Analysis of the AHR gene proximal promoter GGGGC-repeat polymorphism in lung, breast, and colon cancer

Abstract

The aryl hydrocarbon receptor (AhR) regulates expression of numerous genes, including those of the CYP1 gene family. With the goal of determining factors that control AHR gene expression, our studies are focused on the role of the short tandem repeat polymorphism, (GGGGC)n, located in the proximal promoter of the human AHR gene. When luciferase constructs containing varying GGGGC repeats were transfected into cancer cell lines derived from the lung, colon, and breast, the number of GGGGC repeats affected AHR promoter activity. The number of GGGGC repeats was determined in DNA from 327 humans and from 38 samples representing 5 species of non-human primates. In chimpanzees and 3 species of macaques, only (GGGGC)2 alleles were observed; however, in western gorilla, (GGGGC)n alleles with n=2, 4, 5, 6, 7, and 8 were identified. In all human populations examined, the frequency of (GGGGC)n was n=4>5≫2, 6. When frequencies of the (GGGGC)n alleles in DNA from patients with lung, colon, or breast cancer were evaluated, the occurrence of (GGGGC)2 was found to be 8-fold more frequent among lung cancer patients in comparison with its incidence in the general population, as represented by New York State neonates. Analysis of matched tumor and non-tumor DNA samples from the same individuals provided no evidence of microsatellite instability. These studies indicate that the (GGGGC)n short tandem repeats are inherited, and that the (GGGGC)2 allele in the AHR proximal promoter region should be further investigated with regard to its potential association with lung cancer susceptibility.

Keywords: (GGGGC)(n) repeat polymorphism; Aryl hydrocarbon receptor; Breast cancer; Colon cancer; Lung cancer; Short tandem repeat.

Figure 1

Ah-responsiveness in NCI-H292, Caco-2, and MCF-7 cancer cells. Panels A and B: CYP1A1 (A) and CYP1B1 (B) mRNA levels were determined in the three cell lines exposed to 10 nM TCDD (gray bars) or DMSO control (0.1%; black bars) for 48 h; n = 3. Panel C: Western immunoblots of microsomes isolated from the three cell lines after cultures were exposed to 10 nM TCDD or vehicle for 48 h; blots were probed with anti-CYP1A1 or -1B1 antibodies as indicated. Standards (Std) are microsomes containing cDNA-expressed CYP1A1 and CYP1B1 (Gentest, BD Biosciences, San Jose, CA). Panel D: EROD activity was assayed in cancer cells after 48-h exposure to10 nM TCDD (gray bars) or DMSO control (0.1%; black bars); n = 5. Panels E and F: Cancer cells were transfected with luciferase constructs containing the CYP1A1 (E) or CYP1B1 (F) promoters. Cells were then exposed to 10 nM TCDD (gray bars) or DMSO control (0.1%; black bars) for 48 h prior to luciferase assays; n = 4. All determinations are presented as fold relative to the control and are means ± standard error; **, P < 0.01; ***, P < 0.001.

Figure 2

Effects of the number of (GGGGC)_n repeat sequences on AHR promoter activity. Panel A: Depiction of the constructs containing the repeat polymorphisms found in the AHR proximal promoter fused to the luciferase coding sequence (luc). Shown are the five polymorphic forms of the promoter containing (GGGGC)_n, where n = 2, 4, 5, 6, and the rare C/A SNP: gatctgggc(GGGGC)₂GGGGAGGGGCcggtgagggg). Consensus Sp binding sites are shown as vertical black lines. The transcriptional start site is indicated (TSS). Panels B, C, and D: Constructs containing the polymorphisms represented in Panel A, AhRΔ(-120)₂, AhRΔ(-120)₄, AhRΔ(-120)₅, AhRΔ(-120)₆, or AhRΔ(-120)_C/A, indicated by 2, 4, 5, 6, and 4 C/A, respectively, were transfected into the cancer cell lines, as indicated; after 48 h, the luciferase assays were performed, and the data expressed relative to the control vector, AhRΔ(-120)₄; n = 7 or 8. All activities are presented as fold relative to the control and are means ± standard error; *, P < 0.05; ***, P < 0.001.

Figure 3

AHR gene promoter polymorphic repeats and electrophoretic separation of PCR products. Homozygous (GGGGC)_{4 or 5} (4, 5) or heterozygous (GGGGC)_n-repeat standards (2/4, 5/6, 4/5) were separated on a 2.5% agarose gel (A) or 20% polyacrylamide gel (B). C) Homozygous (GGGGC)_{4 or 5} (4, 5) or heterozygous (GGGGC)_4/5 (4/5) standards were separated by PAGE. Upper (~200 bp) and lower (~170 bp) bands of the heterozygous (GGGGC)_4/5 repeat PCR product were excised from the gel, and the DNA was reamplified (Up and Lo, respectively). PCR product of homozygous (GGGGC)_{4 and 5} repeats, in equal amounts, were combined, melted, and cooled (4+5). Lane 1 is a no template control (0). D) All possible homozygous or heterozygous combinations of the polymorphism from constructs containing 2, 3, 4, 5, and 6 numbers of repeats, as indicated, were amplified and separated by PAGE. The 100-bp ladder is indicated (L).

Figure 4

AHR promoter polymorphisms in tumor and non-tumor tissue. DNA from cancer patients (numbers 1 – 16) using either non-tumor (N) or tumor (T) tissue sources was amplified and separated on polyacrylamide gels for heteroduplex analysis. Primary cancers were A) 1, gastric; 2 – 8, lung; 9, tongue; 10, peritoneal; and B) 11 – 16, colon. Electrophoretic standards for (GGGGC)_{4 or 5} (4, 5), (GGGGC)_2/4 (2/4), and (GGGGC)_4/5 (4/5) are boxed. The no-template controls are labeled (0), and the 100-bp ladder is indicated (L).

Figure 5

AHR promoter polymorphisms in tumor tissue. DNA from cancer patients (numbers 1 – 29) using tumor tissue sources was amplified and separated on polyacrylamide gels for heteroduplex analysis. Primary cancers were A) 1, esophagus; 2 – 3, sarcoma; 4, ovary; 5, lung; 6, bladder; 7, gastric; 8 – 9, endometrium; 10 – 14, breast; 15 – 21, colon; and B) 22 – 29, breast. Electrophoretic standards for (GGGGC)_{4 or 5} (4, 5), (GGGGC)_2/4 (2/4), and (GGGGC)_4/5 (4/5) are boxed. The no-template controls are labeled (0), and the 100-bp ladder is indicated (L).

Figure 6

Allele frequencies of the repeat polymorphism in cancer patients and NYS neonates. Represented are the proportion of alleles containing (GGGGC)₂, (GGGGC)₄, (GGGGC)₅, and (GGGGC)₆, as indicated, and (GGGGC)₂GGGGAGGGGC (indicated as C/A SNP) in NYS neonates (black bars), lung cancer patients (white bars), colon cancer patients (medium gray bars), and breast cancer patients (hatched bars). Broken brackets represent P-values for comparison across all sample groups. Solid brackets represent P-values for pair-wise sample group comparisons with P<0.05. For each sequence repeat polymorphism, a test P-value of approximately 0.007 corresponds to a true type-1 error rate of 0.05 (i.e., P=0.05 divided by 7 tests per locus).