Frequent alterations of LOH11CR2A, PIG8 and CHEK1 genes at chromosomal 11q24.1-24.2 region in breast carcinoma: clinical and prognostic implications

Abstract

To understand the importance of frequent deletions at chromosome 11q24.1-24.2 region in breast carcinoma, alterations (deletion/methylation) of the candidate genes LOH11CR2A, ROBO3, ROBO4, HEPACAM, PIG8 and CHEK1 located in this region were analyzed in 106 breast carcinoma samples. Among these genes, LOH11CR2A showed highest frequency of deletion (56%), followed by PIG8 (35%), CHEK1 (31%) and ROBO3/ROBO4/HEPACAM loci (28%). Comparable frequency of promoter methylation (26-35%) was observed for LOH11CR2A, CHEK1 and PIG8. Overall alterations (deletion/methylation) of these genes were in the following order: LOH11CR2A (60%) > PIG8 (46%) > CHEK1 (41%) and showed significant association with each other. Breast carcinoma samples that were estrogen/progesterone receptor negative showed significantly high deletion and overall alterations than estrogen/progesterone receptor positive samples for LOH11CR2A, CHEK1 and PIG8. The methylation and overall alteration of LOH11CR2A were significantly associated with tumor stages in breast carcinoma. However, in early/late onset and estrogen/progesterone receptor positive/negative breast carcinoma, the overall alterations of LOH11CR2A, PIG8 and CHEK1 were differentially associated with advanced stages, tumor grade and lymph node metastasis. Alterations of PIG8 and CHEK1 were significantly associated with poor prognosis in patients with early age of onset of the disease indicating significant prognostic importance. Quantitative mRNA expression analysis detected reduced expression of the genes in the order LOH11CR2A > CHEK1 > PIG8. Immunohistochemical analysis showed reduced protein expression of PIG8 and CHEK1 that was concordant with their molecular alterations. Thus, our study suggests that LOH11CR2A, PIG8 and CHEK1 are candidate tumor suppressor genes associated with breast carcinoma and have significant clinical as well as prognostic importance.

Figure 1

(a) Schematic representation of the candidate genes of Chr.11q24.1‐24.2 and their intragenic/adjoining STS markers. (b) Representative autoradiographs showing deletion (LOH) of BC samples at different marker loci. (c) Representative photograph of HED with PIG8 and CHEK1 exonic marker (EM) performed by multiplex PCR with the control DRD2 EM. (d) Frequency of deletion of candidate genes in overall BC, Early‐onset, Late Onset, ER/PR+ and ER/PR‐ BC. Significant higher deletion in ER/PR‐ than ER/PR + BC is shown by asterisk. T: Tumor DNA; TR: Tumor DNA from right breast, TL: Tumor DNA from left breast N: DNA of the corresponding normal tissue.

Figure 2

(a) Analysis of promoter methylation of candidate genes by MSRA in BC samples. Representative tumor samples (# 3266T, 1099T and 4010T) showing methylated status at different genes, normal breast tissue was unmethylated. M and H: MspI‐ and HpaII digested DNA, respectively. U: undigested DNA. K1 and K2: controls for DNA digestion and integrity, respectively. (b) Frequency of methylation of candidate genes in Early‐onset, Late Onset, ER/PR+ and ER/PR‐ BC. (c) Frequency of overall alterations of candidate genes in Early‐onset, Late Onset, ER/PR+ and ER/PR‐ BC. Significant higher methylation or alteration in ER/PR‐ than ER/PR + BC is shown by asterisk.

Figure 3

Quantitative RT‐PCR analysis showing reduced expression of LOH11CR2A, PIG8 and CHEK1 in BC samples (n = 14) and MCF‐7 cell line. Bars represented the gene expression normalized to β2‐microglobulin and relative to a pool of normal breast tissues. The line illustrates the mean reduction level of respective genes. X axis indicates samples (1–14). T1‐14 represents the BC samples.

Figure 4

(a) Representative histogram of q‐RT‐PCR analysis showing increased mRNA expression of PIG8 and CHEK1 in 5‐aza‐dC treated MCF‐7 cell line with respect to the corresponding untreated control. (b) Western Blot analysis showing PIG8 and CHEK1 protein expression in 5‐aza‐dC treated MCF‐7 cell line and corresponding untreated control. α –tubulin was used as the loading control (c) Representative histogram of Western Blot analysis showing increased PIG8 and CHEK1 protein expression in 5‐aza‐dC treated MCF‐7 cell line with respect to the corresponding untreated control.

Figure 5

Immunohistochemical analysis of CHEK1 and PIG8 in breast carcinoma samples and MCF‐7 cell line. (a) Breast carcinoma sample (#3156) showing reduced expression of CHEK1. (b) Breast carcinoma sample (#374) showing cytoplasmic expression of CHEK1. (c) MCF‐7 cell line showing nuclear expression of CHEK1. (d) Breast carcinoma sample (#5364) showing reduced expression of PIG8. (e) Breast carcinoma sample (#796) showing cytoplasmic expression of PIG8. (f) MCF‐7 cell line showing reduced cytoplasmic expression of PIG8. The slim arrow indicates cytoplasmic expression and bold arrow indicates nuclear expression. D: Deletion, M: Methylation, ER: Estrogen receptor, PR: Progesterone receptor. The original magnifications are indicated in top left corner of the photograph. Scale bar for magnification of 50 μM.

Figure 6

Kaplan Meier 5‐year survival probability curves with cumulative survival of BC patients by alteration status of PIG8 and CHEK1 in early‐onset BC. Survival time was defined as the time from surgery to the patient's death, known recurrence or the last time the patient was known to be alive. The smooth line represents survival probability without overall alterations and the dotted line represents the same probability with molecular alterations.N denotes sample size.