Urological Oncology 168 Urology Journal Vol 7 No 3 Summer 2010 Genetic Aberrations of the K-ras Proto-oncogene in Bladder Cancer in Kashmiri Population Mahoor S. Nanda,1,2 A. Syed Sameer,2 Nidda Syeed,2 Zaffar A. Shah,2 Imtiyaz Murtaza,1 Mushtaq A Siddiqi,2 Arif Ali1 Purpose: To assess the frequency of specific point mutations in the K-ras gene in a group of Kashmiri patients with bladder cancer. Materials and Methods: We analyzed the incidence of K-ras exon 1 gene mutations in tumors and surgical margins in 60 patients with transitional cell carcinoma of varied clinical stages and histological grades using the polymerase chain reaction-single strand conformation polymorphism and DNA sequencing. Results: A significant correlation was found between the K-ras, the lymph node status, and tumor recurrence (P < 0.05). Also, smokers and patients with higher tumor grade showed a significantly higher relative risk of developing K-ras mutations than the normal ones. Conclusion: K-ras exon 1 gene mutations were found with low frequency in the bladder cancer tumors from Kashmir valley, which suggests that K-ras gene might be involved in a sub-set of bladder tumors, but it needs further investigation on a larger cohort sample to authenticate the current findings. Urol J. 2010;7:168-73. www.uj.unrc.ir Keywords: proto-oncogene protein, urinary bladder neoplasm, ras genes, genetics 1Department of Biosciences, Jamia Millia Islamia, Jamia Nagar, New Delhi. India 2Department of Immunology and Molecular Medicine, Sher-I-Kashmir Institute of Medical Sciences, Soura, Srinagar, Kashmir, India Corresponding Author: Arif Ali, PhD Department of Biosciences, Jamia Millia Islamia, Jamia Nagar, New Delhi, 110011 Tel: +91 194 2401 013 Fax: +91 194 240 3470 E-mail: mousavi786@gmail.com Received August 2009 Accepted February 2010 INTRODUCTION Bladder cancer, which is the fourth most incident cancer in the USA and affects more than 350 000 subjects worldwide, is one of the leading causes of mortality.(1,2) In Kashmir, the northern region of India, bladder cancer is considered as the 9th most common cancer.(3) The ras gene family consisting of 4 functional genes, Harvey ras (H-ras), Kristen ras (K-ras) A and B, and Neuroblastoma ras (N-ras), encode highly similar and conserved proteins with a molecular weight of 21 kDa.(4) These closely related proteins are localized in the internal part of the cell membrane and have intrinsic GTPase activity, which regulates their cellular activity. (5) The main function of the ras proteins is to induce activation of downstream kinases belonging to mitogen-activated protein kinase pathway, which in turn results in continuous mitogenic signaling and transformation of immortalized cells.(6) Because of their active involvement in proliferative and/ or differentiative signals within the growing cell, ras genes are the most common targets for somatic gain- of-function mutations in almost all human cancers that lead to the formation of constitutively active proteins due to altered intrinsic GTPase activity.(7) Mutated ras genes are associated with 15% to 30% of all human cancers, with highest frequencies Genetic Aberrations of K-ras in Bladder Cancer in Kashmir—Nanda et al 169Urology Journal Vol 7 No 3 Summer 2010 associated with pancreatic, lung, and colon carcinomas.(8,9) These mutated ras genes encode constitutively active proteins, most commonly with single amino acid substitutions at residues 12, 13, or 61 causing usually the loss of intrinsic GTPase activity of the proteins.(10) The first report of the activating ras mutations in bladder tumors was made by Bos in T24 cell line.(11) Since then, many studies have been carried out on human bladder tumors across the globe, and reports of different types of activating ras mutations involved in the tumorigenesis are well documented in the available literature.(12-19) Because of these observations and the possibility of activating ras mutations to be involved in the tumorigenesis of bladder cancer as well as various suspected etiological factors, the present study was carried out in ethnic Kashmiri population to investigate the frequency of specific point mutations in the K-ras gene and to correlate them with the various etiological parameters to which our population is exposed. MATERIALS AND METHODS Sample Collection Surgically resected specimens of 60 patients were collected from Department of Urology, Sher-I- Kashmir Institute of Medical Sciences, Kashmir, India. The study protocol was approved by the Research Ethics Committee of Sher-I-Kashmir Institute of Medical Sciences. Informed consent was obtained from each patient and/or guardian on pre-designed questionnaire (Available on request). Data regarding age, sex, and smoking history of the patients were obtained. Patients who had received previous chemotherapy for a metastatic disease were excluded. In order to avoid evaluator variability, resected tissue specimens were brought fresh from the theater to Department of Pathology, where they were meticulously examined by two independent and experienced pathologists. The excision of the tumor was histologically proven by examination of the resected margins. All tumors were histologically confirmed to be transitional cell carcinoma. The specimens (both tumor and adjacent normal tissues) were snap-frozen at -70oC immediately until further analysis. Patients were followed up for 1 year after the surgical resection of the tumor. DNA Extraction We extracted DNA from primary tumors and adjacent noncancerous tissues, using the DNA Extraction Kit II (Zymo Research), for examining mutations in the K-ras exon 1 gene. Polymerase Chain Reaction-Single Strand Conformation Polymorphism (PCR-SSCP) Exon 1 of the K-ras (containing hot spot codons 12 and 13) was amplified using the previously described specific primers.(20) Polymerase chain reaction was performed in a 25 μl volume containing 50 ng of genomic DNA, 1 × PCR buffer containing 15 mM MgCl2, 100 μM each of dATP, dGTP, dTTP, dCTP, 1.5 unit of Taq DNA polymerase (Biotools, Spain), and 10 μM of forward and reverse primers. The amplification program was as initial denaturation at 95°C for 5 minutes, 35 cycles of denaturation at 94°C for 30 seconds, annealing at 48°C for 30 seconds, extension at 72°C for 30 seconds, and final extension at 72°C for 7 minutes. In every instance, positive human genomic DNA (Genei, India) was also amplified as internal control. Polymerase chain reaction products were run on 2% agarose gel and analyzed under a ultra violet illuminator (Figure 1). The single strand conformation polymorphism analysis of the amplicons of exon 1 of K-ras was performed on 6% non-denaturing polyacrylamide gel electrophoresis utilizing either non-radioactive silver staining or radioactive procedures.(20) The purified PCR amplicons of the tumor samples showing mobility shift on SSCP analysis (Figure 2) and randomly chosen normal samples were used for direct DNA sequencing (Figure 3), using the automated DNA sequencer ABI prism 310. Statistics Fisher’s exact test (one-tailed) was used to evaluate the association between clinicopathological variables in case of K-ras. P values less than .05 were considered statistically significant. Genetic Aberrations of K-ras in Bladder Cancer in Kashmir—Nanda et al 170 Urology Journal Vol 7 No 3 Summer 2010 RESULTS The mean age of the patients was 61 years and 63.3% of the patients were older than 50 years. About 83.4% were men, 80% were rural, and 75% were smoker (Table 1). The mutational examination of exon 1 of K-ras gene revealed an overall K-ras mutation in 7 subjects aggregating to about 11.67% of the studied sample, which included 6 transitions and 1 transversion. Transition mutation was of only one type G→A, and sole transversion was G→C. Further more, of a total of 7 mutations, 4 affected codon 12 and 3 affected codon 13 (Table 2). Two cases were G12D (GGT > GAT), 2 cases were G13D (GGC > GAC), 2 cases were G12S (GGT > AGT), and 1 case was G13R (GGC > CGC). The mutations in exon 1 of K-ras gene were found relatively more at codon 12 (57.1%) than codon 13 (Table 2), which is consistent with the already available data.(21) No germ line mutations were found, indicating that in every case the change was somatic. Statistical analysis of the mutants with respect to various clinicopathological variables revealed a significant association (P < .05) between the K-ras mutation, the lymph node status, and tumor recurrence. Furthermore, smokers and patients with higher tumor grade showed a significantly higher relative risk of developing a bladder cancer than the normal ones (OR > 2) (Table 1). DISCUSSION A number of different studies carried out on various different cancers have demonstrated some hot spot regions in ras gene family that are susceptible to point mutations, the frequent among them are changes of glycine to valine/ aspartate/serine at codon 12, glycine to arginine/ cysteine at codon 13, and glutamine to arginine/ lysine/leucine at codon 61.(21,22) Figure 1. Amplified DNA fragments of exon 1 of K-ras (162 bp amplicon) gene; First Lane represents 100 bp molecular ladder and rest represent amplicons from different tumor tissues. Figure 3. Partial nucleotide sequences (reverse) of the normal and mutants – T3, T46 in exon 1 of Kirsten ras oncogene. Figure 2. A radioactive SSCP analysis of K-ras exon 1 showing mobility shifts in tumor sample. Genetic Aberrations of K-ras in Bladder Cancer in Kashmir—Nanda et al 171Urology Journal Vol 7 No 3 Summer 2010 The incidence of ras mutation varies and is greatly dependent on the tissue or cell type from which the cancer cells are derived. Although ras mutations occur in 75% to 95% of pancreatic carcinomas and 50% of colon carcinomas, they are rare in several other neoplasms.(23-25) Mutations at codon 12 of K-ras are infrequent in the bladder cancer.(26,27) In a recent study, Jebar and colleagues have found K-ras mutations in 3 of 98 patients with the bladder cancer.(16) Interestingly, experimental studies on transgenic mice have shown that tissue-specific expression of a K-ras transgene in the urothelium leads to urothelial hyperplasia and superficial papillary tumors.(28) These observations suggest that activation of ras may contribute to early steps of carcinogenesis in the bladder.(16,26-28) Results of the present investigation confirmed the role of K-ras mutations in the development of urinary bladder carcinoma, as we found 11.67% tumors (7/60 tumors) having mutations in this gene in Kashmiri population, which is in consistent with many of the previous studies on bladder cancer.(12,16,27) Furthermore, in this study, we also found a significant correlation between the K-ras mutant status and the lymph node involvement and tumor recurrence, suggesting a possible role of K-ras proteins in the metastasis of the cancer. The activating mutations of ras proteins have been previously implicated in all aspects of the malignant tumor, especially cellular proliferation, transformation, invasion as well as metastasis.(29) Furthermore, Campbell and Der(30) demonstrated that activation of ras proteins causes an increase in the transformative, invasive, and metastatic properties of the murine fibroblast cells.(31) Other studies also showed the same concomitant results.(32,33) Since the transformation to a metastatic phenotype requires many changes in cell-cell adhesion, Yan and associates have Variable Total (N = 60) (%) Mutants (M = 7) (%) P; OR; CI (95%) Sex Males: 50 (83.4) Females: 10 (16.6) Males: 6/50 (12.0) Females: 1/10 (10.0) .67, 1.2273; 0.1313 - 11.4727 Age ≤50: 22 (36.7) >50: 38 (63.3) ≤50: 3/22 (13.6) >50: 4/38 (10.5) .50, 1.3421, 0.2713 - 6.6394 Dwelling Rural: 48 (80) Urban: 12 (20) Rural: 5/48 (10.4) Urban: 2/12 (16.7) .42, 0.5814; 0.0982 - 3.442 Smoking status Smokers: 45 (75) Nonsmokers: 15 (25) Smokers: 6/45 (13.3) Nonsmokers: 1/15 (6.7) .43; 2.1538; 0.2379 - 19.5038 Differentiation grade II: 21 (35) III +IV: 39 (65) II: 4/21 (19.1) III + IV: 3/39 (7.7) .18; 2.8235; 0.5676 - 14.0446 Histological type S: 35 (58.3) MI: 25 (41.7) S: 3/35 (8.5) MI: 4/25 (16.0) .31; 0.4922; 0.0999 - 2.4256 Lymph node status NO: 55 (91.7) YES: 5 (8.3) NO: 4/55 (7.3) YES: 3/5 (60.0) .009; 0.0523; 0.0067 - 0.4096 Tumor Recurrence NR: 47 (78.3) R: 13 (21.7) NR: 2/47 (4.3) R: 5/13 (38.5) .003; 0.0711; 0.0117 - 0.432 Stage PT1: 39 (65) PT2: 21 (35) PT1: 4/39 (10.3) PT2: 3/21 (14.3) .46; 0.6857; 0.1383 - 3.4007 Table 1. Clinico-epidemiological variables of the patients with bladder cancer versus the mutant phenotypes of the K-ras exon 1 gene Patient ID Age / Sex Rural/ Urban Histopathological Stage Histopathological Grade Smoking Status Type Codon Number Base Change Amino acid Change T3 80/M R PT1 II Ever MI 12 GGT>GAT Gly>Asp T11 45/M R PT2 III Ever S 12 GGT>AGT Gly>Ser T17 60/M R PT1 II Ever MI 12 GGT>GAT Gly>Asp T22 81/F U PT1 IV Ever MI 13 GGC>CGC Gly>Arg T42 65/M R PT2 II Ever MI 13 GGC>GAC Gly > Asp T46 41/M R PT1 II Never S 12 GGT>AGT Gly>Ser T55 38/M U PT2 IV Ever S 13 GGC>GAC Gly > Asp Table 2. Details and nature of K-RAS exon 1 gene mutations in patients with bladder cancer from Kashmir valley M, indicates male; F, female; R, rural; U, urban; MI, muscle invasive; S, superficial; and Base change, mutated or inserted nucleotide underlined. Genetic Aberrations of K-ras in Bladder Cancer in Kashmir—Nanda et al 172 Urology Journal Vol 7 No 3 Summer 2010 reported that mutant K-ras, but not H-ras, causes disruption of the adhesive qualities of the mutant cell due to oncogene’s ability to interfere with the maturation of cell surface integrins.(34) Furthermore, in our study, a significantly higher relative risk of K-ras mutant status was observed in patients with history of smoking and/or having higher tumor grade than the normal ones (OR > 2) (Table 1). This observation is in harmony with the other studies, where smoking has been implicated as one of the risk factors of the bladder cancer.(35) In our study, almost 75% of the patients with the bladder cancer were smoker and they were almost two times more prone to K-ras mutations than the normal ones (OR = 2.15). The K-ras activating mutations that were identified in this study were missense in nature, 57.15% were found at codon 12 and 42.85% at codon 13; these results were consistent with other reported studies.(12,20) The mutations of codons 12 and 13 are the most common genetic aberration in K-ras, involving the substitution of the active site of glycine residue, which in turn changes the functionality of K-ras protein.(20) The substitution at codons 12 and 13 can either alter intrinsic GTPase activity of K-ras protein or change its ability to interact with wide variety of regulators.(33) The effectiveness of the substitution depends on the amino acid that replaces the original glycine (at 12/13). In this study, we also found that all K-ras mutants were G>R or G>S or G>D variants. It has been reported that replacement of glycine residues with valine or arginine has the aggressive transforming capability followed closely by serine and glutamine variants. (7,21,36,37) All of the above variants have the diminished GTPase activity due to which they remain activated under basal conditions, too, as there is no switching back to inactive GDP-bound form.(7) Substitution by proline makes K-ras protein resistant to GAP activity and hence is less aggressive in phenotypic expression.(37) Of a total of 7 missense mutations, 6 (85.7%) were transitions and only 1 (14.3%) was transversion. All transistions were A>G type, 4 affecting codon 12 and 3 affecting codon 13. The sole transversion of G>C occurred at codon 13 causing replacement of glycine to arginine. The G>A transitions have been already reported to be the most common genetic change in K-ras and incidentally these aberration also score second in the human gene mutation database after C>T conversion.(38) CONCLUSION To sum up, we can say that K-ras exon 1 gene mutations were found with low frequency in bladder cancer tumors from Kashmir valley, which suggests that K-ras gene is involved in a sub-set of bladder tumors. Nevertheless, these observations need further investigations in a bigger cross section of the patients with bladder cancer and relevant controls. CONFLICT OF INTEREST None declared. REFERENCES 1. Jemal A, Siegel R, ward E, Hao Y, Xu J, Thun MJ. Cancer statistics, 2009. CA Cancer J Clin. 2009;59:225-49. 2. Ferlay J, Randi G, Bosetti C, et al. Declining mortality from bladder cancer in Europe. BJU Int. 2008;101: 11-9. 3. Dhar GM, Shah GN, Naheed B, Hafiza. Epidemiological trend in the distribution of cancer in Kashmir Valley. 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