Alterations in RD(INK4/ARF) -mediated en bloc regulation of the INK4-ARF locus in human squamous cell carcinoma of the head and neck

Abstract

The presence of RD(INK4/ARF) (RD) enhancer in the INK4-ARF locus provides a novel mechanism to simultaneously increase the transcription of p15(INK4b) (p15), p14ARF (p14), and p16(INK4a) (p16). While such upregulation can be repressed through interactions between RD and oncoproteins CDC6 and BMI1, little is known about the involvement of RD in cancer. In this study we investigated RD deletions in 30 squamous cell carcinoma of the head and neck (SCCHN) and the patient-matched High At-Risk Mucosa specimens (HARM, "phenotypically normal" tissues neighboring SCCHN foci but beyond the surgical resection margin). RD was deleted (homozygously/heterozygously) in SCCHN and HARM at the incidence of 36.7% (11/30) and 13.3% (4/30), respectively. In comparison, no RD deletion was detected in 26 oral buccal brush biopsy specimens from healthy donors. Both p16 and p14 were lowly expressed in SCCHN and HARM, and their mRNA expression levels were positively associated with each other (P < 0.01). Moreover, BMI1 was highly expressed in both SCCHN and HARM, and BMI1 overexpression was associated with p16 downregulation in SCCHN (P < 0.05). These results indicate that RD deletion and BMI1 overexpression frequently occur in the early stage of oral carcinogenesis and BMI1 overexpression may downregulate the transcription of p16 and p14 through interfering with RD.

Keywords: BMI1; RD enhancer; en bloc regulation; genetic deletion; the INK4-ARF locus.

Figure 1

Deletions in the INK4-ARF locus. (A) The schematic structure of the INK4-ARF locus. Promoters for p14, p15, and p16 genes are indicated by horizontal arrows. Boxes represent exons, and the empty circle represents the regulatory element RD. Sizes of coding regions and non-coding regions are not in proportion strictly. The (−) sign indicates negative regulation; red lines represent the locations of dual-labeled fluorogenic probes used in the deletion analyses. This figure was adapted from Reference . (B) The calibration curves for qPCR-based deletion assays. qPCR reactions using primers and fluorogenic probes against a target gene and β-actin (ACTB, the reference gene) were carried out separately with a series of mixtures of genomic DNAs from TE-1177 (with intact INK4-ARF locus) and Panc-1 (with homozygously-deleted INK4-ARF locus) cells at various ratios (100:0, 75:25, 50:50, 25:75, 0:100) as previously described [12]. The calibrations curves were generated through plotting the ΔΔCq values against known ratios of RD (in exponential form). The correlation coefficients of these curves are around −0.99, indicating that the dosage of a target gene in a sample can be accurately measured using this technique [13]. The genetic status of a target gene was determined as follows: dosage <25%, −/−; dosage between 25% and 75%, +/−; and dosage >75%, +/+. RD, red circle; p15, green square; p14, yellow triangle; p16, blue diamond.

Figure 2

mRNA expression of CDC6 and BMI1 in SCCHN, HARM, and healthy control tissues. A, CDC6 expression; B, BMI1 expression; C, BMI1 expression in tissues with different RD statuses. RT-qPCR-based assays using pre-validated primers and probes (Supplementary Table 1) were used to determine the mRNA expression levels of target genes. HPRT1 was used as an endogenous reference, and the relative expression level of a target gene was defined using the 2^−ΔCq method [16]. Each assay was conducted in triplicate and the average value was used for statistical analyses. Data were presented in box plots. Differences in CDC6 or BMI1 mRNA expression between the control group and the SCCHN or HARM group were analyzed using two-sided Welch two-sample t-tests with the significant level of p =0.05, whereas the difference between the SCCHN group and the HARM group was analyzed using two-sided paired t-tests. RD− HARM, HARM specimens with homozygous and heterozygous RD deletions; RD− Tumor, SCCHN specimens with homozygous and heterozygous RD deletions; RD+ HARM, HARM specimens with intact RD; RD+ Tumor, SCCHN specimens with intact RD. *, p <0.05; **, p <0.01.

Figure 3

mRNA expression of p16 (A), p14 (B), and p18 (C) in SCCHN, HARM, and healthy control tissues. Legends are similar as in Fig. 2. For p16 and p14, differences between different groups were analyzed using two-sided Welch two-sample t-tests with the significant level of p =0.05. For p18, differences between the control group and the SCCHN or HARM group were analyzed as p16 or p14, whereas the difference between the SCCHN group and the HARM group was analyzed using two-sided paired t-tests. *, p <0.05; **, p <0.01.