Upregulation of minichromosome maintenance complex component 3 during epithelial-to-mesenchymal transition in human prostate cancer

Abstract

Metastasis is often associated with epithelial-to-mesenchymal transition (EMT). To understand the molecular mechanisms of this process, we conducted proteomic analysis of androgen-repressed cancer of the prostate (ARCaP), an experimental model of metastatic human prostate cancer. The protein signatures of epithelial (ARCaPE) and mesenchymal (ARCaPM) cells were consistent with their phenotypes. Importantly, the expression of mini-chromosome maintenance 3 (MCM3) protein, a crucial subunit of DNA helicase, was significantly higher in ARCaPM cells than that of ARCaPE cells. This increased MCM3 protein expression level was verified using Western blot analysis of the ARCaP cell lineages. Furthermore, immunohistochemical analysis of MCM3 protein levels in human prostate tissue specimens showed elevated expression in bone metastasis and advanced human prostate cancer tissue samples. Subcutaneous injection experiments using ARCaPE and ARCaPM cells in a mouse model also revealed increased MCM3 protein levels in mesenchymal-derived tumors. This study identifies MCM3 as an upregulated molecule in mesenchymal phenotype of human prostate cancer cells and advanced human prostate cancer specimens, suggesting MCM3 may be a new potential drug target for prostate cancer treatment.

Keywords: androgen receptor; epithelial-to-mesenchymal transition; metastasis; minichromosome maintenance complex proteins; proteomics.

Figure 1. Western blot of MCM3 in ARCaP_E and ARCaP_M cells

Total and nuclear proteins were analyzed for MCM3 expression. Beta-actin was used as a control.

Figure 2. MCM3 expression profiling in ARCaP_E cells

(A), ARCaP_M cells (B), ARCaP_M-Bone cells (C) and patient bone metastasis (D). MCM3 expression is higher in primary tumor induced by subcutaneous injection of ARCaP_M than by ARCaP_E (A-B). MCM3 is highly expressed in ARCaP_M-bone (C) and in three out of four human bone metastatic tissues from prostate cancer patients (D). Images were taken at 400X magnification.

Figure 3. MCM3 expression in human prostate cancer

MCM3 expression was visually absent in 12 normal prostate tissues (A), barely expressed in 6 benign prostatic hyperplasia tissues (B), and significantly increased in 12 prostate cancer clinical specimens (C-D). Images were taken at 200X magnification.

Figure 4. Semi-quantitative analysis of MCM3 expression in human samples

The number of MCM3 positive nuclei from normal (n = 12), benign (n = 6), and cancerous (n = 12) patient tissue samples were counted in ten random 20X microscopic fields. Differences between normal, benign, and cancerous samples were determined using a Poisson regression model. Normal prostate and benign prostatic hyperplasia showed no significant difference in expression (p = 0.0866). MCM3 differential expression is significant between benign and cancer (p=0.01) and normal and cancer (p = 0.0001). Mean and standard deviations are shown.