Abstract 2421: The role of p53 mutational status and SOX9 suppression in chemotherapy response of ovarian cancer

Ovarian cancers are highly heterogeneous where platinum based chemotherapy which induces DNA crosslinking resulting in apoptosis of the cell is the preferred treatment. However, many patients are intrinsically resistant or quickly develop resistance. The Sox factors are a large family of transcription factors that play important roles in tumor development and progression in a variety of human malignancies and diverse developmental processes, but their impact in clinical tumorigenesis is still unclear. An analysis of genomic changes in ovarian cancer has provided the most comprehensive and integrated view of cancer genes for any cancer type to date. Ovarian serous adenocarcinoma tumors from 500 patients were examined by The Cancer Genome Atlas (TCGA) Research Network and analyses are reported in a recent issue of Nature. This evidence suggests that epigenetic deregulation, such as methylation, may be a key factor in the onset and maintenance of chemoresistance. Previous microarray analysis results in our lab correctly identified a subset of about 300 genes that when methylated altered the chemoresistance of the ovarian epithelium cells in culture. Of the genes identified in the analysis we further set out to characterize oncogenes and tumor suppressor genes that interact with the guardian of the genome, TP53, to determine if we could elucidate the mechanism by which it increased resistance. Using several in-vitro assays, we determined that the loss of p53 in conjunction with SOX9 decreased the level of apoptosis in response to carboplatin. Furthermore, in cells with mutated p53/SOX9 show an increase in tumorigenesis. Regulation of several pathways with p53 mutations in ovarian cancer might represent a therapy response prediction and could be a future therapeutic target for ovarian cancer. In addition, the crosstalk between p53/SOX9 and epigenetic regulators may present a valid treatment option for increasing carboplatin sensitivity in resistant patients

Molecular Analysis of T2/GT3/tTA Gene Disruptions

<div><p>(A) RT-PCR analysis of wild-type (−/−), hemizygous (−/+), and homozygous (+/+) carriers of insertions 03A-0033 and 03A-0063 are compared. Primers to the housekeeping <i>Gapdh</i> gene were used as an internal control for sample quality.</p><p>(B) Immunohistochemical staining of wild-type (wt), carrier, and null soleus muscles for MyHC type IIa (top) and type I (bottom). Samples were co-stained with Anti-Laminin to outline individual fibers.</p></div

Distribution of Chromosome 11 Insertions

<p>The insertions over the entire Chromosome 11 and the gene-dense, balanced region between <i>Trp53</i> and <i>Wnt3</i> are shown as a histogram over the ENSEMBL <i>ContigView</i> (<a href="http://feb2006.archive.ensembl.org/Mus_musculus/contigview?region=11&vc_start=69.3M&vc_end=103.6M&h=11" target="_blank">http://feb2006.archive.ensembl.org/Mus_musculus/contigview?region=11&vc_start=69.3M&vc_end=103.6M&h=11</a>). The number of insertions over the whole chromosome is shown in 1-Mbp bins, while the balanced region is shown in 100-kb bins.</p

Design of a Forward-Genetic Screen

<div><p>(A) The T2/GT3/tTA gene-trap tTA transposon was designed with splice acceptors (SA) in both orientations and the bidirectional SV40 polyadenylation signal (pA) to truncate expression of an endogenous gene after insertion into an intron. LoxP recombination sites (gray arrowheads) flank the mutagenic core of the transposon to potentially rescue a transposon-induced mutation.</p><p>(B) Southern blot and PCR analysis (+/–, top) was used to identify G1 animals that inherited the concatemer, but not the transposase transgene (asterisk).</p><p>(C) Insertions (red rectangle) genetically linked to the concatemer donor site (black rectangles) on Chromosome 11 (—) are homozygosed in a three-generation breeding scheme using the Inv(11)8Brd^Trp53–Wnt3 strain balancer chromosome (↔) with its engineered <i>Wnt3</i> mutation and visible <i>Agouti</i> marker conferring a yellowish color to the ears and tail. G1 animals were crossed to mice that carry a balanced <i>Rex</i> (curly coat) mutation (gray outline). Animals inheriting two copies of the balancer die in utero.</p></div

Defined Genomic Rearrangements Caused by Transposition

<div><p>(A) Moving average plots of ROMA data of deletion in pedigree W and amplification (due to insertion) in pedigree AG.</p><p>(B) Summary of rearrangements as determined by FISH (black bars) or ROMA (blue bars). The green box represents the concatemer, though its position relative to the deletions is speculative. ROMA detects loss (---) of chromosomal material, and defines the minimal overlapping regions for complementation groups 1 (blue box) and 2 (orange box), as well as amplification (+++) of genomic sequences. Where the FISH method was used (black bars) the true extent of each rearrangement could not be determined.</p></div

Molecular Evidence of Chromosomal Rearrangements after Transposition

<div><p>(A) Five BAC probes (red bars) were designed to the Chromosome 11 region from approximately 89.6–90.6 Mb (ENSEMBL m34 build, May 17, 2005 freeze). The transposon donor site (green) is presumed to be within the bracketed area based on the accumulation of insertions in this region. Single copies of the transposon were not detectable in this assay. Representative FISH hybridizations to metaphase preparations are shown.</p><p>(B) Evidence for deletion of BAC signals 424I8 and 107H16 in pedigree W (white arrows).</p><p>(C) Translocation of transposons (white arrowheads) along with distal Chromosome 11 sequence (yellow arrows) in pedigree AG.</p><p>(D) Evidence for inversion of a large region of Chromosome 11 is detected by BAC probes 367E18 and 297L3 (red arrows) in pedigree BC, likely involving multiple copies of the transposon.</p></div

Visible and Behavioral Phenotypes as a Result of Transposon Mutation

<div><p>(A) Dominant polydactyly (extra digits) or polysyndactyly (extra, fused digits) is evident in the fore (top) and hind limbs (bottom) of animals in pedigree BM.</p><p>(B) A recessive hyperactive phenotype is measured by the number of squares visited in the SHIRPA arena (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020156#s4" target="_blank">Materials and Methods</a>) in homozygous animals in the viable pedigree BG (<i>p</i> = 0.0113 by unpaired t-test).</p></div

De Novo Rearrangements in Somatic Cells of a GT3A; RosaSB11 Mouse

<p>Metaphase and interphase FISH images of normal (A, C, E) and abnormal (B, D, F) splenic lymphocytes from a doubly transgenic mouse are shown using the same probes as <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020156#pgen-0020156-g006" target="_blank">Figure 6</a>. Evidence for deletion (white arrows) and translocated Chromosome 11 sequences (yellow arrows) were evident for these three probes. These data, including other probes, are further summarized in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020156#pgen-0020156-st002" target="_blank">Table S2</a>.</p

Complementation Testcrosses

<p>For each testcross, heterozygous animals from independent lethal pedigrees (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020156#pgen-0020156-t001" target="_blank">Table 1</a>) were intercrossed to obtain offspring that inherited both copies of their respective mutagenized chromosomes as detected by PCR genotyping or balancer screening (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020156#pgen-0020156-g001" target="_blank">Figure 1</a>B). Noncomplementation (−) and complementation (+) divided the lethal pedigrees into at least six complementation groups, with two major groups labeled I and II. Pedigrees highlighted in pink complemented every other pedigree tested except one case, where AX failed to complement AS. Pedigrees in blue failed to complement pedigrees other than AG, AX, and BC (purple).</p