). (C) Transcriptional upregulation of SREBP2 target genes was higher in the context of extracellular acidic pHe (pH) than under H and NS conditions after 24 hr in PANC-1 and AsPC-1 cells. Data are presented as mean ± SEM of at least three independent experiments. The relative expression of each transcript to β-actin in response to exposure to the culture conditions for 24 hr was determined by real-time qPCR analysis. Student’s t tests were performed for the indicated comparisons (∗∗∗p < 0.005). (D) Anti-K27ac ChIP-seq and FAIRE-seq profiles of open chromatin specific to low pH after 24 hr. (E) Anti-K27ac ChIP-seq and FAIRE-seq profiles of open chromatin specific to hypoxia after 24 hr. (F) Anti-K27ac ChIP-seq and FAIRE-seq profiles of open chromatin specific to nutrient starvation after 24 hr. (G) Venn diagram representation of nucleosome-free regions under hypoxia, nutrient starvation, and low pH in response to exposure to the culture conditions for 24 hr. The top 2,000 acidic pH-specific nucleosome-free peaks determined by FAIRE-seq were used for the motif analysis. (H) Sequence motifs enriched in nucleosome-free regions under acidic pH. See also Figures S2 and S3. Cell Reports 2017 18, 2228-2242DOI: (10.1016/j.celrep.2017.02.006) Copyright © 2017 The Author(s) Terms and Conditions

Figure 3 Extracellular Acidic pH Activated SREBP2 and Induced Its Nuclear Translocation (A) siRNA-mediated knockdown of SREBP2 suppressed extracellular acidic pH-induced upregulation of IDI1, MSMO1, and INSIG1 mRNA expression after 24 hr of exposure to extracellular acidic pH in PANC-1 and AsPC-1 cells. (B) siRNA-mediated knockdown of SREBP1 did not suppress extracellular acidic pH-induced upregulation of IDI1, MSMO1, and INSIG1 mRNA expression in PANC-1 and AsPC-1 cells. (C) Activated SREBP2 in the nuclear extract fraction (Nuc. Ext.) was increased compared with that in the membrane fraction (Membranes) after 24 hr of exposure to extracellular acidic pH. P and N denote the precursor and cleaved nuclear forms of SREBP2. (D) Low pH-induced binding of SREBP2 on the promoters of IDI1, MSMO1, and INSIG1 was quantified by ChIP-qPCR after 24 hr of exposure to extracellular acidic pH. (E) Cholesterol/25OH-cholesterol inhibited low pH-induced upregulation of SREBP2 target genes after 24 hr of exposure to extracellular acidic pH. Data are presented as mean ± SEM of at least three independent experiments. The expression of each transcript relative to that of β-actin was determined by real-time qPCR analysis. Student’s t tests were performed for the indicated comparisons (∗∗p < 0.01; ∗∗∗p < 0.005). See also Figure S4. Cell Reports 2017 18, 2228-2242DOI: (10.1016/j.celrep.2017.02.006) Copyright © 2017 The Author(s) Terms and Conditions

Figure 4 Decreased Intracellular pH Upregulated the Expression of SREBP2 Target Genes in Response to Extracellular Acidification (A) Changes in intracellular pH as the extracellular pH was decreased after 24 hr of exposure to different pH. (B) Upregulation of SREBP2 target genes in response extracellular acidification. (C) Effects of the Na+/H+ exchanger inhibitor HMA on intracellular pH under extracellular acidic culture conditions. PANC-1 and AsPC-1 cells were treated with 1–50 μM HMA at pH 6.8. (D–F) Effects of decreased intracellular pH on the upregulation of IDI1 (D), MSMO1 (E), and INSIG1 (F). The expression of each transcript relative to that of β-actin was determined by real-time qPCR analysis. Data are presented as mean ± SEM of at least three independent experiments. Student’s t tests were performed for the indicated comparisons (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.005; n.s., not significant). Cell Reports 2017 18, 2228-2242DOI: (10.1016/j.celrep.2017.02.006) Copyright © 2017 The Author(s) Terms and Conditions

Figure 5 SREBP2 Upregulated Genes Related to Biogenesis of Cholesterol under Extracellular Acidic Conditions (A) Heat map showing the 50 probes identified as pH-responsive SREBP2 target genes. (B) Venn diagram representation of genes upregulated under extracellular low pH (2-fold upregulation under pH 6.8 after 24 hr) and downregulated by SREBP2 knockdown (0.75-fold downregulation in the presence of two independent siRNAs targeting SREBP2) in both PANC-1 and AsPC-1 cells. (C) Gene ontology analysis of the 50 acidic pH (pH 6.8) upregulated and SREBP2 siRNA downregulated genes, as determined in Figure 5B. (D) Extracellular acidic pH-induced transcriptional upregulation of HMGCS1, FDFT1, and SQLE was reversed by SREBP2 knockdown, as determined by real-time qPCR. The expression of each transcript relative to β-actin is presented as the mean ± SEM (n = 3). (E) Extracellular acidic pH-responsive SREBP2 target enzymes involved in cholesterol biosynthesis. pH-responsive SREBP2 target genes are shown in red. (F) Effects of SREBP2 inhibition on total cholesterol levels (nmol/106 cells) in response to acidic pH. Data are presented as mean ± SEM (n = 3). Student’s t tests were performed for the indicated comparisons (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.005). Cell Reports 2017 18, 2228-2242DOI: (10.1016/j.celrep.2017.02.006) Copyright © 2017 The Author(s) Terms and Conditions

Figure 6 Integration of Transcription and SREBP2 ChIP-seq Analysis Revealed 12 Low pH-Responsive Genes, including ACSS2, under Extracellular Acidic pH (A) Nucleosome-free regions, histone H3K27ac modification, and SREBP2 binding on IDI1, MSMO1, and INSIG1 genes under pH 7.4 or pH 6.8 culture conditions in PANC-1 cells. (B) Genome-wide distribution of SREBP2-binding sites in PANC-1 cells under low pH (pH 6.8). (C) Sequence motifs enriched in extracellular acidic pH-responsive SREBP2-binding sites. (D) Venn diagram representation of common SREBP2 target genes determined by integration of transcription analysis and ChIP-seq analysis in PANC-1 cells after 24 hr of exposure to acidic pH. 12 pH-responsive SREBP2 target genes, including ACSS2, were identified. (E) SREBP2 binding to the promoter regions of ACSS2 under acidic pH (pH 6.8) in PANC-1 cells. (F) ACSS2 mRNA expression in PANC-1 cells. (G) Effects of SREBP2 knockdown on extracellular acidic pH-induced upregulation of ACSS2 mRNA expression in PANC-1 cells. The expression of each transcript relative to that of β-actin is presented as the mean ± SEM (n = 3). (H) Effects of ACSS2 knockdown on cancer cell growth. Relative cell growth was determined by SRB cell proliferation assays under pH 6.8 culture conditions in PANC-1 cells. The data are presented as mean ± SEM (n = 3). Student’s t tests were performed for the indicated comparisons (∗p < 0.05; ∗∗∗p < 0.005; §§§p < 0.005). See also Figures S5 and S6. Cell Reports 2017 18, 2228-2242DOI: (10.1016/j.celrep.2017.02.006) Copyright © 2017 The Author(s) Terms and Conditions

Figure 7 SREBP2- and Acidic pHe-Regulated Genes, including ACSS2, Contributed to In Vivo Tumor Growth and Patient Survival (A) Effects of siRNA-mediated SREBP2 knockdown on tumor growth in vivo. (B) Effects of siRNA-mediated ACSS2 knockdown on tumor growth in vivo. Data are presented as mean ± SEM (n = 3 per group). Student’s t tests were performed for the indicated comparisons (∗p < 0.05; §p < 0.05). (C) Overall survival of patients with pancreatic cancer, glioma, kidney renal clear cell carcinoma, sarcoma, breast invasive carcinoma, esophageal carcinoma, rectal adenocarcinoma, and thyroid carcinoma. Overall patient survival was determined in patients with high risk (log risk > 0) and low risk (log risk < 0) categorized by the Cox proportional hazard model based on expression of 12 pH-responsive SREBP2 direct target genes (ACSS2, DHCR7, ELOVL5, FDFT1, HMGCR, HMGCS1, IDI1, LDLR, MSMO1, MTHFR, SQLE, and SREBP2). See also Figure S7. Cell Reports 2017 18, 2228-2242DOI: (10.1016/j.celrep.2017.02.006) Copyright © 2017 The Author(s) Terms and Conditions