PROL1 is essential for xenograft tumor development in mice injected with the human prostate cancer cell-line, LNCaP, and modulates cell migration and invasion

Abstract

Background and objective: A growing body of literature suggests modulated expression of members of the opiorphin family of genes (PROL1, SMR3A and SMR3B) is associated with cancer. Recently, overexpression of PROL1 was shown to be associated with prostate cancer, with evidence of a role in overcoming the hypoxic barrier that develops as tumors grow. The primary goal of the present studies was to support and expand evidence for a role of PROL1 in the development and progression of prostate cancer.

Material and methods: We engineered knock-out of the opiorphin gene, PROL1, in LNCaP, an androgen-sensitive, human prostate cancer derived, cell-line. Using xenograft assays, we compared the ability of injected LNCaP PROL1 knock-out cell-lines to develop tumors in both castrated and intact male mice with the parental LNCaP and LNCaP PROL1 overexpressing cell-lines. We used RNAseq to compare global gene expression between the parental and LNCaP PROL1 knock-out cell-lines. Wound closure and 3D spheroid invasion assays were used to compare cell motility and migration between parental LNCaP cells and LNCaP cells overexpressing of PROL1.

Results: The present studies demonstrate that LNCaP cell-lines with consisitutive knock-out of PROL1 fail to develop tumors when injected into both castrated and intact male mice. Using RNAseq to compare global gene expression between the parental and LNCaP PROL1 knock-out cell-lines, we confirmed a role for PROL1 in regulating molecular pathways associated with angiogenesis and tumor blood supply, and also identified a potential role in pathways related to cell motility and migration. Through the use of wound closure and 3D spheroid invasion assays, we confirmed that overexpression of PROL1 in LNCaP cells leads to greater cell motility and migration compared to parental cells, suggesting that PROL1 overexpression results in a more invasive phenotype.

Conclusion: Overall, our studies add to the growing body of evidence that opiorphin-encoding genes play a role in cancer development and progression. PROL1 is essential for establishment and growth of tumors in mice injected with LNCaP cells, and we provide evidence that PROL1 has a possible role in progression towards a more invasive, metastatic and castration resistant prostate cancer (PrCa).

Keywords: Cell invasiveness; Cell motility; Opiorphin; PROL1; Prostate cancer.

FIG. 1.. Overexpression of PROL1 in LNCaP cells results in a more invasive phenotype.

(A) A representative wound healing assay for LNCaP-ProL1+ and LNCaP cells is shown (upper panel). The width of the wound was measured at at least eight points per image. Data shown (lower panel) represents the mean rate of wound healing (μm per 24 hours) ± Std. Dev. of five independent experiments (with measurements performed in triplicate for each experiment). * = p-value < 0.001. (B) A representative 3D spheroid invasion assay for LNCaP-ProL1+ and LNCaP cells is shown (upper panel). Spheroids were determined to be invasive if a 2D layer of cells was observed growing around the spheroid. Data shown (lower panel) represents the total number of invasive and noninvasive spheroids formed by LNCaP-ProL1+ and LNCaP after 2-weeks. LNCaP-ProL1+ cells generated significantly higher invasive according to the Fisher’s exact test (p < 0.0001).