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

Aus der Klinik für Frauenheilkunde und Geburtshilfe
der Charité Campus Virchow-Klinikum
(Direktor: Prof. Dr. med. W. Lichtenegger)


Medizinische Fakultät der Humboldt-Universität zu Berlin

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

Habilitationsschrift

zur Erlangung der Venia legendi
für das Fach

Gynäkologie und Geburtshilfe

vorgelegt von

Dr. med. Angela Reles

Präsident: Prof. Dr. rer. nat. J. Mlynek

Dekan: Prof. Dr. med. Joachim W. Dudenhausen

Eingereicht im: 28.03.2001

Öffentlich-wissenschaftlicher Vortrag: 04.12.2001

Gutachter:
Frau Prof. Dr. med. M. Kiechle
Herr Prof. Dr. med. D. Kieback


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Inhaltsverzeichnis

TitelseiteMOLECULAR GENETIC ALTERATIONS IN OVARIAN CANCER The Role of the p53 Tumor Suppressor Gene and the mdm2 Oncogene
Abkürzungsverzeichnis ABBREVIATIONS
1 INTRODUCTION
1.1Clinical and histopathological aspects of ovarian tumors
1.1.1Diagnosis and clinical aspects
1.1.2Histological classification of epithelial ovarian tumors
1.1.3Therapeutic aspects of epithelial ovarian tumors
1.1.4Clinical course and prognosis
1.2Molecular genetic alterations in ovarian tumors
1.2.1Hereditary ovarian tumors
1.2.2Sporadic ovarian tumors
1.2.3Gene therapy
1.3The p53 tumor suppressor gene
1.3.1p53 domains: structure and function
1.3.2p53 response to cellular stress: upstream events
1.3.3Downstream mediators of p53-dependent cell cycle arrest
1.3.4Mechanisms of p53-dependent apoptosis
1.3.5p53 and genomic stability
1.3.6The role of p53 in tumorigenesis
1.3.7Clinical implications
1.3.8p53 and efficacy of chemotherapeutic agents
1.4The mdm2 (murine double minute 2) gene
1.4.1Molecular structure of the mdm2 gene
1.4.2mdm2 expression in tumors
1.4.3The MDM2/p53 autoregulatory feedback-loop
1.4.4Clinical implications of mdm2 alterations
2 OBJECTIVES
3 PATIENTS, MATERIALS, AND METHODS
3.1Patients and clinical data
3.1.1Asservation and storage of tissue
3.1.2Study population and surgical therapy
3.1.3FIGO-stage, histology and grade of differentiation
3.1.4Adjuvant chemotherapy
3.1.5Clinical follow-up information
3.2Materials
3.2.1Plastic ware, chemicals, and consumables
3.2.2Standard buffers and solutions
3.2.3Standard gel electrophoresis components
3.2.4Chemicals
3.2.5Radio-chemicals
3.2.6Standard kits
3.2.7Monoclonal and polyclonal antibodies
3.2.8Laboratory equipment and other materials
3.3Methods
3.3.1Immunohistochemistry
3.3.1.1Sections of tissues and control cell lines
3.3.1.2Incubation with primary and secondary antibodies
3.3.1.3Peroxidase-antiperoxidase reaction
3.3.1.4Microscopic evaluation
3.3.2Molecular biology techniques
3.3.2.1Isolation of genomic DNA from frozen tumor tissue
3.3.2.2Isolation of genomic DNA from paraffin embedded tissue
3.3.2.3Polymerase Chain Reaction (PCR) for p53
3.3.2.4Single Strand Conformation Polymorphism (SSCP)
3.3.2.5DNA sequence analysis
3.3.2.6Automated DNA sequencing
3.3.2.7Isolation of total RNA from tumor tissue
3.3.2.8Northern hybridization
3.3.2.9Southern hybridization
3.3.2.10cDNA synthesis
3.3.2.11Polymerase Chain Reaction (PCR) for mdm2 with nested primers
3.3.2.12Subcloning of cDNA into a TA cloning vector
3.3.2.13Growth and storage of bacteria
3.3.2.14Generation of electro-competent bacteria
3.3.2.15Small scale (mini-prep) and medium scale preparation (midi-prep) of plasmid DNA
3.3.2.16Restriction enzyme digestion of plasmid DNA and PCR product
3.3.2.17Sequencing of DNA fragments cloned into the pCRTM2.1 vector
3.3.2.18Gel extraction of PCR products for sequencing
3.3.2.19Sequencing of cDNA PCR products after gel purification
3.3.2.20Primer design and PCR for in vitro protein expression using the pcDNA3-Vector
3.3.2.21Purification and restriction enzyme digestion of PCR Product and pcDNA3 vector
3.3.2.22Ligation of mdm2 splice variant DNA into the pcDNA3 expression vector
3.3.2.23Transformation of pcDNA3/cDNA constructs into electro-competent E. coli bacteria
3.3.2.24Restriction enzyme digestion of the pcDNA3 vector containing the cDNA insert
3.3.2.25Transient transfection of Hela-cells with the cDNA containing plasmid
3.3.3Biochemical techniques
3.3.3.1Preparation of cell lysates for SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
3.3.3.2SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
3.3.3.3Western blotting of p53 and MDM2 proteins fractionated by SDS-PAGE
3.4Statistical tests
4 RESULTS
4.1Alterations of the p53 tumor suppressor gene in ovarian cancer
4.1.1p53 mutations
4.1.2p53 polymorphisms and intron alterations
4.1.3Confirmation of p53 polymorphisms in normal tissue DNA
4.1.4p53 protein overexpression
4.1.5Comparison of p53 overexpression with p53 mutations
4.1.6p53 polymorphisms and p53 protein overexpression
4.1.7Correlation of p53 alterations with histopathological and clinical data
4.1.8p53 alterations and response to chemotherapy
4.1.9p53 alterations as a predictor of time to progression and overall survival
4.1.10Univariate and multivariable analysis of prognostic factors
4.2Alterations of the mdm2 gene in ovarian cancer
4.2.1Expression and absence of amplification of mdm2 in ovarian carcinomas
4.2.2mdm2 alternative and aberrant RNA splicing in ovarian carcinomas
4.2.3Loss of p53 binding sequence in mdm2 splice variants
4.2.4mdm2 splice sites and repeat sequences
4.2.5mdm2 alterations in ovarian cystadenomas and borderline tumors
4.2.6mdm2 alterations in normal ovarian tissue
4.2.7In vitro expression of p53 and MDM2 splice variant proteins
4.2.8Correlation of mdm2 and p53 alterations
4.2.9Correlation of mdm2 alterations with histopathological and clinical data
4.2.10mdm2 alterations and clinical outcome in ovarian cancer
5 DISCUSSION
5.1Alterations of the p53 tumor suppressor gene in ovarian cancer
5.1.1p53 mutations
5.1.2p53 mutations in evolutionary highly conserved domains
5.1.3p53 polymorphisms and intron alterations
5.1.4p53 protein overexpression
5.1.5Correlation of p53 overexpression with p53 mutations
5.1.6p53 alterations and response to chemotherapy
5.1.7p53 alterations as a predictor of time to progression and overall survival
5.2Alterations of the mdm2 gene in ovarian cancer
5.2.1mdm2 expression and absence of amplification in ovarian cancer
5.2.2mdm2 alternative and aberrant splicing in ovarian carcinomas
5.2.3Loss of p53 binding sequence in mdm2 splice variants
5.2.4mdm2 splice sites and repeat sequences
5.2.5mdm2 alterations in ovarian cystadenomas and borderline tumors
5.2.6mdm2 alterations in normal ovarian tissue
5.2.7In vitro expression of p53 and MDM2 proteins
5.2.8Correlation of mdm2 and p53 alterations
5.2.9Correlation of mdm2 alterations with histopathological and clinical data
5.2.10mdm2 alterations and clinical outcome in ovarian cancer
6 SUMMARY AND CONCLUSIONS
Bibliographie References
Danksagung
Selbständigkeitserklärung

Tabellenverzeichnis

Table 1: Histologic classification of common epithelial tumors of the ovary
Table 2: Genetic alterations in hereditary ovarian cancer syndromes
Table 3: Molecular genetic alterations in sporadic ovarian cancer
Table 4: List of all chemicals used in the experiments
Table 5: Primers and PCR conditions for amplification of p53 exon 2-11
Table 6: mdm2 external and internal primers for nested PCR
Table 7: Summary of p53 sequence alterations in 178 ovarian carcinomas
Table 8: p53 mutations and protein overexpression in ovarian carcinomas
Table 9: p53 polymorphisms and overexpression in ovarian carcinomas
Table 10: Normal tissue sequencing results for p53 polymorphisms
Table 11: p53 protein overexpression in ovarian cancer cases with p53 polymorphisms
Table 12. Clinico-pathologic characteristics of patients with epithelial ovarian cancer
Table 13: Response to platinum-based chemotherapy in correlation to p53 alterations
Table 14: Multivariable proportional hazards Cox regression analysis for the identification of independent prognostic factors in time to progression of patients with ovarian carcinoma.
Table 15: Multivariable proportional hazards Cox regression analysis for the identification of independent prognostic factors in overall
survival of patients with ovarian carcinoma.
Table 16: mdm2 RNA splicing in ovarian cancer as analyzed by reverse transcriptase PCR and DNA Sequencing
Table 17: mdm2 RNA splice variants in ovarian cancer identified by RT-PCR and DNA sequencing. The table shows sizes of splice variants, splice sites and tissues in which the splice variant was identified. (First nucleotide of coding sequence is #312, last nucleotide of coding sequence is #1784). The length of the splice variants includes only the coding sequence but not the flanking primer sequence. (> indicates exon/intron boundary and < indicates intron/exon boundary)
Table 18: Loss of p53 binding sequence in mdm2 splice variants
Table 19: mdm2 alternative and aberrant splicing in benign ovarian cystadenomas
Table 20: mdm2 alternative and aberrant splicing in ovarian tumors of borderline malignancy
Table 21: mdm2 alternative and aberrant RNA splicing in normal ovarian tissue
Table 22: p53 alterations in comparison to mdm2 RNA splicing
Table 23: Clinico-pathologic characteristics of patients with ovarian cancer and mdm2 alterations
Table 24: Response to platinum-based chemotherapy in ovarian cancer depending on mdm2 alternative splicing

Abbildungsverzeichnis

Fig. 1: Structural and functional regions of the p53 protein (redrawn from May and May, 1999) Functional regions and corresponding amino acid residues are shown on top. In the middle, evolutionary highly conserved domains I-V are shown. The bottom represents the tertiary structure of the site-specific DNA-binding. L1, L2, L3 indicate loops, and LSH indicates a loop-sheet-helix structure. The tertiary structure is shown in more detail in Fig. 2.
Fig. 2: Topological diagram of the secondary structure elements of the core domain of the p53 protein (redrawn from Cho et al. 1994). The residues at the start and the end of each secondary structure element are indicated. The DNA-binding regions of the protein (L1-S2-S‘, L2, L3, S10-H2) correspond to the conserved regions of the p53 gene and are colored yellow for region II (residue 117-142), red for region III (residue 171-181), blue for region IV (residue 234-258) and green for region V (residue 270-286). The boundaries of the two ß-sheets that make up the ß-sandwich are shaded. The scaffolding regions are white.
Fig. 3: p53 pathways. Upstream activators are shown above in red. Multiple downstream effectors of p53 which play a role for cell cycle arrest and apoptosis are shown. Arrows indicate a positive effect, while a flat line indicates inhibition. Quadrangles indicate specific functions in p53/MDM2 interaction.
Fig. 4: Response to cellular stresses in cells with wildtype p53 versus cells with mutated p53
Fig. 5: Structure and functional regions of the MDM2 protein. Above functional domains with corresponding amino acid residues are shown (see Freedman et al. 1999). DNA-PK sites comprise aa 388/389, 395/396 and 407/408. The p53 binding domain and conserved region I are almost identical. Since the human mdm2 exon boundaries are not fully known yet, mouse exons 1-12 are shown with the first codon of each exon indicated. Exon 1 and 2 are noncoding (Montes de Oca Luna et al. 1996).
Fig. 6: Autoregulatory feedback loop of the p53 tumor suppressor gene and the mdm2 gene. p53 at basal and at increased levels activates the mdm2 gene. The MDM2 protein binds to p53, inhibits its DNA binding ability, and promotes nuclear export and protein degradation.
Fig. 7: Hypothetic model about the effects of impaired MDM2 function on p53 protein accumulation in the cell. Cellular stresses cause p53 activation, which is downregulated by functional MDM2 in an autoregulatory feedback loop. In case of nonfunctional MDM2, wildtype p53 accumulates in the nucleus.
Fig. 8a: pcDNA3 expression vector (Invitrogen). cDNA of p53, full length mdm2 and mdm2 splice variants was ligated into the pcDNA3 expression vector.
Fig. 8b: mdm2 splice variant cDNA after NcoI restriction enzyme digestion of the pcDNA3 expression vector. The expression vector has three NcoI restriction sites at positions 610, 1976, and 2711 and the mdm2 cDNA (654 bp) has one NcoI restriction site at position 533. The cDNA analyzed on the agarose gel shows three fragments of the vector and two fragments of the cDNA insert, one with additional vector sequence. All clones (lanes 1-6) contain the mdm2 splice variant cDNA.
Fig. 9: Transient transfection of pcDNA constructs into vaccinia virus infected HeLa cells. The cDNA of interest was inserted into the pcDNA3 plasmid such that it came under the control of the T7 RNA polymerase promoter (pT7). Using liposome-mediated transfection, this recombinant plasmid is introduced into the cytoplasm of HeLa-cells infected with the vTF7-3 strain, a recombinant vaccinia virus encoding bacteriophage T7 RNA polymerase. During incubation, the cDNA is transcribed with high efficiency by T7 RNA polymerase. Proteins were detected by immunoprecipitation and Western blotting.
Fig. 10 A-C: p53 mutation and overexpression
A:
Single Strand Conformation Polymorphism (SSCP) analysis of the p53 gene, exon 5 in ovarian carcinomas.
Arrows in lanes 7 and 8 indicate band shifts suspicious for mutations.
B: DNA sequence analysis of exon 5 of the p53 gene in endometrioid ovarian carcinoma #2335.
Missense mutation in codon 141, TGC to TGG (amino acid exchange cystein to tryptophan).
C: Immunostaining shows overexpression of the p53 protein in the tumor nuclei (case #2335).
Fig. 11 A-C: p53 mutation and overexpressio
A: Single Strand Conformation Polymorphism (SSCP) analysis of p53, exon 9 in ovarian carcinomas. Arrows in lanes 3 and 10 (case #3402) indicate band shifts suspicious for mutations.
B: DNA sequence analysis of exon 9 of the p53 gene in endometrioid ovarian carcinoma #3402. A deletion mutation of one basepair in codon 320 causes a frameshift.
C: Immunostaining shows overexpression of the p53 protein in the tumor nuclei (case #2335.)
Fig. 12: p53 mutations in ovarian cancer. Mutations of the p53 gene were identified in 99/178 epithelial ovarian carcinomas. The majority of mutations were missense mutations.
Fig. 13: p53 mutations in evolutionary highly conserved domains of the gene. The upper part of the figure shows the number of mutations in codons 36 through 324 of the p53 gene. Mutations in evolutionary highly conserved regions are marked in yellow, mutations in non-conserved regions in dark blue and splice site mutations in white. The lower part of the figure shows the corresponding DNA interacting regions of the p53 protein.
Fig. 14: CGC rarr CCC (Arg rarr Pro) polymorphism at codon 72 of the p53 gene
A: SSCP analysis of p53, exon 4 in ovarian carcinomas. DNA in lanes 2,4,5,6,and 8 shows
band shifts suspicious for sequence alterations and was analyzed by DNA sequencing.
B: DNA sequence analysis of p53, exon 4 shows a CGC to CCC poymorphism at codon 72,
resulting in an arginine to proline amino acid exchange.
Fig. 15: Intron 3 polymorphism of the p53 gene
A: Single Strand Conformation Polymorphism (SSCP) of p53, exon 3 with the adjacent intron
sequence shows a band shift in lanes 2,4,8 and 9 indicating the intron 3 polymorphism.
B: Wild type (lanes 3 and 4) and the polymorphism sequence (lanes 1 and 2) of p53, intron 3
with a 16 bp insertion (lanes 1 and 2) and adjacent sequence are shown as
5‘ to 3‘: GCTggggacctggagggctGGGGGG
Fig. 16: Intron 10 polymorphism of the p53 gene
at nucleotide 17708
A novel polymorphism of the p53 gene was identified
at nucleotide 17708 (a>t) of intron 10 by SSCP analysi
and DNA sequencing in the ovarian cancer case #3402.
Fig. 17: Intron 10 polymorphism of the p53 gene at nucleotide 18550
A: SSCP analysis of p53 exon 11 and flanking intron sequence in ovarian carcinomas.
The band shift in lane 5 (case #3394) is suspicious for DNA sequence alterations.
B: DNA sequence analysis of p53 exon 11/ intron 10 reveals a novel c>t polymorphism
at nucleotide 18550 of intron 10 in the ovarian cancer cases #2660 and #3344.
The polymorphism was confirmed in normal tissue DNA.
Fig. 18a: p53 protein overexpression according to type of p53 mutation.
Overexpression is found in a high percentage of cases with missense mutations,
while the frequency of overexpression in nonmissense mutations is overall only 46%.
Fig. 18b: p53 protein overexpression according to p53 mutation or wildtype p53.
A high proportion of cases (38%) shows p53 protein accumulation (immunostaining in
ge10% of the nuclei) despite p53 wildtype sequence.
Fig. 19 A-B: Estimated probability of not progressing in epithelial ovarian cancer
patients (n=74), who received platinum-based chemotherapy. (A) Ovarian carcinomas with p53 mutations versus wildtype p53. (B) Ovarian carcinomas with p53 proteinoverexpression versus normal p53 expression.
Fig. 19 C-D:: Estimated probability of (C) not progressing, and (D) overall
survival respectively in epithelial ovarian cancer patients (n=74), who received
platinum-based chemotherapy.
Ovarian cancer with p53 alterations (mutation or
overexpression or both) versus normal p53 status.
Fig. 20 A-B: Estimated probability of overall survival in patients with epithelial
ovarian cancer (n=178) according to p53 alterations.
(A) p53 mutations versus
wildtype p53. (B) p53 mutations in evolutionary highly conserved domains versus
wildtype p53 or mutations in non-conserved domains.
Fig. 20 C-D: Estimated probability of overall survival in epithelial ovarian
patients (n=178) according to p53 alterations. (C) p53 mutations in DNA-
interacting regions versus wildtype p53 or mutations in scaffolding regions of the p53
protein. (D) p53 protein overexpression versus normal expression of the p53 protein.
Fig. 20 E-F: Estimated probability of (E) not progressing, and (F) overall survival
respectively in epithelial ovarian cancer patients (n=178). Ovarian cancer with p53
alterations (p53 mutation or p53 overexpression or both) versus normal p53 status.
Fig. 21: Southern hybridization of genomic DNA with the mdm2 cDNA probe. The sarcoma cell line SA1 which was used as a positive control shows amplification of
mdm2 DNA, while none of the ovarian cancer tissues shows DNA amplification.
Fig. 22: Northern hybridization of total mRNA with the mdm2 cDNA probe. The
sarcoma cell line SA1 which was used as a positive control shows overexpression of
mdm2 RNA, while none of the ovarian cancer tissues shows mdm2 overexpression.
Fig. 23: mdm2 RNA splice variants in ovarian carcinomas. Total RNA was
ana-lyzed by RT-PCR, and PCR-products were separated on a 1.2% agarose gel. Cases
in lanes 1,2,6,7,9, and 10 show splice variants of different sizes. The case in lane 3
shows no mdm2 expression. (cDNA bands on the gel contain additional 53 bp of flanking primer sequence)
Fig. 24: A-B: RT-PCR for ß2 microglobulin as a positive control for quality and
amount of RNA.
(A) RNA was extracted from normal ovarian tissue. All cases show
equal amplification of a 898 bp ß2M PCR product. Lane 21 shows the PCR product of
cDNA from the SA1 sarcoma cell line. Lane 22 is the negative control. (B) RT-PCR
for ß2 microglobulin expression in ovarian cancer RNA. Except for case #2341 in lane
7 all cases show approximately equal expression of the ß2 microglobulin RNA.
Fig. 25: Loss of functional regions in mdm2 splice variants in comparison to the full length mdm2 gene Above, functional domains of the MDM2 protein with corresponding amino acid residues are shown (according to Freedman et al. 1999). Below, splice variants of the mdm2 gene are shown. Black lines indicate the missing part of the sequence.
Fig. 26: A-D: Sequence overlaps in mdm2 RNA splice variants. (A-C) Splice site sequences of the 391 bp and 221 bp mdm2 splice variants show overlaps of several basepairs. The capital letters refer to transcribed bases that remain in the final mRNA products, while the lower case letters refer to untranscribed bases which are spliced out of the final mRNA product. The green boxes enclose the transcribed sequence at the donor site and the blue boxes enclose the transcribed sequence at the acceptor site. The yellow boxes indicate sequence homologies. D) As opposed to many of the aberrant splice variants which show overlapping sequences at the splice site, no such sequence homology is notable in the 654 bp mdm2-b splice variant which splices at exact exon/intron boundaries of exon 3 and exon 12.
Fig. 27: mdm2 RNA splice variants in ovarian cystadenomas and tumors of
borderline malignancy.
Total RNA was analyzed by RT-PCR and PCR-products were
separated on a 1.2% agarose gel. Cases in lanes 1, 2, 5-8, and 10 show splice variants
of different sizes. The case in lane 3 shows no mdm2 expression despite normal
expression of the ß2-microglobulin control gene. (cDNA bands on the gel contain additional 53 bp of flanking primer sequence).
Fig. 28: mdm2 RNA splice variants in normal ovarian tissue. Total RNA of
ovarian tissue was analyzed by RT-PCR and PCR-products were separated on a 1.2%
agarose gel. Cases in lanes 3, 5, 7, and 8 show splice variants of different sizes. (cDNA bands on the gel contain additional 53 bp of flanking primer sequence).
Fig. 29: Cytoplasmic expression of p53 and MDM2 proteins by transient transfection of pcDNA3 vector constructs into vaccinia virus infected HeLa cells
A: Expression of the full length p53 protein (53 kDa). B: Expression of the full length MDM2 protein (90 kDa).
C: Expression of a 40 kDa protein of the splice variant mdm2-b (654 bp). No protein could be expressed from the mdm2 splice variants of 351 bp and 52 bp. Proteins were analyzed by Western blotting and anti-myc respectively anti-HA immunodetection.
Fig. 30: A-B: Clinical outcome of ovarian cancer patients dependent on mdm2
alternative RNA splicing (n=92) as analyzed by RT-PCR and DNA sequencing.
A)
Overall survival in patients with expression of the full length mdm2 RNA transcript,
versus mdm2 splice variants, versus absence of mdm2 RNA expression.
B) Overall survival in patients with aberrant splicing in the donor and acceptor or only
the acceptor site, versus patients with full length mdm2 and/or alternative splicing at
exon/intron boundary of exon 3 and 9 (mdm2-a, 888 bp) or exon 3 and 11 (mdm2-b, 654 bp).
Fig.30 C-D: Clinical outcome of ovarian cancer patients dependent on mdm2 RNA alternative splicing (n=92) as analyzed by RT-PCR and DNA sequencing.
C)
Overall survival in patients with expression of small splice variants of <300 bp versus expression of the mdm2-b splice variant (654 bp) or full length mdm2
D) Overall survival in patients with expression of small splice variants (<300 bp) versus expression of mdm2 splice variants of >300 bp in the presence or absence of the full length mdm2 transcript. Cases with expression of the the mdm2-b splice variant (654 bp) were not included in this analysis.
Fig.30 E-F: Clinical outcome of ovarian cancer patients dependent on mdm2 RNA alternative splicing (n=92) as analyzed by RT-PCR and DNA sequencing.
E)
Overall survival in patients with expression of the mdm2-b splice variant (654 bp) versus absence of the mdm2-b splice variant.
F) Overall survival in patients with expression of a small splice variant of 221 bp was correlated with early stage (FIGO I/II) and marginally significant with a better clinical outcome.

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