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. 2011 Oct 15;10(20):3571–3597. doi: 10.4161/cc.10.20.17842

Figure 2.

Figure 2

Figure 2

Figure 2

Figure 2

Expression of PCS-snpRNAs induces resistance to androgen depletion (RAD) and castration-resistant (CR) phenotypes of human prostate cancer. Expression of 8q24 PCS-snpRNAs (red arrows) is induced by NLRP1-locus snpRNAs (top gel image) and is increased in highly metastatic PC cells and circulating tumor cells (CTC) from orthotopic human PC xenografts (bottom gel images). Prostate cancer susceptibility snpRNAs A6 (rs16901979) manifest increased expression in highly metastatic human prostate carcinoma cells (LNCapLN3, PC321) and assert biologically and clinically relevant effects on growth of hormone-dependent human prostate carcinoma cells LNCap. RT-PCR analysis demonstrates low expression of prostate cancer susceptibility snpRNAs A6 in normal human prostate epithelial cells RWPE1 and markedly higher expression level in LNCapLN3 cells, which were selected in vivo for increased metastatic potential by serial orthotopic implantation of parental, poorly metastatic LNCap cells. Similarly, expression of multiple PCS-snpRNAs is elevated in blood-borne human prostate carcinoma metastasis precursor cells PC-3-21 compared with the parental cell line PC-3. p values of the two-tailed t-tests between corresponding experimental settings and control experiments are shown (Fig. 2B). Hormone-dependent human prostate carcinoma LNCap cells engineered to stably express the prostate cancer susceptibility snpRNA A6 acquired the ability to survive and grow in androgen-depleted media (set of bars in the top right part). Note that growth patterns in androgen-depleted media of LNCap cells expressing prostate cancer susceptibility snpRNA allele became similar to highly metastatic LNCapLN3 cells (set of bars in the bottom right part). Set of bars in the bottom left part shows the increased survival and growth in androgen-depleted media of LNCap cells constitutively expressing A alleles of NLRP1-locus snpRNAs. Set of bars in the top left part shows the results of the Q-PCR analysis of the expression of PCS-snpRNA A6 in RWPE1 normal prostate epithelial cells engineered to stably express distinct allelic variants of the NLRP1-locus snpRNAs. Hormone-dependent human prostate carcinoma LNCap cells engineered to stably express the prostate cancer susceptibility snpRNA A6 acquired markedly higher anchorage-independent clonogenic growth potential in agar (top) and the ability to survive and grow in androgen-depleted media (bottom). Note that growth potentials in both agar cultures and androgen-free media of LNCap cells expressing prostate cancer susceptibility A allele of snpRNA A6 are significantly higher compared with LNCap cells expressing ancestral C allele. Transfection of antisense alleles of PCS-SNP RNA A6 (asA6) diminishes clonogenic growth in agar (inset in top figure) and survival and growth in androgen-depleted media of highly metastatic LNCapLN3 human prostate carcinoma cells (inset in bottom figure). Inoculation of hormone-dependent, low-malignancy human prostate carcinoma LNCap cells engineered to stably express prostate cancer susceptibility snpRNA A6 (LNCap-A6 cells) induces rapidly growing tumors in castrated mice (left). Note that tumors in castrates appear to grow faster be larger and compared with the sham-operated mice (right). No tumors were detected in either castrate or sham-operated groups within the indicated observation periods after inoculation of parental or control-transfected LNCap (insets). Bottom set of figures show images of cultured in vitro in androgen-depleted media of LNCap-A6 cells that were recovered from tumors in castrated mice.