Reles, Angela : MOLECULAR GENETIC ALTERATIONS IN OVARIAN CANCER The Role of the p53 Tumor Suppressor Gene and the mdm2 Oncogene

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Kapitel 6. SUMMARY AND CONCLUSIONS

Background: The p53 tumor suppressor gene, the ’guardian of the genome‘ (Lane, 1992), is involved in key responses to genotoxic stress and plays a central role in the maintenance of genomic stability. Activation of p53 in response to DNA damage is associated with a rapid increase in its level and with an increased ability of p53 to bind DNA and mediate transcriptional activation. Upon low or repairable levels of DNA damage, p53 mediates delay or arrest of replication to give the cell the opportunity to repair the damage before its fixation and propagation. Upon high or irreparable DNA damage, p53 promotes apoptosis. Mutations of the gene which inactivate some or all of p53´s functions provide a selective advantage for clonal expansion of neoplastic cells.

Functional p53 must be tightly regulated and the mdm2 (murine double minute 2) gene plays an important role in this regulation. The transcription of the mdm2 oncogene is induced by the p53 gene after DNA damage. The MDM2 protein then binds to p53 and downregulates it in an autoregulatory feedback loop. MDM2 inhibits p53 transcriptional activity, promotes nuclear export to the cytoplasm and rapid degradation of the p53 protein. It, therefore, plays a crucial role as a regulator of p53 in early embryonic development and for recovery of cellular proliferation after repair of DNA damage.

The aim of this study was to analyze the frequency and type of p53 and mdm2 alterations in ovarian cancer and to correlate the results with clinico-pathological variables, response to chemotherapy and clinical outcome of the patients.

Methods: 178 cases of primary epithelial ovarian cancer, snap frozen and stored at -80°C, were used in this study. Mutations of the entire p53 coding sequence (exons 2-11) were analyzed by SSCP (Single Strand Conformation Polymorphism) and DNA-sequencing. p53 protein expression was analyzed immunohistochemically with the monoclonal Ab DO7 in frozen tissue sections. p53 was considered to be overex-pressed, if more than 10% of the nuclei in the sections showed immunostaining. Clinical follow-up of the ovarian cancer patients was documented up to 12 years. In the total cohort, the median time of follow-up was 31 months and among the survivors, 52 months. 74 patients who received Cis- or Carboplatin in combination with Cyclophos-phamide were evaluated for platinum-sensitivity.

Analysis of the mdm2 gene for alternative splicing was performed by using PCR amplification of full length mdm2 cDNAs. Total RNA was reverse transcribed into cDNA, and then the cDNA was amplified by nested PCR. Cases with RT-PCR products other than the full length mdm2 cDNA were cloned into a pCR 2.1 vector and


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sequenced or the PCR products were directly sequenced. p53 and mdm2 splice variant cDNAs were cloned into a pcDNA3 expression vector. p53 and MDM2 full length and mdm2 splice variant proteins were expressed in vitro by transient transfection of the pcDNA3 constructs into HeLa-cells. The HeLa cells had been previously infected with the vTF7-3 virus, a recombinant vaccinia virus encoding bacteriophage T7 RNA polymerase.

Results: p53 mutations were seen in 56% (99/178) of the ovarian cancer cases of which 72% were missense mutations. p53 overexpression (> 10% positively stained nuclei) was seen in 62% (110/178) of cases. 62% of the p53 mutations were located in the evolutionary highly conserved domains of the gene. p53 mutations were significantly correlated with shortened time to progression (p=0.028) and poor overall survival of the patients (p=0.015). Comparing clinical outcome of patients with mutations in highly conserved domains with those who had either wildtype p53 or a mutation in a nonconserved domain, the difference in time to progression (p=0.007) and overall survival was even more obvious (p=0.005). Furthermore, time to progression and overall survival were seen to be shorter in patients with p53 overexpression as compared to normal p53 expression, but the results only reached marginal statistical significance (p=0.073, p=0.058). Patients with p53 overexpression (p=0.001) or p53 missense mutations (p=0.008) were significantly more often platinum resistant than patients without p53 alterations. Overall, the most favorable prognosis in terms of overall survival was seen in patients who had wildtype p53 sequence and normal p53 expression, as opposed to those who had either one or both alterations of the p53 gene and protein (p=0.007).

Besides p53 mutations 45 sequence alterations were identified and considered polymorphisms. The codon 72 argininerarrproline polymor-phism was identified in 5% (9/178) of the cases. Furthermore, alterations were found at codon 36 (exon 4), codon 213 (exon 6), codon 224 (exon 6), and codon 231 (exon 7). The 16 basepair repeat polymorphism of intron 3 was identified in 14% (24/178) of the cases. Three novel intron alterations were identified by sequencing the introns 6 and 10. A grarrc nucleotide exchange at position 13964 in intron 6 was found in two cases. An ararrt polymorphism was found at nucleotide 17708 of intron 10 in a frequency of 1.1% and a crarrt polymorphism at nucleotide 18550 of intron 10 in a frequency of 2.3%. Both intron 10 polymorphisms were confirmed in normal tissue of the patients. Most of the cases (7/8) with intron 6 or intron 10 polymorphisms showed p53 over-expression.

The mdm2 gene was neither amplified nor overexpressed in any of the ovarian cancer cases. An mRNA transcript of 7.4 kb, 5.5 kb and 2.8 kb was seen at normal levels in all ovarian carcinomas. However, mdm2 alternative or aberrant splicing was


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seen in 66/92 (72%) of the ovarian carcinomas, 7/9 (78%) of borderline tumors, 5/6 (83%) of benign ovarian cystadenomas and 11/20 (55%) of the normal ovarian tissues. A total of 30 splice variant sequences were identified. 67% of these had a partial and 7% a complete loss of the p53 binding site. 28/30 splice variants do not splice at exon/intron boundaries but use cryptic splice sites within exons and were therefore considered aberrant splice variants.

A 654 bp splice variant (mdm2-b) was expressed in 41% of ovarian carcinomas, but only in 1/9 (11%) borderline tumors, none of the benign cystadenomas, and 1/20 (5%) normal ovaries. This splice variant splices out exon 4-11, including 90% (81 of 90 amino acids) of the 3‘end of the p53 binding domain as well as the nuclear localization signal (NLS) and the nuclear export signal (NES). Expression of this splice variant in ovarian carcinomas was significantly correlated with poor grade of differentiation (p=0.004), residual tumor after surgery (p=0.004), and high S-phase fraction (p=0.016) of ovarian carcinoma. Presence of mdm2-b was furthermore correlated with p53 protein overexpression (p=0.018). A small splice variant of only 221 bp, which has lost most of the functional mdm2 domains was present in only 16% of the ovarian carcinomas but in 5/9 (56%) of borderline tumors, 1/6 of the cystadenomas, and interestingly also in 8/20 (40%) of the normal ovarian tissues. The 221 bp splice variant was correlated with early stage of ovarian cancer (FIGO I and II) and longer overall survival (p=0.048).

The absence of the full length mdm2 transcript respectively, the presence of mdm2 splice variants in general were correlated with a higher proportion of ovarian carcinomas, which were resistant or refractory against a platinum-based chemotherapy but were not correlated with survival.

Depending on the diagnosis for which the patient underwent surgery, different mdm2 RNA splicing patterns were seen in normal ovarian tissue. The presence of splice variants in the unaffected normal ovary was significantly correlated with the diagnosis of gynecological cancer, either in the contralateral ovary or in the uterine cervix or endometrium (p=0.017).

Conclusions: p53 mutations are present in more than 50% of epithelial ovarian carcinomas and are associated with early progression and shortened overall survival. Mutations in evolutionary conserved domains correlate with significantly shorter survival, compared to mutations in nonconserved regions and p53 wildtype sequence. p53 protein overexpression reached only marginal statistical significance as a predictor of clinical outcome. With multivariable analysis however, p53 alterations were not an independent prognostic factor. Evaluation of adjuvant treatment showed that p53 overexpression as well as p53 missense mutations were correlated with resistance to


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platinum-based chemotherapy. This provides further clinical evidence that the sensitivity of ovarian cancer cells for Cis- or Carboplatin depends on the efficient induction of apoptosis mediated by a functional p53 protein.

mdm2, which inhibits p53‘s transcriptional activity and downregulates it in an autoregulatory feedback-loop by promoting nuclear export and rapid degradation of the protein, was found to be frequently altered in ovarian carcinomas. mdm2 alternative and aberrant splicing was found in the majority of ovarian carcinomas and borderline tumors, but also in cystadenomas and normal ovarian tissue. A distinct difference in patterns of splice variants was notable in comparing benign and malignant tissues. While the mdm2-b splice variant was found in nearly 50% of the ovarian carcinomas and was associated with more advanced and histologically more aggressive tumors, smaller size variants were typically seen in early stage ovarian carcinomas and benign tissues.

The mdm2 splice variants mostly lack part, or all of the p53 binding sequence. mdm2 alterations appear to stabilize p53 protein and may cause p53 accumulation in the absence of p53 mutation. If these results are corroborated by other investigators, alterations of mdm2 may be considered to be an important step in carcinogenesis and may have important implications for the response to chemotherapy and clinical course of ovarian carcinomas.

Since most of the mdm2 splice variants lack various functional domains of the gene, such as the p53 binding site and the nuclear localization signal, the purpose of the alternative splicing remains unclear. But the fact that splicing is found in the majority of ovarian carcinomas and splice variants of identical sequence occur throughout this cancer cohort and in benign tissues, suggests that these splice variants have distinct, as yet unknown functions and are not simply a by-product of RNA processing.


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