Suppression of cancer stemness p21-regulating mRNA and microRNA signatures in recurrent ovarian cancer patient samples

Abstract

Background: Malignant ovarian disease is characterised by high rates of mortality due to high rates of recurrent chemoresistant disease. Anecdotal evidence indicates this may be due to chemoresistant properties of cancer stem cells (CSCs). However, our understanding of the role of CSCs in recurrent ovarian disease remains sparse. In this study we used gene microarrays and meta-analysis of our previously published microRNA (miRNA) data to assess the involvement of cancer stemness signatures in recurrent ovarian disease.

Methods: Microarray analysis was used to characterise early regulation events in an embryonal carcinoma (EC) model of cancer stemness. This was then compared to our previously published microarray data from a study of primary versus recurrent ovarian disease. In parallel, meta-analysis was used to identify cancer stemness miRNA signatures in tumor patient samples.

Results: Microarray analysis demonstrated a 90% difference between gene expression events involved in early regulation of differentiation in murine EC (mEC) and embryonic stem (mES) cells. This contrasts the known parallels between mEC and mES cells in the undifferentiated and well-differentiated states. Genelist comparisons identified a cancer stemness signature set of genes in primary versus recurrent data, a subset of which are known p53-p21 regulators. This signature is present in primary and recurrent or in primary alone but essentially never in recurrent tumors specifically. Meta-analysis of miRNA expression showed a much stronger cancer stemness signature within tumor samples. This miRNA signature again related to p53-p21 regulation and was expressed prominently in recurrent tumors. Our data indicate that the regulation of p53-p21 in ovarian cancer involves, at least partially, a cancer stemness component.

Conclusion: We present a p53-p21 cancer stemness signature model for ovarian cancer. We propose that this may, at least partially, differentially regulate the p53-p21 mechanism in ovarian disease. Targeting CSCs within ovarian cancer represents a potential therapeutic avenue.

Figure 1

Validation of mEC and mES microarray data. Microarray data was validated through qPCR (TaqMan) analysis of a group of 36 genes. Data presented represents gene expression change in differentiated cells compared to undifferentiated and shows good correlation for mES (A) and PSA-SCC1 (B) datasets.

Figure 2

Expression of SCC-PSA1 stemness signature mRNAs in recurrent tumors. Sixteen genes were identified as differentially expressed in SCC-PSA1 mEC cells and in tumor data. Data is presented as the percentage change in gene expression in differentiated compared to undifferentiated SCC-PSA1 cells (blue) and in primary tumor samples compared to recurrent (red). Gene Expression values are detailed in table 3 (Microarray data p-Value ≤ 0.05).

Figure 3

Expression of Nulli-SCC stemness signature mRNAs in recurrent tumors. Fourteen genes were identified as differentially expressed in Nulli-SCC mEC cells and in tumor data. Data is presented as the percentage change in gene expression in differentiation-stimulated compared to undifferentiated Nulli-SCC cells (blue) and in primary tumor samples compared to recurrent (red). Gene Expression values are detailed in table 3 (Microarray data p-Value ≤ 0.05).

Figure 4

Expression of NTera2 stemness signature miRNAs in recurrent tumors. Twenty one miRNAs were identified as differentially expressed in differentiated NTera2 hEC cells and in tumor data. Data is presented as the percentage change in miRNA expression in differentiated NTera2 hEC cells (blue) and in recurrent tumors compared to primary (red). Values represent the mean of at least n = 3 and error bars the standard error of the mean.

Figure 5

Expression of 2102Ep stemness signature miRNAs in recurrent tumors. Twenty six miRNAs were identified as differentially expressed in undifferentiated 2102Ep compared to undifferentiated NTera2 hEC cells and in tumor data. Data is presented as log₁₀ (fold change). miRNAs presented showed altered expression in undifferentiated 2102Ep cells compared to undifferentiated NTera2 (blue) and in recurrent tumors compared to primary (red). Values represent the mean of at least n = 3 and error bars the standard error of the mean.

Figure 6

Suppression of cancer stemness p53-p21 regulation in recurrent tumors. Primary disease is characterised by the expression of p53-regulating stemness signature genes Sox4 and Sdsl, which is continued and enhanced with Dusp26 expression in the recurrency. In contrast, p21 regulating stemness signature genes Pak6 and Cask are expressed in primary disease and suppressed in recurrency. This process is paralleled by recruitment of stemness signature miRNAs by recurrent disease.