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. 2010 Jun 1:10:248.
doi: 10.1186/1471-2407-10-248.

Pim1 promotes human prostate cancer cell tumorigenicity and c-MYC transcriptional activity

Jongchan Kim  1 , Meejeon Roh, Sarki A Abdulkadir
Affiliations

Pim1 promotes human prostate cancer cell tumorigenicity and c-MYC transcriptional activity

Jongchan Kim et al. BMC Cancer. .

Abstract

Background: The serine/threonine kinase PIM1 has been implicated as an oncogene in various human cancers including lymphomas, gastric, colorectal and prostate carcinomas. In mouse models, Pim1 is known to cooperate with c-Myc to promote tumorigenicity. However, there has been limited analysis of the tumorigenic potential of Pim1 overexpression in benign and malignant human prostate cancer cells in vivo.

Methods: We overexpressed Pim1 in three human prostate cell lines representing different disease stages including benign (RWPE1), androgen-dependent cancer (LNCaP) and androgen-independent cancer (DU145). We then analyzed in vitro and in vivo tumorigenicity as well as the effect of Pim1 overexpression on c-MYC transcriptional activity by reporter assays and gene expression profiling using an inducible MYC-ER system. To validate that Pim1 induces tumorigenicity and target gene expression by modulating c-MYC transcriptional activity, we inhibited c-MYC using a small molecule inhibitor (10058-F4) or RNA interference.

Results: Overexpression of Pim1 alone was not sufficient to convert the benign RWPE1 cell to malignancy although it enhanced their proliferation rates when grown as xenografts in vivo. However, Pim1 expression enhanced the in vitro and in vivo tumorigenic potentials of the human prostate cancer cell lines LNCaP and DU145. Reporter assays revealed increased c-MYC transcriptional activity in Pim1-expressing cells and mRNA expression profiling demonstrated that a large fraction of c-MYC target genes were also regulated by Pim1 expression. The c-MYC inhibitor 10058-F4 suppressed the tumorigenicity of Pim1-expressing prostate cancer cells. Interestingly, 10058-F4 treatment also led to a reduction of Pim1 protein but not mRNA. Knocking-down c-MYC using short hairpin RNA reversed the effects of Pim1 on Pim1/MYC target genes.

Conclusion: Our results suggest an in vivo role of Pim1 in promoting prostate tumorigenesis although it displayed distinct oncogenic activities depending on the disease stage of the cell line. Pim1 promotes tumorigenicity at least in part by enhancing c-MYC transcriptional activity. We also made the novel discovery that treatment of cells with the c-MYC inhibitor 10058-F4 leads to a reduction in Pim1 protein levels.

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Figures

Figure 1
Figure 1
Overexpression of Pim1 in human prostate cell lines. (A) Western blots demonstrated Pim1 expression in benign human prostate cell line (RWPE1) and human prostate cancer cell lines (LNCaP and DU145). In addition, endogenous levels of c-MYC were upregulated in two cancer cell lines. Phosphorylation of p21 was increased in Pim1-expressing RWPE1 cells compared to control cells (Neo). (B) Cell sorting analyses showed that there was no difference between control (Neo) and Pim1 cells in cell cycle at the time when the cells were grafted.
Figure 2
Figure 2
Increase in c-MYC activity due to Pim1 overexpression. (A) Luciferase assay using c-MYC responsive reporter construct demonstrated that Pim1 overexpression induced transcriptional activity of c-MYC but its kinase dead mutant Pim1 (K67 M) showed dramatically repressed c-MYC activity compared to control. *P < 0.05. (B) Western blots showed c-MYC expression is undetectable in RWPE1 cells.
Figure 3
Figure 3
Pim1 expression is insufficient to convert benign human prostate cells (RWPE1) to malignancy. (A) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed there was no difference in in vitro cell proliferation between control and Pim1-expressing RWPE1 cells. (B) Representative H&E images of grafts. RWPE1-Neo (N = 5); RWPE1-Pim1 (N = 7). Scale bars: 100 μm. (C) Quantitation of proliferation and apoptosis in xenografts after immunostaining for Ki67 and activated Caspase 3, respectively. *P < 0.05.
Figure 4
Figure 4
Pim1 enhances tumorigenicity of androgen-dependent human prostate cancer cells (LNCaP) in vitro and in vivo. (A) Soft agar assay showed increased in vitro tumorigenicity of Pim1-expressing LNCaP cells. When control or Pim1-expressing LNCaP cells were subcutaneously grafted in nude mice, the latter developed larger tumors in size (B) and weight (C). (D) Kaplan-Meier survival analysis demonstrates slightly accelerated tumor onset by Pim1 expression. Numbers in the parentheses indicate the number of replicates or grafts in each group. (E) H&E stains demonstrated that Pim1 expression caused more hemorrhagic phenotype than control. *P < 0.05.
Figure 5
Figure 5
Pim1 enhances tumorigenicity of aggressive human prostate cancer cells (DU145) in vitro and in vivo. (A) Soft agar assay shows increased in vitro tumorigenicity of Pim1-expressing DU145 cells. When control or Pim1-expressing DU145 cells were subcutaneously grafted in nude mice (nu/nu), the latter developed larger tumor in size (B and C). (D) Kaplan-Meier survival analysis demonstrates significantly accelerated tumor onset by Pim1 expression. Numbers in the parentheses indicate the number of replicates or grafts in each group. *P < 0.01, **P < 0.05.
Figure 6
Figure 6
Role of androgen in proliferation and transcriptional activity of androgen receptor in control and Pim1-expressing LNCaP cells. (A) Effect of androgen receptor signaling on cell proliferation. Cell proliferation with or without DHT (5α-Dihydrotestosterone) treatment was analyzed in control and Pim1-expressing LNCaP cells. (B) PSA mRNA levels were measured by RT-PCR analysis after the treatment of various dose of DHT. Neo vs. Pim1 or Neo vs. K67M was compared. *P < 0.05. (C) Western blots for Pim1, androgen receptor (AR) and Actin in the indicated cell lines.
Figure 7
Figure 7
Examination of gene expression profile using MYC-ER inducible system in RWPE1 cells and its validation of selected genes in LNCaP and DU145 cells. (A) A MYC-ER inducible system was established in RWPE1 prostate cells and shown is western blot analysis of stable protein expression of MYC-ER, Pim1 and Actin (arrows) in RWPE1 cell lines. * marks a non-specific band. (B) RT-PCR confirmed mRNA expression of several MYC/Pim1 target genes selected from Table 1 in LNCaP-Pim1 and DU145-Pim1 cells. Arrows indicate selected genes that are consistently regulated by Pim1 in DU145 and LNCaP cells.
Figure 8
Figure 8
c-MYC inhibition either by a small molecule inhibitor (10058-F4) or by RNA interference (RNAi) causes in vitro growth arrest and alters target gene expression. (A) c-Myc inhibitor (10058-F4) abrogated colony formation of LNCaP and DU145 cells in soft agar assay. # indicates no colonies. *P < 0.0001, **P < 0.002. (B) Control and Pim1-expressing LNCaP and DU145 cells were treated with different doses (0, 50, 100 and 200 uM) of 10058-F4. Note that 10058-F4 inhibits protein expression of Pim1, but not Pim1 mRNA. (C) Control and two small hairpin RNA constructs (sh1 and sh2) against c-MYC were transfected in LNCaP-Pim1 cells to knock down c-MYC expression. Relative c-MYC expression is shown. (D) Expression of some target genes (LAMC2 and VAV3) in LNCaP cells was reversed by repression of c-MYC levels in a dose-dependent manner. * indicates altered target gene expression by c-MYC knock-down.
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