Pim1 kinase is required to maintain tumorigenicity in MYC-expressing prostate cancer cells

Abstract

PIM1 kinase and MYC are commonly co-expressed in human prostate cancer and synergize to induce rapidly progressing prostate cancer in mouse models. Deficiency of the Pim kinase genes is well tolerated in vivo, suggesting that PIM1 inhibition might offer an attractive therapeutic modality for prostate cancer, particularly for MYC-expressing tumors. Here we examine the molecular consequences of Pim1 and MYC overexpression in the prostate as well as the effects of depleting Pim1 in prostate carcinoma cells with high levels of MYC. Overexpression of Pim1 in the mouse prostate induces several pro-tumorigenic genetic programs including cell cycle genes and Myc-regulated genes before the induction of any discernible pathology. Pim1 depletion by RNA interference in mouse and human prostate cancer cells decreased cellular proliferation, survival, Erk signaling and tumorigenicity even when MYC levels were not significantly altered. These results indicate that PIM1 may be necessary to maintain tumorigenicity, and further support efforts aimed at developing PIM1 inhibitors for prostate cancer therapy.

Figure 1. Gene expression profiling identifies pathways dysregulated in Pim1 and MYC-expressing prostate tissue recombinant grafts

A, GSEA analysis was performed to identify ‘gene sets’ significantly enriched in Pim1, MYC and Pim1/MYC grafts relative to control prostate grafts. The False discovery rates (FDR) are indicated for each gene set. TR (tissue recombinant graft); IDX (Insulin/Dexamethasone/Isobutylmethylxanthine); TSA (Trichostatin A); Adip_Diff (Adipocyte differentiation); Yu_MYC (MYC target genes); Dox_Resist_Gastric (Doxorubicin resistant gastric carcinoma cells). See also Figure S1 and Tables S1 and S2. B, Limited overlap among leading edge genes across the top gene sets enriched in Pim1, MYC and Pim1/MYC prostate tissue recombinant grafts. The numbers indicate percent overlap of leading edge genes in each gene set. These results indicate that the 5 gene sets are distinct. C, Overlap of leading edge genes in gene sets enriched by expression of Pim1, MYC or Pim1/MYC. The numbers indicate percent overlap of leading edge genes in each experimental group. These data indicate that Pim1, MYC and MYC/Pim1 regulate a similar set of genes within each gene set.

Figure 2. Knockdown of Pim1 expression in MYC/Pim1-overexpressing prostate tumor derived (MPT) cell lines

A, Establishment of MYC/Pim1 Tumor (MPT) prostate cell line from a MYC/Pim1-expressing tumor graft. The right panel shows the image of established cell line from 6-week MYC/Pim1 overexpressing tumor. YFP fluorescence can be seen under fluorescence microscope as the cells contain lentivirus construct bicistronically expressing YFP in addition to MYC or Pim1 (Scale bar: 100 μm). B, Western blot analysis of parental MPT cells or MPT cells stably expressing shRNAmir against Pim1 (shPim1#1), non-silencing shRNAmir (shControl#1), shRNA against Pim1 (shPim1#2) or control (shControl#2). MPT cells express NSE and MYC, the levels of which were not altered by Pim1 knockdown. Actin served as loading control. C, MPT2 cells were derived from 4-week MYC/Pim1 overexpressing prostate grafts (see Figure S2) and Pim1 expression stably knocked down using shPim1#2. MPT2 cells expressed androgen receptor (AR) in early passage, but not in late passage. NSE was also expressed in MPT2 cells. Tubulin served as loading control. D, Growth curve were generated by counting shPim1#1 and shControl#1 MPT cells in duplicates. *P<0.05 E, Cell proliferation was measured using the CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS, Promega). The absorbance is directly proportional to the number of living cells in culture. Results represent the mean ± s.d. of quadruplicate wells after 3 days of incubation of 2000 cells. *P<0.05. F, Analysis of apoptosis by activated caspase-3 staining (red), with nuclei counterstained with DAPI (blue) (Original magnification: 400×). G, Quantification of apoptosis rate by counting active capase-3 positive cells. The results represent the mean ± s.d. of two independent experiments *P<0.05.

Figure 3. Pim1 expression is required to maintain tumorigenicity of MYC/Pim1 tumor cells

A and B, Stable knockdown of Pim1 by shRNA reduces colony formation in MPT (A) and MPT2 (B) cells. C and D, Pim1 knockdown (shPim1#1) abrogates tumorigenicity of MPT cells when injected subcutaneously into nude mice. E, Representative H&E images of graft sections. Control group showed high-grade tumor consistent with the original MYC/Pim1 tissue recombinant graft tumor while Pim1 knockdown group showed fat and blood vessel cells with absence of tumor cells. Scale bar: 100 μm. Insets: Higher-magnification images. F, Immunohistochemical staining showed that sections from control grafts were synaptophysin (SYP) positive and androgen receptor (AR) negative. Scale bar: 100 μm. Pim1 knockdown grafts could not be analyzed because no tumor cells were found by H&E staining. Insets: AR positive control.

Figure 4. Pim1 knockdown in DU145 cells reduces proliferation, survival and tumorigenicity

A, Western blot analyses for PIM1, MYC and NSE in DU145 cells with stable PIM1 knockdown by shRNA. Tubulin served as a loading control. B, Cell proliferation was measured by MTS assay. The results are mean ± s.d. of quadruplicate wells after 3 days of incubation of 4000 cells. *P<0.05. C, Quantitation of apoptosis rate by counting active capase-3 positive cells after DU145 cells were treated with serum free medium overnight. The results are mean ± s.d. of two independent experiments. *P<0.05. D, Representative images of soft agar assay show that PIM1 knockdown in DU145 cells decreased the size and number of colonies (Original magnification: 4×). E, Quantitation of colonies from soft agar assay of Pim1 knockdown and control DU145 cells. The results are mean ± s.d. of triplicates. *P<0.05.

Figure 5. Pim1 is required to maintain Erk signaling in prostate carcinoma cells

A-C. Western blots showed that Pim1 knockdown in MPT and MPT2 cells by two different sets of shPim1 constructs decreased phosphorylation of Erk1/2 compared to control shRNA. D, PIM1 knockdown in DU145 cells decreases ERK1/2 phosphorylation of Erk1/2 E and F, Western blots showed that Pim1 overexpression did not increase ERK phosphorylation in LNCaP and PC3 cells compared to vector control cells (Neo). G, Western blot analysis of 6-week tissue recombinant grafts generated from cells expressing control or Pim1 lentivirus. Note increased phosphorylation of Erk1/2 in Pim1 graft compared to control.