Frequent ras gene mutations in squamous cell cervical cancer

Cancer Letters 95 (1995) 29-32

ELSEVIER

CANCER LETTERS

Frequent ras gene mutations in squamous cell cervical cancer Y.F. Wonga,*, Tony K.H. Chunga, T.H. Cheunga, SK. Lama, Y.G. Xub, Allan M.Z. Changa aDepamnent of Obstetrics and Gynaecology, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong bShanghai Institute of Biochemistry, Sinica Academia, Shanghai, China

Received24 February1995;revisionreceived24 May 1995;accepted25 May 1995

Abstract

Eighty samplesof cervical invasive squamouscell carcinoma were examined for ICZS gene mutations using polymerase chain reaction (PCR) followed by restriction enzyme digestion. We found 28 (35%) cervical cancerscontained rus mutations at H-ras codon 12,49 (61%) at K-ras codon 12, and 5 (6%) at K-ras codon 13. There were no significant differences in incidenceof the ras gene mutations amongdifferent histologic gradesor clinical stagesof the cancer(P > 0.05). This result suggeststhat ras mutation may be an important step involved in a substantial number of cervical carcinoma. The interaction of ras with other genesand/or events may also be involved in pathogenesisof this malignancy. Keywords:

Ras

gene;Mutation; Cervical cancer;Squamous

1. Introduction Carcinoma of the uterine cervix is one of the most common gynecological cancers in Hong Kong. Epidemiologically, the occurrence of the cancer is associated with human papillomavirus (HPV) infection. However, transfection in vitro with HPV alone is insufficient to cause transformation, and in vivo only a small proportion of women who are HPV-infected are estimated to develop cervical carcinoma over 25 years [ 11. The evidence suggests that other events must occur in host cellular genes. Since HPV 16 DNA has been shown experimentally to be capable of incorporating activated H-rus in transforming baby rat kidney cells [2], rus activation is a possible additional event in the pathogenesis of these cancers. * Corresponding author.

Mutations of the rus family oncogenes are a common occurrence in human cancers. Up to 90% of pancreatic adenocarcinomas have an activated K-ras gene, while rates of at least 30% have also been reported for non-small cell lung cancer, colon cancer, seminoma, melanoma, and thyroid cancer [3,4]. We have investigated involvement of H-ras and K-ras gene mutations in cervical cancers. 2. Materials and methods and samples Tumor tissue from a total of 80 women with cervical squamous cell carcinoma was obtained, frozen in liquid nitrogen and stored at -70°C pending analysis. The samples were confirmed histologically to contain tumor tissue. Their matched blood was also collected and stored.

2.1. Patients

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2.2. DNA extraction High molecular weight DNA was prepared from the tumor specimen and blood using conventional procedures [5]. Tumor tissue and blood were digested by Proteinase K and DNA was isolated after phenol/chloroform extraction and ethanol precipitation. 2.3. Determination of ras gene mutations Oligonucleotide primers used for PCR amplification of H-ras and K-ras are as follows: H-ras 5’:5’-GAG ACC CTG TAG GAG GAC CC 3’; H-ras 3’:5’-GGG TGC TGA GAC GAG GGA CT 3’; K-ras 5’:5’-ACT GAA TAT AAA CTT GTG GTA G‘IT GGA CCT 3’; K-ras 3’:5’-TCA AAG AAT GGT CCT GGA CC 3’. The primers were synthesized by Operon Technologies Inc., Alameda, USA. PCR amplification was performed in a volume of 50~1 with 100 ng of specimen DNA containing 10 mM Tris-HCI (pH 8.3), 1.5 mM MgClz, 50 mM KCl, 1.25 mM dNTPs, 250 ng of each primer, and 2.5 units of Taq DNA polymerase. A total of 30 cycles of PCR were carried out with a Perkin-Elmer Cetus Thermal Cycler, each cycle consisting of denaturation for 2 min at 94°C annealing for 1 min at 55°C and elongation for 1 min at 72”C, which was preceded by heating for 5 min at 94°C and followed by a final extension for 10 min at 72°C. Aliquots of 15~1 of PCR amplified samples were digested with the restriction enzymes MspI (H-ras codon 12), BstNI (K-ras codon 12) or HphI (K-ras codon 13) in a total volume of 20~1 under conditions recommended by the manufacturer (New England Biolabs, Beverly, MA, USA). Digestions were incubated at appropriate temperatures (MspI and HphI, 37°C; BstNI, 60°C) for 3 h. DNA was electrophoresed through 3% agarose. Gels were

Table 1 Expected size of fragments in PCR analysis of human H-rus gene codon 12, K-r-us gene codons 12 and 13 sequences Gene codon

Enzyme

Size of fragments

Normal allele

Mutant allele

H-rus codon 12 K-ras codon 12 K-rus codon 13

MspI BstNl HpHl

312 157 157

21.55236 29, 114, 14 157

21,291 143, 14 114.43

stained with ethidium bromide and photographed on an ultraviolet light transluminator. A detailed summary of the expected fragments generated after the analytical restriction enzyme digestion step is shown in Table 1. Statistical analysis was performed by Pearson’s chi-square test using the STATXACT program (CYREL Software Cooperation, Cambridge, UK). 3. Results and discussion There were no ras mutations in any of the matched blood samples in the 80 women with squamous cell cervical cancer. As shown in Table 2, 28 of 80 (35%) cervical cancers contained ras mutations at H-ras codon 12,49 (61%) at K-ras codon 12, and 5 (6%) at K-ras codon 13. Fifty-two cervical cancers contained K-ras codon 12 and/or 13 mutations, while 69 contained at least one of the three codon mutations. Histologic grading was according to WHO criteria. There were 26 grade 1 tumors, 42 grade 2 tumors, and 12 grade 3 tumors. Clinical staging was determined by the criteria of the International Federation of Gynecology and Obstetrics (FIGO). There were 57 early stage (I-II) tumors, and 23 advanced stage (III-IV) tumors. There were no significant differences in incidence of the ras gene mutation among different histologic grades or clinical stages of the cancer (P > 0.05). A representative example obtained from two tumor samples is shown in Fig, 1. One predicted MspI-digested fragment of Table 2 Incidence of ras gene mutations in cervical carcinoma Cases

No. examined

H-ras 12

K-ras 12

K-ras 13

Total

80

28

49

5

Histologic grade 1 26 42 2 12 3 P value

9 15 4 1.000

12 19 8 0.403

3 1 1 0.342

Clinical stage I-II 57 II-IV 23 P value

24 4 0.066

36 13 0.766

4 1 0.552

Y.F. Wang et al. I Cancer Lerrers 9.5(I 995) 29-32

tant rmal

1

2

3

4

5

Fig. 1. Determination of H-ras codon 12 mutations in cervical cancer using PCR amplification followed by MspI digestion. Lane 1, Hae III digested @X 174 DNA marker. Numbers on the left denote the sizes of DNA marker bands. Lane 2, normal female blood DNA containing non-mutated ras sequence.Lane 3, tumor sample with rus mutation. When both non-mutated and mutated sequencesare present, two bands of 291 and 236 bp are seen. Lane 4, tumor sample without mutation at H-ras codon 12. Lane 5 control without template DNA.

291 bp with H-rus at codon 12 mutation was observed in lane 3, it is interpreted as indicative of heterozygous H-r-us 12 mutation in that cervical cancer. The role of the rus genes in human cancer was first identified more than a decade ago when it was noted that some human cancer cells contained DNA sequences capable of inducing neoplastic transformation of established rodent cell lines. The rus gene family consists of three genes, K-, H- and N-rus, which encode similar 21 000 Da membrane-bound proteins. The rus genes can be activated by point mutations at codon 12, 13 or 61, resulting in an enhanced ability to retain its guanosine triphosphatebound form and oncogenic potential. Genetic disruption of the activated K-rus allele in each of two different human colorectal cancer cell lines has been shown to inhibit the tumorigenic properties of the cells [6], which indicates the critical role of mutant rus genes in human tumorigenesis. Mutations of the rus gene have been found in a wide variety of tumors although the incidence varies greatly. In this study a simple non-radioactive method was used for the detection of mutations of H-ras gene at codon 12 and K-rus gene at codons 12 and 13 in a total of 80 invasive cervical cancers. Previous studies

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have shown that PCR amplification followed by enzymatic cleavage is a highly sensitive method for detecting single point mutations in sequences of the rus family [7,8]. This method can detect activated rus genes in a tumor sample containing as few as lo3 cells [7]. The sensitivities of assay for K-rus 12 and H-rus 12 were 1: 16 and 1:9 dilution of mutated tumor cells to normal cells, respectively [7,8]. We have also verified the sensitivity of assay for K-rus 13 mutation as 1:lO. These results have demonstrated that activation of rus, especially K-rus gene, is a common event in cervical cancer. Unfortunately, we have not checked for K-rus mutation at codon 61 as this mutation was not as common as that of codon 12 and 13. The fact suggests that these changes may represent a genetic alteration involved in the tumorigenesis of cervical squamous cells. In addition we correlated our findings with histologic grade and clinical stage of the cancer. However an association between rus mutation and histologic grade was not observed in cervical cancer. It seems that rus mutation does not play an important role in the cellular pathway of differentiation which has been shown in ovarian and colorectal epithelial cells [9, lo]. We found rus mutations were commonly observed in early (I-II) as well as late (III-IV) stages of invasive cervical cancer. This suggests that these alterations may not confer progression potential to cervical cancer. The data reported here also lend some insight into a seeming discrepancy in the literature concerning the extent of rus gene mutations in cervical cancer. Riou et al. [ 1l] reported a relatively high incidence of H-rus mutation and this also correlated with the clinical stage of the disease. One mutation at codon 12 was detected in 47 early stage tumors (stage I and II) where as 7 mutations were detected in 29 late stage tumors (stage III and IV), and statistical significance was present. The findings of our study were completely different. More early stage tumors exhibited codon 12 mutation than that of tumors of late stage disease. It is difficult to explain such discrepancies as the distribution of clinical stage and histology were similar in both studies. However, there are no distinguishing clinical features of cervical cancers in these populations compared with subjects in the west. To complicate the picture even further, Bos [ 121 and Wilczynski et al. [ 131 found no rus point

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mutations in cervical carcinoma. Ito et al. have reported K-rus mutations in two out of seven cervical adenocarcinoma [ 141. While many studies show that rus gene mutations have a critical role in the development of some cancers, the role of these mutations in the development of cervical cancers remain unclear. Further investigation will be needed to define the functional role and clinical significance of rus mutations in human cervical cancer. Further studies of ras mutations obtained from patients with premalignant lesions of cervical cancer are needed to elucidate the role of ras mutations as initiating genetic events in this cancer. Acknowledgments We thank Mr. Albert Cheung for assistance with statistical analysis, Ms. Panty Tam and Ms. Flora Tam for technical assistance, Gynaecologic Cancer Laboratory, Department of Obstetrics and Gynaecology, The Chinese University of Hong Kong for providing reagents and equipment. References [I] [2]

[3]

Zur Hausen, H. (1988) Papillomaviruses in human cancers. Mol. Carcinogenesis, 1, 147-1.50. Matlewsheski, L., Schneider, J., Bnaks, L., Jones, N., Murray, A. and Crawford, L. (1987) Human papillomavirus type 16 cooperates with activated ras in transforming primary cells. EMBO J., 6, 1741-1746. McCormick, F. (1989) rus oncogenes. In: Oncogenes and the Molecular Origins of Cancer, pp. 125-145. Editor: R. Weinberg. Cold Spring Harbor Press, Cold Spring Harbor, NY.

r41 Bos, J.L. (1989) r~ls oncogenes

in human cancer: a review, Cancer. Res., 49.4682-4689. [51 Sambrook, J., Fritch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. L61 Shirasawa, S., Furuse, M., Yokoyama, N. and Sasazuki, T. (1993) Altered growth of human colon cancer cell lines disrupted at activated Ki-ras. Science, 260, 85-88. [71 Jiang, W., Kahn, S., Guillem, J., Lu, S. and Weistain, I.B. (1989) Rapid detection of nzs oncogenes in human turnouts: applications to colon. esophageal and gastric cancer. Oncogene, 4.923-928. A., Gill, R., Thomas, D.M., GilF31 Levi, S., Urbano-lspizua, bertson, J., Foster, C. and Marshall, C.J. (1991) Multiple Kras codon 12 mutations in cholangiocarcinomas demonstrated with a sensitive polymerase chain reaction technique. Cancer Res., 5 1, 923-928. P., Olschwang, S., Delattre, 0.. Validire, P., 191 Laurent-Puig, Melot, T., Mosseri, V., Salmon, R.J. and Thomas, G. (1991) Association of Ki-ras mutation with differentiation and tumor-formation pathways in colorectal carcinoma. Int. J. Cancer, 49, 220-223. ttu1 MO, S.C.H.S., Bell. D.A., Knapp, R.C.. Fishbaugh, P.M., Welch, W.R., Muto, M.G., Berkowwitz, R.S. and Tsao, S.W. (1993) Mutation of K-rus protooncogene in human ovarian epithelial tumors of borderline malignancy. Cancer Res., 53, 1489-1493. [I II Riou, G., Barrois, M., Sheng, Z., Duvillard, P. and Lhomme, C. (1988) Somatic deletions and mutations of c-Ha-rus in human cervical cancers. Oncogene, 3,329-333. 1121Bos, J.L. (1988) The rus gene family and human carcinogenesis. Mutat. Res., 195, 255-27 1. S., Cook, N. and Srivastan, E. (1992) Altera[I31 Wilczynski, tions of RAS oncogenes in cervical cancers. Lab. Invest., 66, 70A. [I41 Ito, K., Sasano, H.. Ozawa, N., Sate, S. and Yajima, A. (1992) K-ms point mutation and immunolocalization of EGFR, c-myc and c-e&2 oncogene products in human endometrial and cervical adenocarcinoma. Lab. Invest.. 66. 65A.