Role of the prorenin receptor in endometrial cancer cell growth

Figures

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  Figure 1. ATP6AP2 mRNA and protein levels in the endometrial epithelial cancer cell lines Ishikawa, AN3CA and HEC-1-A. (A) Messenger RNA expression of ATP6AP2 relative to a human term placenta control. (B) Immunoblotting on RIPA extracted cell lysates confirmed the expression of the (pro)renin receptor ((P)RR) protein product (36kDA; red arrow). Mean ± SEM values are plotted in histograms. *** P < 0.001 and **** P < 0.0001.

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  Figure 2. siRNA knockdown of ATP6AP2 gene and protein ((P)RR) expression in endometrial epithelial cancer cell lines. Messenger RNA expression of ATP6AP2 following (P)RR siRNA transfection in Ishikawa (A), AN3CA (B) and HEC-1-A (C) cell lines. Complementary immunoblotting analysis of protein expression following transfection examining the Ishikawa (D), HEC-1-A (E) and AN3CA (F) cell lines. Mean ± SEM densitometric values are plotted in histograms as determined by pixel intensity analysis. * P < 0.05, ** P < 0.01 and **** P < 0.0001.

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  Figure 3. Analysis of cellular viability and cellular proliferation following siRNA knockdown of ATP6AP2 in the endometrial epithelial cancer cell lines. Cell viability was assessed using the fluorometric resazurin assay for the Ishikawa (A), HEC-1-A (B) and AN3CA (C) cell lines. Cell viability was determined as the mean ± SEM fluorescence intensity value measured as a relative conversion fold change of non-fluorescent blue to fluorescent pink dye (with excitation emission wavelengths of 530 nm and 590 nm). (D–F) Proliferative capacity was assessed using xCelligence technology whereby electrical impedance acted as a surrogate measure of a cell’s proliferative rate in the three cell lines. Mean ± SEM values are plotted in histograms. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001.

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  Figure 4. Proteomic assessment of the functional consequences of ATP6AP2 knockdown in the Ishikawa endometrial epithelial cancer cell line. Tandem Mass Tags (TMT) mass spectrometry-based proteomics was used to quantitatively assess differences in the protein composition of the Ishikawa cell line following siRNA knockdown of ATP6AP2. (A) Pearson multi-scatter plot displaying the correlation among the three biological replicates of the transfected (top) and control (bottom) samples. (B) Ingenuity Pathway Analysis (IPA) of the overall distribution and (C) protein class of each of the proteins identified. Scatter plots were constructed using Perseus (version 1.6.10.43) and the distribution and class assessment using licensed Ingenuity Pathway Analysis software (IPA®, Qiagen) and the Ingenuity Knowledge Base. (D) Quantitative expression profiles (heat map) indicated that all transfected samples cluster as do the vehicle control samples. In addition, the heat map displays the protein identifications with most significant abundance changes.

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  Figure 5. Proteomic assessment of the functional consequences of ATP6AP2 and Gene ontology based functional annotation. (A) Volcano plot displaying the total proteome and differentially expressed proteins following transfection; 3 proteins were upregulated (FC ≥ 1.5 and P ≤ 0.05) and 19 were down regulated (FC ≤ 0.667 and P ≤ 0.05). (B) Histogram confirming the loss of (P)RR expression following transfection. (C) Broad biological function and molecular and cellular function (D) as determined by licensed Ingenuity Pathway Analysis software (IPA®, Qiagen) utilizing the ‘canonical pathway, disease and function’ analyses, which returned P-values (an enrichment measurement based on the number of proteins that map to a particular pathway, function or regulator), and Z-score, which is a prediction scoring system that assesses activation or inhibition of a given pathway or function based upon statistically significant patterns in the dataset and prior biological knowledge previously manually curated in the Ingenuity Knowledge Base. C. Cancer = cervical cancer. Mean ± SEM values are plotted in the histogram. *** P < 0.001.

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  Figure 6. Soluble prorenin receptor (s(P)RR) expression in endometrial epithelial cancer cell culture supernatants and in endometrial cancer patient plasma and uterine fluid samples. Soluble (P)RR expression was measured by a commercially available human ELISA in three endometrial cancer cell lines prior to (A) and after transfection (B–D) with (P)RR siRNA. In addition, s(P)RR was measured in human plasma (E) and uterine fluid/lavage samples (F) donated by endometrial cell cancer patients. Mean ± SEM values are plotted in scatterplots. * P < 0.05, ** P < 0.01 and *** P < 0.0010.