Functional role of BTB and CNC Homology 1 gene in pancreatic cancer and its association with survival in patients treated with gemcitabine

Abstract

Genetic variation (rs372883C/T) in the 3'-untranslated region of BTB and CNC homology 1 (BACH1) has been associated with pancreatic ductal adenocarcinoma (PDAC) risk in our previous genome-wide association study; however, the action roles of this genetic variation in PDAC remains unknown. Methods:BACH1 expression was measured by quantitative real-time PCR, Western blot and immunohistochemistry. The effects of BACH1 on cell proliferation and sensitivity to gemcitabine were examined by alteration of BACH1 expression in PDAC cells. Angiogenesis was determined in vitro using a human umbilical vein endothelial cell model. Reporter gene assays were conducted to compare the effects of microRNA-1257 on rs372883 variation. The associations between rs372883 variants and survival time in patients treated with gemcitabine were estimated by logistic regression. Results: We found substantially lower BACH1 expression in PDAC compared with normal pancreatic tissues and the rs372883T allele had significantly lower BACH1 levels than the rs372883C allele in both tumor and normal tissues. Knockdown of BACH1 expression provoked proliferation of PDAC cells and angiogenesis, which might result from upregulation of hemeoxygenase-1 that evokes oncogenic AKT and ERK signaling. The rs372883T>C change inhibits interaction of BACH1 with microRNA-1257, resulting in increased BACH1 expression. PDAC patients with the rs372883T allele were more resistant to gemcitabine and had shorter survival time compared with those with the rs372883C allele. Conclusion: These results shed light on the mechanism underlying the associations of BACH1 rs372883 variation with risk of developing PDAC and differential gemcitabine sensitivity in patients.

Keywords: BACH1; HO-1; genetic variation; pancreatic cancer; survival.

Figure 1

Expression of BACH1 in PDAC. (A) BACH1mRNA levels determined by qRT-PCR were significantly lower in PDAC compared with paired adjacent normal tissues (N=75). Results represent mean ± SEM normalized to GAPDH and P-values are for Student's t-test. (B) BACH1 protein levels determined by Western blot were significantly lower in PDAC (T) than in paired normal tissues (N). Shown is a representative picture selected from 35 pairs of clinical specimens. (C) Representative IHC images (200×) showing BACH1 protein expression in normal pancreatic tissues (left) and PDAC (right). Bar scale, 100 μm. (D-E) Expression of BACH1 in published data sets of Pei et al. and Badea et al. (Oncomine, https://www.oncomine.org/resource/main.html). The line in the middle of the box represents the median; bars represent 10th and 90th percentiles and greater values were plotted as individual points. P-values are for unpaired Wilcox rank-sum test.

Figure 2

BACH1 inhibits PDAC cells proliferation and angiogenesis. (A) Stable overexpression or knockdown of BACH1 in CFPAC-1 and BXPC-3 cells. (B) Effect of BACH1 overexpression or knockdown on colony formation of CFPAC-1 and BXPC-3 cells. Results represent colony formation ability relative to Control or shControl set to 100% from three experiments. P-values are for Student's t-test. *, P<0.05; **, P<0.01 compared with Control or shControl. (C) Overexpression of BACH1 substantially reduced proliferation of both CFPAC-1 and BXPC-3 cells determined by CCK-8 assay. Results are mean ± SEM from three experiments and each experiment had six replicates. *, P<0.05 compared with control. (D) Knockdown of BACH1 significantly enhanced proliferation of both CFPAC-1 and BXPC-3 cells. Results are mean ± SEM from three experiments and each experiment had six replicates. *, P<0.05 compared with shControl. (E-F) Xenograft tumor formation and growth of CFPAC-1 and BXPC-3 cells with BACH1 overexpression or knockdown in nude mice. Results are mean ± SEM from 6 animals in each group. *, P<0.05 compared with Control or shControl. (G) Effect of BACH1 expression on VEGF levels quantified by ELISA in the culture medium of CFPAC-1 or BXPC-3 cells. Results are mean ± SEM from three measurements. *, P<0.05; **, P<0.01 compared with Control or shControl. (H) Effects of culture medium of CFPAC-1 or BXPC-3 cells with BACH1 overexpression or knockdown on HUVEC tube formation. Results are mean ± SEM from three measurements. *, P<0.05 compared with Control or shControl.

Figure 3

BACH1 negatively regulates HO-1 expression in PDAC cells. (A) MA plot of the gene expression profile. Eight genes directly regulated by BACH1 were marked: red, upregulated; blue, downregulated; gray, not significantly changed. The dotted lines indicate the cut-off value of log₂ fold change (± 0.58). (B) BACH1 binding peaks in the HMOX1 gene locus based on ChIP-sequencing data (GSM693953 and GSM693952). (C) Chromatin immunoprecipitation assays showing binding of BACH1 to two enhancers EN1 (-9.0kb) and EN2 (-4.0kb) in the upstream of HMOX1 in CFPAC-1 and BXPC-3 cells. Overexpression of BACH1 in these cells substantially enhanced enrichment of BACH1 in EN1 and EN2, while knockdown of BACH1 substantially decreased the enrichment in these two enhancers. Fold enrichment (mean ± SEM) represents DNA levels associated with BACH1 or IgG relative to an input control from three independent experiments. IgG served as negative control. ***, P<0.001 compared with Control or shControl. (D) Overexpression of BACH1 suppressed HO-1 expression while knockdown of BACH1 elevated HO-1 expression in both mRNA and protein levels in CFPAC-1 and BXPC-3 cells. *, P<0.05; ***, P<0.001 compared with Control or shControl. (E) HMOX1 mRNA expression was significantly higher in PDAC compared with their adjacent normal tissues (N=75). Results are mean ± SEM normalized to GAPDH and the P-values are for Student's t-test. (F) Correlation between BACH1 and HMOX1 mRNA levels in PDAC and paired normal tissues (N=75). The RNA levels were determined by qRT-PCR and expressed relative to GAPDH. The r- and P-values are for Pearson's correlation analysis. (G-H) Correlation between BACH1 and HMOX1 mRNA levels in PDAC and paired normal tissues. Data are from Oncomine database generated by Pei et al. and Badea et al. .

Figure 4

BACH1 regulates signaling pathway downstream of HO-1. (A-B) Western blot analysis of AKT and ERK signaling modules downstream of HO-1 in CFPAC-1 and BXPC-3 cells with overexpression or knockdown of BACH1. (C-E) A reverse correlation between mRNA levels (N=75) of BACH1 and mRNA levels of HIF1A (C) or VEGF (D) and a positive correlation between mRNA levels of BACH1 and mRNA levels of PTEN (E) in normal tissues (upper panel) and paired PDAC (lower panel). The RNA levels were determined by qRT-PCR relative to GAPDH. The r- and P-values are for Pearson's correlation analysis.

Figure 5

Functional relevance of rs372883 variants. (A) Relative reporter gene activity bearing BACH1 3'UTR fragment with the rs372883T or rs372883C allele in CFPAC-1 and BXPC-3 cells. Results are mean ± SEM from three experiments and each had six replicates. The P-values are for Student's t-test. (B) In silico prediction of interaction between miR-1257 and BACH1 3'UTR showing differences in binding within the seed region. (C) Relative reporter gene activity of the psiCHECK2-rs372883T and psiCHECK2-rs372883C constructs cotransfected with 1.0, 5.0 and 10.0 pmol of miR-1257 or its inhibitor in CFPAC-1 and BXPC-3 cells. Results are mean ± SEM from three experiments and each had six replicates; P-values were for Student's t-test. (D-F) Levels of BACH1 mRNA, miR-1257 and HMOX1 mRNA in normal pancreatic tissues adjacent to tumors of subjects with the rs372883 CC (N=11), CT (N=38) or TT (N=26) genotype. Results are mean ± SEM relative to GAPDH or U6. (G) Western blot analysis of AKT and ERK signaling modules downstream of HO-1 in surgically removed PDAC specimens from subjects with the rs372883CC, CT or TT genotype.

Figure 6

Gemcitabine sensitivity in PDAC cells depends on BACH1 in an allele-specific manner. (A) Effects of BACH1 on gemcitabine sensitivity in CFPAC-1 and BXPC-3 cells. Results represent mean ± SEM from three independent experiments and each had four replications. *, P<0.05 and **, P<0.01. (B-C) Effect of HO-1 expression on gemcitabine sensitivity in CFPAC-1 and BXPC-3 cells with overexpression (B) or knockdown (C) of BACH1. Results are mean ± SEM from three independent experiments and each had four replications. *, P<0.05; **, P<0.01 and ***, P<0.001. (D-F) Effect of miR-1257 on gemcitabine sensitivity in BXPC-3 (D), CFPAC-1 (E) and Capan-2 (F) cells carrying the rs372883 CC, CT or TT genotype, respectively. Cells transiently transfected with miR-1257 or its inhibitor were exposed to gemcitabine at final concentrations ranging from 10^-3 to 10³ nM. Cells were enumerated with the CCK-8 assay at 48h after drug exposure. IC₅₀ represents gemcitabine concentration that inhibits cell proliferation by 50%. Results are mean ± SEM from three experiments and each had four replicates. *, P<0.05 and ***, P<0.001.

Figure 7

Kaplan-Meier estimation of PDAC survival time in 102 patients. (A) Survival curves by disease stage. (B) Survival curves by response to gemcitabine therapy. (C) Survival curves by BACH1 3'UTR rs372883 genotypes.