Heterozygous deletions are main cause of expression alterations of PPM 1 M and PRICLE 2 genes in human clear cell renal cell carcinomas

Aim. To discover the mechanisms of the PPM1M and PRICKLE2 genes expression alterations in clear cell renal cell carcinomas. Methods. Study of the copy number was performed, using the quantitative PCR (Q-PCR). Results. Deletions of PPM1M were found in 55 % of cases, amplifi cations – in 17 % and in 28 % of samples there were no changes of the copy number. We found the deletions of PRICKLE2 in 50 % of samples, no changes of the copy number in 39 %, and amplifi cations in 11 % of cases. Conclusions. By the analysis of the gene copy number we have shown that homoand heterozygous deletions are the main reason of changes in expression of the PPM1M and PRICKLE2 genes.


Introduction
Annual incidence of tumors of the genitourinary system is over 200 thousand worldwide.During 2011 the kidney cancer was diagnosed in 5622 patients in Ukraine; 2469 of them died.For a quarter of these patients a period from the time of diagnosis to death is very short, less than one year (see http://www.ncru.inf.ua/publications/BULL_14/PDF_E/49-50-poch.pdf).Among urologic tumors, clear cell renal cell carcinoma (ccRCC later on in the text) takes the third place in incidence, following prostate and bladder cancer.ccRCC is the fi rst by the number of death.ccRCC is the prevalent malignancy of the kidney, accounting for about 85 % of kidney cancers [1].Kidney carcinoma is associated with the most negative prognosis and responds poorly to medical treatment, as a rule [2].The course of disease and treatment depends on the molecular profi le of tumors.Very important is to study the expression pattern of tumor suppressor genes (TSG) that play an important role in cancerogenesis.The TSG expression is usually downregulated in tumors by different mechanisms, such as genetic and epigenetic alterations.One of the most frequent aberrations in solid tumors is inactivation of TSG in chromosome 3 [3].Using the NotImicroarray to study ccRCC samples, we have found that the PPM1M and PRICLE2 genes showed genetic and/or epigenetic changes [4].It has been already shown that these genes play the role of TSG in cervical, ovarian and lung cancers [5][6][7].
Earlier we have shown that a reduction in the PPM1M and PRICKLE2 expression is typical for ccRCC; no promoter methylation was found for these genes, however [8].It is known that these pro teins are involved in the signalling pathways associated with carcinogenesis.In particular, PPM1M inhibits the IL-1-NF-kappaB signalling pathway by selective dephosphorylating of IKKbeta [9].PRICKLE2 is involved in the WNT pathway of planar cell polarity determination (PCP), which regulated the polarity and movement of cells [10].Therefore, there is a possibility that these two genes might be TSGs in ccRCC.
In the present work we performed a detailed study of heterozygous deletions of the PPM1M and PRICKLE2 genes in ccRCC and propose the new mechanism of deletion of these genes during carcinogenesis.

Materials and Methods
Tissue samples.Samples of surgically removed tumors and surrounding tissues (considered as normal) were obtained from Kyiv National urology cen ter (Kyiv, Ukraine).All tissue samples were cha rac terized according to the International System of Clinical and morphological classifi cation of tumors (TNM) [11] and WHO classifi cation criteria [12].The average age of patients was 48.59 (in the range of 30-68 years).Normal tissues were obtained at a distance of at least 2 cm from the tumor and histologically confi rmed as unchanged renal epithelial cells.Altogether, 18 samples of ccRCC were investigated, i.e. 12 samples at stage 1-2 and 6 samples at stage 3-4.

Isolation of genomic DNA
Genomic DNA was isolated, using DNA purifi cation Kit (Fermentas, Lithuania), according to the manufacturer's re-commendations.Quality of genomic DNA was as- Samples, which had ratio >0.85 and where genes showed unchanged copy number are marked regular text Samples, which displayed ratio >1.5 and where genes were amplifi ed marked bold.
sessed by agarose gel electrophoresis; DNA concentration was measured, using a spectrophotometer Nano-Drop ND-1000 (NanoDrop Technologies Inc., USA).The samples used for the Q-PCR reactions were of high molecular weight (OD 260/280 was in range 1.6-1.8).
Analysis of gene copy number.The Q-PCR method and Bio-Rad iQ5 machine to quantify the number of gene copies were used.It was shown that this method is accurate enough to determine the number of allele copies.Each Q-PCR reaction contained 12.5 ul 2xSYBR Green PCR Master Mix (Fermentas, Lithuania), forward and reverse primers at a concentration of 400 nM, genomic DNA -10 ng/ml, and sterile water to make a fi nal volume 25 ul.Gene TBP was used as the reference [13].The primer design was carried out, using a program Primer3 (http://frodo.wi.mit.edu/primer3/) and In te grated DNA Technologies, http:// eu.idtdna.com/analyzer/Applications/Oli goAnalyzer/).The follo wing primers were used (Invitrogen):
Analysis of the number of copies of the PPM1M and PRICKLE2 genes was performed by 2-ΔΔCP relative quantitative analysis [14].
According to the histological analysis, the contamination of tumour samples by normal stroma lymphocytes could be up to 30-40 %.Therefore, the homozygous deletions were considered when the calculated values were below 0.35; the heterozygous deletions were proposed if the calculated values were below 0.85.The locus was considered as amplifi ed if the smallest of the range exceeded 1.5 [15].

Results and Discussion
An analysis of the gene copy number was perfor med for 18 samples of ccRCC and corresponding to them normal tissues (Table 1).
The PPM1M gene was deleted in 55.6 % of cases (nine cases of heterozygous deletions and one homozygous deletion).Amplifi cation were observed in 16.7 % (three samples), while 27.8 % cases were unchanged (fi ve samples).Previously we have reported that the expression level of PPM1M altered in 50 % of cases in ccRCC tumors [8].
The product of PPM1M gene is a serine threonine phosphatase from the PPM family (Mg 2+ -and Mn 2+dependent).Phosphorylation is an important post-translation modifi cation that regula tes many signaling systems and it is an effective way to control cellular internal and external signal responses [16].Phosphatases together with kinases regulate phosphorylation and are involved in many physiological processes, such as cell migration, pro liferation, intracellular substrate localization, apo p tosis, differentiation, metabolism, and immune sys tem response [17].
Phosphatases are also involved in the cell cycle regulation [18], therefore they are considered as suppressors of tumor growth.Most probably, they should be inactivated in the process of carcinogenesis.One of the functions of PPM1M is the inhibition of the IL-1-induced activation of NF-kappa-B.The IL-1 is a cytokine that has different functions in control of infl ammation.[9].It is yet un known how PPM1M is involved in cancerogenesis [19].
Deletions in the PRICKLE gene were found in 50 % of samples (eight samples heterozygous deletions and one homozygous deletion).In 38.9 % (se ven samples) there were no changes in the copy num ber, while amplifi cation was observed in 11.1 % of cases (two samples).According to our previous da ta, the expression level of PRICKLE2 altered in 83 % of cases [8].
The PRICKLE2 protein is a member of WNT-cascade that activates Fz-receptors on the cell surface.This pathway regulates the planar polarization of the cells that differ from the basal-apical polarization and is, actually, perpendicular to the latter [20].Apart that the PRICKLE2 is attached to the inner surface

631 [1.575; 1. 686]
N o t e: Samples, which displayed ratio <0.35 and where gene was considered as homozygously deleted are marked bold italic.Samples, which displayed ratio <0.85 and ≥0.35 and where genes were considered as heterozygously deleted are shown in italic.