Pim1 kinase synergizes with c-MYC to induce advanced prostate carcinoma

Abstract

The oncogenic PIM1 kinase has been implicated as a cofactor for c-MYC in prostate carcinogenesis. In this study, we show that in human prostate tumors, coexpression of c-MYC and PIM1 is associated with higher Gleason grades. Using a tissue recombination model coupled with lentiviral-mediated gene transfer we find that Pim1 is weakly oncogenic in naive adult mouse prostatic epithelium. However, it cooperates dramatically with c-MYC to induce prostate cancer within 6-weeks. Importantly, c-MYC/Pim1 synergy is critically dependent on Pim1 kinase activity. c-MYC/Pim1 tumors showed increased levels of the active serine-62 (S62) phosphorylated form of c-MYC. Grafts expressing a phosphomimetic c-MYCS62D mutant had higher rates of proliferation than grafts expressing wild type c-MYC but did not form tumors like c-MYC/Pim1 grafts, indicating that Pim1 cooperativity with c-MYC in vivo involves additional mechanisms other than enhancement of c-MYC activity by S62 phosphorylation. c-MYC/Pim1-induced prostate carcinomas show evidence of neuroendocrine (NE) differentiation. Additional studies, including the identification of tumor cells coexpressing androgen receptor and NE cell markers synaptophysin and Ascl1 suggested that NE tumors arose from adenocarcinoma cells through transdifferentiation. These results directly show functional cooperativity between c-MYC and PIM1 in prostate tumorigenesis in vivo and support efforts for targeting PIM1 in prostate cancer.

Figure 1

Coexpression of c-MYC and PIM1 in human prostate tumors. A, Representative micrographs of human prostate tumor samples showing co-expression of c-MYC (green) and PIM1 (red) by double immunofluorescence staining. DAPI was used as a nuclear counter stain. B, Venn diagram showing overlap between samples positive for c-MYC and PIM1 overexpression. C, Gleason grade distribution of samples coexpressing MYC and PIM1 (MYC+PIM1+) compared to samples without coexpression of MYC and PIM1 (other). MYC+PIM1+ samples are associated with higher Gleason grades. *P < 0.05.

Figure 2

Tissue recombination coupled with lentiviral-mediated gene transfer for expression of MYC and Pim1 in regenerated mouse prostate. A, Schematic of the bicistronic lentiviral vector FM-1 used to target transgene expression together with YFP/Venus. B, Scheme used for prostate recombination. Primary mouse prostate epithelial cells were infected with the indicated lentiviruses and recombined with fetal rat urogenital mesenchyme to regenerate prostates. C, Representative images of sub-renal capsule grafts (arrows) after 6 weeks. Scale bar, 5mm. D, Western blot analyses from 6-week graft tissue lysates with the indicated antibodies.

Figure 3

MYC/Pim1 coexpression leads to high-grade prostate cancer within 6 weeks. A, H&E stained sections of 6-week grafts show normal-looking prostatic glands in control, Pim1 and K67M grafts. MYC and MYC/K67M samples show HGPIN lesions and hypercellular stroma. The MYC/Pim1 samples show high-grade tumor consistent with neuroendocrine carcinoma. Scale bar, 50 µm. Insets: Higher-magnification images. B, Human prostate samples showing LGPIN (a), HGPIN (b) and neuroendocrine (small cell) carcinoma (c) are shown for comaparison. The small cell carcinoma shows classic features of this tumor type with nuclear molding, focal “salt and pepper” nuclei, and abundant nuclear debris and apoptosis. (Original magnifications for a and b 600×; c, 400×).

Figure 4

Increased cellular proliferation and MYC activity in MYC/Pim1 tumors. A, Ki67 proliferative index in graft tissues. B, Apoptotic index in graft tissues determined by staining for activated caspase 3. Data shown as mean ± SD, N=3. *, P <0.05 relative to control. **, P < 0.05 relative to all samples. C, Western blot for the indicated proteins in graft tissue lysates. D, Quantitation of Cyclins D1, D2 and E levels from “C” normalized to actin. E, Upper panel: Western blot for serine-62 phosphorylated MYC (MYCS62P) and total MYC. Lower panel: Quantitation of western blot data. F, Six-week grafts expressing the MYCS62D phosphomimetic mutant are similar by H&E and SMA staining to wild type MYC grafts. Scale bars, 50 µm. G, Higher mitotic index (% phospho-histone H3 positive MYC-expressing cells) in grafts expressing the MYCS62D mutant (n=4) compared to those expressing wild type MYC (n=5). *, P < 0.05.

Figure 5

MYC/Pim1 coexpression shows evidence of neuroendocrine (NE) differentiation. Immunohistochemical analysis of 6-week tissue recombinants for expression of androgen receptor (AR), E-cadherin, p63, cytokeratin 8 (CK8), synaptophysin (SYN) and smooth muscle actin (SMA). MYC/Pim1 tumors showed reduced expression of AR, E-cadherin, p63 and CK8 expression and strong expression of synaptophysin. HGPIN lesions in MYC group strongly express SMA in the hypercellular stroma. Note loss of SMA staining in the MYC/Pim1 tumors, consistent with their invasive nature. SMA positive cells surrounding blood vessels in MYC/Pim1 tumor served as internal positive controls. Scale bars, 50 µm.

Figure 6

Chronic (12-week) overexpression of Pim1 results in LGPIN while MYC expression leads to a combination of adenocarcinoma and carcinoma with neuroendocrine differentiation. A, H&E sections show normal-looking prostate glands in K67M grafts and epithelial hyperplasia and LGPIN (arrow) in Pim1 grafts. Twelve-week MYC grafts resulted in two types of lesions: MYC (I) showed HGPIN with invasive adenocarcinoma (arrows), while MYC (II) had high-grade tumor consistent with neuroendocrine carcinoma. N=3 each. Scale bars, 50 µm. B, Immunohistochemistry for synaptophysin (SYN) shows strong expression in MYC (II) neuroendocrine tumor and isolated focal expression (arrows) in MYC (I) adenocarcinoma. Scale bars, 50 µm. C, Proliferative (Ki67) and apoptotic (activated caspase 3) indices in 12-week graft tissues. *, P <0.05 relative to K67M or Pim1. **, P < 0.05 relative to all other groups. N=3. D, Western blot analyses for the indicated proteins in 12-week graft tissues. Note increased Pim1 and reduced AR expression in MYC (II) sample.

Figure 7

Evidence that c-MYC/Pim1-induced neuroendocrine tumors arise by transdifferentiation. a-a”, MYC/Pim1 graft costained for androgen receptor (AR, red), synaptophysin (SYN, green) and DNA (DAPI, blue). b-b”, An adjacent section to that in ‘a’ stained for MYC (green), synaptophysin (SYN, red) and DNA (DAPI, blue). Arrows point to nest of tumor cells coexpressing AR, SYN and MYC. c-c”, MYC/Pim1 graft costained for androgen receptor (AR, red), synaptophysin (Ascl1, green) and DNA (DAPI, blue). d and d’, An adjacent section to that in ‘c’ stained for MYC (green), and DNA (DAPI, blue). Note coexpression of AR, Ascl1 and MYC. d”, Ascl1 expression (brown) in a MYC/Pim1 neuroendocrine tumor. Scale bars, 50 µm.