Expression ratio of CCND1 to CDKN2A mRNA predicts RB1 status of cultured cancer cell lines and clinical tumor samples

Abstract

Background: The retinoblastoma product (RB1) is frequently deregulated in various types of tumors by mutation, deletion, or inactivation through association with viral oncoproteins. The functional loss of RB1 is recognized to be one of the hallmarks that differentiate cancer cells from normal cells. Many researchers are attempting to develop anti-tumor agents that are preferentially effective against RB1-negative tumors. However, to identify patients with RB1-negative cancers, it is imperative to develop predictive biomarkers to classify RB1-positive and -negative tumors.

Results: Expression profiling of 30 cancer cell lines composed of 16 RB1-positive and 14 RB1-negative cancers was performed to find genes that are differentially expressed between the two groups, resulting in the identification of an RB1 signature with 194 genes. Among them, critical RB1 pathway components CDKN2A and CCND1 were included. We found that microarray data of the expression ratio of CCND1 and CDKN2A clearly distinguished the RB1 status of 30 cells lines. Measurement of the CCND1/CDKN2A mRNA expression ratio in additional cell lines by RT-PCR accurately predicted RB1 status (12/12 cells lines). The expression of CCND1/CDKN2A also correlated with RB1 status in xenograft tumors in vivo. Lastly, a CCND1/CDKN2A assay with clinical samples showed that uterine cervical and small cell lung cancers known to have a high prevalence of RB1-decifiency were predicted to be 100% RB1-negative, while uterine endometrial or gastric cancers were predicted to be 5-22% negative. All clinically normal tissues were 100% RB1-positive.

Conclusions: We report here that the CCND1/CDKN2A mRNA expression ratio predicts the RB1 status of cell lines in vitro and xenograft tumors and clinical tumor samples in vivo. Given the high predictive accuracy and quantitative nature of the CCND1/CDKN2A expression assay, the assay could be utilized to stratify patients for anti-tumor agents with preferential effects on either RB1-positive or -negative tumors.

Figure 1

Identification of a gene expression signature to classify RB1 status. The expression profiles of 16 RB1-positive and 14 RB1-negative cancer cell lines were analyzed. One hundred and ninety-four genes which were differentially expressed between the two groups were extracted as described in Materials and Methods. For the selected genes and 30 cell lines, two-dimensional hierarchical clustering was conducted. Each row represents a cell line. Each column represents a gene. Red, up-regulated genes; green, down-regulated genes.

Figure 2

Expression pattern of CCND1 and CDKN2A in RB-positive and -negative cell lines. Correlation between RB1 status and CDKN2A (A) or CCND1 (B) expression level derived from microarray data. (C) Relationship between RB1 status and the ratio of CCND1 to CDKN2A expression derived from microarray data.

Figure 3

CCND1/CDKN2A expression predicts RB1 status. (A) RB1 status of 12 cell lines determined by RB1 functional assay. Each cell line was transfected with E2F-regulatory reporter SEAP plasmid with or without the CDKN2A expression vector. The inhibition level of the SEAP reporter gene activity in response to CDKN2A induction was normalized to luciferase activity. (B) RB1 status of 12 cell lines predicted by the expression ratio of CCND1/CDKN2A. mRNAs from the 12 cell lines were analyzed with quantitative RT-PCR analysis for CCND1 and CDKN2A. The threshold of CCND1/CDKN2A to determine RB1 status was established as 0.404 by discriminate analysis. The RB1 statuses determined by CCND1/CDKN2A coincided with those determined by RB1 function assay. (C) Relative RB1 mRNA expression level in 12 cell lines measured with quantitative RT-PCR assay.

Figure 4

CCND1/CDKN2A expression predicts RB1 status in animal tumor models. (A) mRNAs from five xenograft tumors were purified from FFPET and analyzed with quantitative RT-PCR for CCND1 and CDKN2A. NCI-H358, NCI-H441, and HCT116 cell lines are known to be RB1-positve. HeLaS3 and H1048 cell lines are known to be RB1-negative. The cut-off value of log₁₀ (CCND1/CDKN2A) ratio to classify RB1 status is shown: 0.404. (B) CCND1/CDKN2A expression was analyzed in osteoblast cells derived from RB1 wild-type and RB1 knockout mice using publicly available microarray expression data (GEO: GSE19299).

Figure 5

CCND1/CDKN2A expression in clinical tumor and normal tissues. FFPETs of clinical samples were obtained from the Kanagawa Cancer Research and information Association. mRNAs from normal and tumor tissues were purified and analyzed with quantitative RT-PCR for CCND1 and CDKN2A. The tissue type is designated in each figure; (A) cervical, (B) endometrial, (C) small cell lung, (D) stomach cancers and corresponding normal tissues. The cut-off value of log₁₀ (CCND1/CDKN2A) ratio to classify RB1 status is shown: 0.404.