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. 2012 Jan 19;31(3):333-41.
doi: 10.1038/onc.2011.227. Epub 2011 Jun 13.

Identification and functional analysis of 9p24 amplified genes in human breast cancer

J Wu  1 , S Liu, G Liu, A Dombkowski, J Abrams, R Martin-Trevino, M S Wicha, S P Ethier, Z-Q Yang
Affiliations

Identification and functional analysis of 9p24 amplified genes in human breast cancer

J Wu et al. Oncogene. .

Abstract

Previously, our group identified a novel amplicon at chromosome 9p24 in human esophageal and breast cancers, and cloned the novel gene, GASC1 (gene amplified in squamous cell carcinoma 1, also known as JMJD2C/KDM4C), from this amplicon. GASC1 is a histone demethylase involved in the deregulation of histone methylation in cancer cells. In the current study, we aimed to comprehensively characterize the genes in the 9p24 amplicon in human breast cancer. We performed extensive genomic analyses on a panel of cancer cell lines and narrowed the shortest region of overlap to approximately 2 Mb. Based on statistical analysis of copy number increase and overexpression, the 9p24 amplicon contains six candidate oncogenes. Among these, four genes (GASC1 UHRF2, KIAA1432 and C9orf123) are overexpressed only in the context of gene amplification while two genes (ERMP1 and IL33) are overexpressed independent of the copy number increase. We then focused our studies on the UHRF2 gene, which has a potential involvement in both DNA methylation and histone modification. Knocking down UHRF2 expression inhibited the growth of breast cancer cells specifically with 9p24 amplification. Conversely, ectopic overexpression of UHRF2 in non-tumorigenic MCF10A cells promoted cell proliferation. Furthermore, we demonstrated that UHRF2 has the ability to suppress the expression of key cell-cycle inhibitors, such as p16(INK4a), p21(Waf1/Cip1) and p27(Kip1). Taken together, our studies support the notion that the 9p24 amplicon contains multiple oncogenes that may integrate genetic and epigenetic codes and have important roles in human tumorigenesis.

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Figures

Figure 1
Figure 1
Genomic analysis of the 9p24 region in human cancer cell lines. (a) Genome view of chromosome 9p analyzed on the Agilent oligonucleotide array (Agilent Technology) in Colo824, HCC1954 and KYSE150 cells. (b) Schematic representation of the 9p24-amplified region in four breast cancer cell lines (HCC70, HCC1954, Colo824 and SUM-149), one esophageal cancer cell line (KYSE150) and the recently published genomic data of basal-like primary breast tumor (P), brain metastasis (BM) and xenograft (X) samples (Ding et al., 2010). Localization of the 9p21-24 genes is shown to the right of the chromosome 9 ideogram. The lines at far right represent the amplified region of each sample based on our array CGH data and Ding et al.’s published data. SRO, shortest region of overlap.
Figure 2
Figure 2
(a) Expression level of six genes in the 9p24 amplicon. Gene expression was examined in cancer cells with 9p24 gain/amplification (KYSE150, Colo824, HCC1954, SUM-149 and HCC70) or without the gain/amplification (SUM-44, SUM-52, SUM-190 and SUM-225). mRNA expression levels in the MCF10A cells, an immortalized but non-tumorigenic breast epithelial cell line without 9p24 gain/amplification, were arbitrarily set as 0. Relative expression levels were shown as log2 values. (b) UHRF2 and GASC1 protein levels were analyzed by western blot in eight breast cancer cell lines with or without 9p24 amplification, as well as in MCF10A control cells.
Figure 3
Figure 3
Effect of UHRF2 knockdown on cancer cell growth. (a) Knockdown of UHRF2 mRNA in HCC1954 cells with two different shRNAs was confirmed by real-time RT–PCR. The real-time RT–PCR data were normalized with a GAPDH control and is shown as the mean±s.d. of triplicate determinations from two independent experiments. The baseline for the cells infected with control shRNA was arbitrarily set as 1. (b) Top panel shows TurboGFP images of HCC1954 cells after viral infection with control shRNA and UHRF2 shRNA#2. After seeding the same number of HCC1954 cells with or without UHRF2 knockdown, cells were stained with crystal violet at day 7 (bottom panel). (c) Relative cell growth after knocking down UHRF2 in five cell lines: HCC1954 and HCC70 with 9p24 amplification, SUM-52 and SUM-102 without the amplification as well as non-tumorigenic MCF10A cells. The same number of cells were seeded and allowed to grow for 7 days after attachment. Relative growth is shown as the mean±s.d. of triplicate determinations (**P<0.01).
Figure 4
Figure 4
(a) Stable overexpression of UHRF2 in MCF10A cells with the pLenti6/V5-UHRF2 construct (MCF10A-UHRF2). Overexpression of UHRF2 protein in two cell clones (UHRF2#1 and UHRF2#2), and knockdown of UHRF2 in clone #2 cells were confirmed by western blot. (b) Ectopic overexpression of UHRF2 confers a growth advantage to MCF10A cells, which can be reversed by UHRF2 shRNA (*P<0.05).
Figure 5
Figure 5
UHRF2 influences expression of p16INK4a, p21Waf1/Cip1, p27Kip1 and pRB. (a) mRNA levels of p21Waf1/Cip1, p16INK4a and p27Kip1 were examined by real-time RT–PCR after knocking down UHRF2 in HCC1954 cells. The baseline for the cells infected with control shRNA was arbitrarily set as 1. (b) Protein levels of p21Waf1/Cip1, p16INK4a, p27Kip1 and pRB in HCC1954 cells stably expressing control shRNA, UHRF2 shRNA#1 or shRNA#2 were analyzed by western blot. The migration control for the hypophosporylated (p) form of RB protein is shown in Supplentmentary Figure S7. (c) Overexpression of UHRF2 in MCF10A cells results in reduced protein levels of p16INK4a, p21Waf1/Cip1 and p27Kip1, but not of pRB as determined by western blot.
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