Genetic and epigenetic alterations of human chromosome 3, investigated by NotI-microarrays in seven types of epithelial cancers

Aim. To identify common and specific genetic/epigenetic changes of human chromosome 3, using the data of NotI-microarrays in seven types of epithelial cancers. Methods. We used descriptive statistics for the comparative analysis of NotI-microarray data from seven types of epithelial cancers. Results. The analysis of the NotI-microarrays showed significant changes (deletion or methylation) in 74 genes/loci in seven different epithelial cancers, namely colorectal, ovarian, renal, lung, breast, cervical and prostate. Five genes from the 3p14-3p24 region ( FOXP1, LRRC3B, NKiRAS1, RBSP3, ZIC4) were altered in all cancer types. For fifteen genes deletion/methy lation was found in a majority of tumors. For example, ITGA9, GORASP1, IQSEC1, CGGBP1, NBEAL2 and VHL are localized in the 3p12-3p26 region; PPP2R3A, FGF12, ALDH1L1, GATA2 and PLCL2 are localized on the 3q13-3q28 region. Twenty-two genes out of 74 studied showed alterations specific for a single type of tumor. The largest number, 13 genes/loci was found in the prostate cancer. This suggests specific mechanisms of prostate cancer development. Conclusions. NotI-microarrays for human chromosome 3 al-lowed to identify several common genetic/epigenetic alterations and also tumor-specific changes in seven types of epithelial cancer.


Introduction
E. Zabarovsky and V. Kashuba groups investigated genetic/epigenetic alterations in human cancers by a large-scale method, named the NotI-microarray, for more than fifteen years. This method represents a comparative genome hybridization technology (Karolinska Institute International Patent WO02/086163 and PCT/ SE02/00788 [1]), based on hybridization of the NotI-linking libraries, produced from tumor and normal genomic DNA [2]. It makes ISSN 1993-6842 (on- possible, to determine both, the genetic (deletions, amplifications) and epigenetic (methylation, demethy lation) changes in the genomic DNA of the NotI-linked genes / loci, due to the sensitivity of the NotI restriction enzyme to a methy lation status of CpG islands. Using this technology, 181 NotI-linking clones from different regions of human chromosome 3 were analyzed in more, than 250 malignant tumor samples, derived from different organs and tissues. [2,3]. It is known that genetic and epigenetic disturbances of chromosome 3 have very important influence on carcinogenesis of different human cancers [4][5][6]. On chromosome 3 several well-known and putative tumor suppressor genes (TSG) as well as many cancer-associated genes are situated [3][4][5][6][7]. The 3p25-p26 region is harboring the well-known TSG, such as VHL; 3p12-p14.2 region contains the FHIT gene; 3p24 possesses the RARB gene and 3p21-p22 region includes the RASSF1A gene [8,9]. However, a function and a role of many other genes of chromosome 3, which show alterations in different human cancer types, were largely unknown, before the NotImicroarray study. The aim of the present work is to identify common and specific genetic/epigenetic changes of human chromosome 3, using the data of NotI-microarrays in seven types of epithelial cancer.

Materials and Methods
We have performed comparative analysis of the NotI-microarray data for 7 types of epithelial cancers [2, 10-18] using methods of descriptive statistics. Fisher's exact test and Chisquare criteria were used for analysis of methylation and/or deletion frequencies in groups of tumors with different patho-morphological characteristics [2,[10][11][12][13][14][15][16][17][18]. The cases with pvalue below 0.05 were considered statistically significant. The Benjamini-Hochberg procedure with false discovery rate (FDR) 0.20 was used to correct p-value under multiple comparisons detection [19].
Notably, the greatest number of alterations is hetero-and homozygous deletions or methylation, in all reported data sets. Amplifications and demethy lation were quite a rare event in epithelial tumors in comparison with leukemia [20]. Hence, deletions and methy lation were in the focus of the present paper. Altogether, we found that 74 genes / loci of chromosome 3 exhibited significant changes in seven types of epithelial tumors. These results are presented in Table 1. It was found 40 genes/ loci with changes from 3p arm and 34 genes/ loci from 3q arm of chromosome 3. Five genes, namely FOXP1, LRRC3B, NKIRAS1, RBSP3 and ZIC4 altered in all se ven studied tumor types. They are located in the 3p14-3p24 region.
Noti-microarrays alterations in seven cancer types   Noteworthy, earlier many investigations have been focused on studying the genes of the 3p arm of the chromosome 3 [2,5,6], whereas little attention has been paid to the genes of the 3q arm. The results of NotImicroarrays show the involvement of 3q arm genes / loci in the carcinogenesis of epithelial tumors of all seven localizations. For example, the ZIC4 gene encodes the Zic family member 4 that is important in the development.
It participates in the regulation of transcription by RNA-polymerase II, but it has very low expression levels. It has deletion/me thylation changes in all seven tumor localization. Our data are confirmed by other researchers on another type of epithelial cancer (bladder cancer) [21]. Importantly, these epigenetic changes could be detected in biological fluids, such as urine, while it is impossible to detect the ZIC4 expression levels.
Another gene from 3q arm with deletion/ methy lation changes in 6 tumor localizations is PPP2R3A. This gene encodes one of the regulatory subunits of the protein phosphatase 2, which is implicated in the negative control of cell growth and division [22]. However, the genetic/epigenetic changes of this gene in epithelial cancers were not known until our studies.
Four genes from 3q arm, which have deletion/methy lation in 5 localizations of epithelial tumors are FGF12, ALDH1L1, GATA2, EPHB1. FGF12 is a member of the FGF family which is involved in a variety of biological processes, including cell growth, morpho genesis, tissue repair, tumor growth, and invasion [23]. The methy lation of FGF12 in colorectal cancers was shown [24]. Our study has confirmed this type of the FGF12 epigenetic changes in prostate cancer [18]. It is revealed as a putative biomarker in esophageal cancer [25]. The ALDH1L1 gene encodes the aldehyde dehydrogenase 1 family member L1. Loss of function (epigenetic silencing) or expression of ALDH1L1 is associated with increased cell motility, decreased apoptosis and cancer progression [26]. On the other hand, ALDH1L1 is the indicative gene of cancer cell stemness and it is a biomarker in colon cancer, which is associated with worth prognosis [27].
GATA2 encodes a member of the GATA family of zinc-finger transcription factors. It conducts transcriptional signals in particular from the androgen receptor [28]. GATA2 has a multifaceted function in prostate cancer aggressiveness and is a highly attractive target for treatments of lethal prostate cancer [29]. The GATA2 expression is associated with poor prognosis in acute myeloid leukemia [30]. The EPHB1 gene encodes a transmembrane protein which is a receptor for ephrin-B1. Loss of the ephrin receptor (EphB1) expression may be associated with aggressive cancer phenotypes in acute myelogenous leukemia [31]. The tumor suppressor function of EPHB1 in breast, colon and lung cancers was shown [32].
Moreover, we have investigated genetic/ epigenetic changes and expression of some genes, which have no NotI-site, from wellknown TSG RASSF1A 3p21.31 region. We have shown deletion/methy lation changes by NotImicroarray in some tumor localization of genes from this region (3p21.31) named NBEAL2, GNAI2, TOPAZ1. Our study has confirmed genetic/epigenetic changes and loss of expression for GPX1 and SEMA3B in renal and lung cancers [35][36][37]. Data of other investigators have revealed the down regulation of HYAL1, HYAL2, RASSF1A (3p21.31 region) in non-small cell lung cancer [34]. These data indicate the multiple inactivation of TSG and potential TSG clusters in human chromosome 3.

Conclusions
The analysis of the data, obtained with NotImicroarrays for human chromosome 3, identified several common genetic/epigenetic alterations in seven types of epithelial cancer and tumor-specific changes as well. These data make a basis for the creation of special sets of markers for early diagnostics, prediction of a course of disease, and evaluation of efficacy and a choice of therapy.