Evidence that p53-mediated cell-cycle-arrest inhibits chemotherapeutic treatment of ovarian carcinomas

Abstract

Gene expression profiles of malignant tumors surgically removed from ovarian cancer patients pre-treated with chemotherapy (neo-adjuvant) prior to surgery group into two distinct clusters. One group clusters with carcinomas from patients not pre-treated with chemotherapy prior to surgery (C-L), while the other clusters with non-malignant adenomas (A-L). We show here that although the C-L cluster is preferentially associated with p53 loss-of-function (LOF) mutations, the C-L cluster cancer patients display a more favorable clinical response to chemotherapy as evidenced by enhanced long-term survivorships. Our results support a model whereby p53 mediated cell-cycle-arrest/DNA repair serves as a barrier to optimal chemotherapeutic treatment of ovarian and perhaps other carcinomas and suggest that inhibition of p53 during chemotherapy may enhance clinical outcome.

Figure 1. (A) Unsupervised hierarchical clustering of the gene expression pattern of all 9,106 expressed probe sets detected on the HG-U95Av2 GeneChip in 43 ovarian tumor samples.

Samples beginning with AD are adenomas, with CA are carcinomas, and CC are patients pre-treated with chemotherapy. Samples divided into two major clusters termed adenoma-like and carcinoma-like. (B) Hierarchical clustering of the 1,527 probe sets that were significantly altered (>2-fold, FDR<1%) between the carcinoma-like and adenoma-like samples.

Figure 2. qRT-PCR validation of microarray results.

The expression patterns of BCL2, BAX and CCND1 in 4 tissue samples were determined using quantitative RT-PCR. In all cases, the qRT-PCR results of the analyses confirmed the differences detected in the microarray studies.

Figure 3. Two-dimensional hierarchical clustering of 43 samples is shown using a set of 36 probe sets that were employed in at least one KNN predictive model and two or more KNNxVal models.

To the right of the clustering pattern are given the probe set ID, HUGO Gene Symbol, number of models that each probe set appeared, and the fold change for each probe set for carcinoma-like vs. adenoma-like samples. Also shown are the number and percent correct predictions made by the 43 KNNxVal models and the four KNN models (upper right panel).

Figure 4. Gene interaction network of altered genes associated with cell cycle regulation and cancer as determined by Ingenuity Pathways Analysis.

Genes colored red showed increased expression in carcinomas relative to adenomas and in green had decreased expression in carcinomas relative to adenomas. Increased levels of cyclin D1 (CCND1), cyclin E1 (CCNE1), cyclin B1 (CCNB1), cyclin B2 (CCNB2), cyclin A2 (CCNA2), E2F3, E2F5, cyclin-dependent kinases (CDC2 and CDK7) as well as decreased levels of p57 cyclin-dependent kinase inhibitor (CDKN1C) and growth arrest and DNA-damage-inducible, gamma (GADD45G) are all consistent with carcinomas having higher proliferation rates than adenomas. Interactions of these genes with p53 are highlighted as green lines.

Figure 5. Apoptosis pathway with genes displaying increased expression (colored red) and decreased expression in carcinoma-like samples (colored green).

Carcinoma-like samples had higher expression of pro-apoptotic genes such as BAX, BID, CASP8, and AIF. These samples also expressed higher levels of anti-apoptotic factors such as IKK, Bcl-xl, and c-IAP2. Adenoma-like samples had higher Bcl-2 expression than carcinoma-like samples.

Figure 6. p53 immunohistochemical staining of a serous papillary adenocarcinoma (sample CA99) displaying strong diffuse p53 immunoreactivity.

Intense staining of tumor cells is typically associated with the presence of point mutational damage in p53 exons 5–8 (magnification X 400).

Figure 7. Cumulative survival curves of ovarian carcinoma patients treated with neo-adjuvant chemotherapy.

Seventy percent of neo-adjuvant cancer patients whose tissue gene expression profiles clustered with those of cancer patients not pre-treated with chemotherapy have survived five years after surgery. In contrast, only thirty percent of patients whose expression arrays clustered with benign adenoma patients prior to surgery survived five years after surgery. Although the trends are clear, because of the limited sample size of the cohort of patients for which follow-up data were available (A-L group n = 13; C-L group n = 11), the differences are marginally insignificant (p<0.191, logrank test). The survivorship data used in the computation of the Kaplan-Meier plot can be found in the supplementary material (Figure S2).