Genes upregulated in prostate cancer reactive stroma promote prostate cancer progression in vivo

Abstract

Purpose: Marked reactive stroma formation is associated with poor outcome in clinically localized prostate cancer. We have previously identified genes with diverse functions that are upregulated in reactive stroma. This study tests the hypothesis that expression of these genes in stromal cells enhances prostate cancer growth in vivo.

Experimental design: The expression of reactive stroma genes in prostate stromal cell lines was evaluated by reverse transcriptase (RT)-PCR and qRT-PCR. Genes were knocked down using stable expression of short-hairpin RNAs (shRNA) and the impact on tumorigenesis assessed using the differential reactive stroma (DRS) system, in which prostate stromal cell lines are mixed with LNCaP prostate cancer cells and growth as subcutaneous xenografts assessed.

Results: Nine of 10 reactive stroma genes tested were expressed in one or more prostate stromal cell lines. Gene knockdown of c-Kit, Wnt10B, Bmi1, Gli2, or COMP all resulted in decreased tumorigenesis in the DRS model. In all tumors analyzed, angiogenesis was decreased and there were variable effects on proliferation and apoptosis in the LNCaP cells. Wnt10B has been associated with stem/progenitor cell phenotype in other tissue types. Using a RT-PCR array, we detected downregulation of multiple genes involved in stem/progenitor cell biology such as OCT4 and LIF as well as cytokines such as VEGFA, BDNF, and CSF2 in cells with Wnt10B knockdown.

Conclusions: These findings show that genes upregulated in prostate cancer-reactive stroma promote progression when expressed in prostate stromal cells. Moreover, these data indicate that the DRS model recapitulates key aspects of cancer cell/reactive stroma interactions in prostate cancer.

Figure 1. Differential reactive stroma cell lines variably enhance LNCaP tumorigenesis in a 10-day DRS model

LNCaP cells were injected subcutaneously in nude mice alone, with Matrigel or with the indicated prostate stromal cell line. After 10 days the number of palpable tumors was scored. The percent tumor formation is shown and the number of injections evaluated is indicated over each bar.

Figure 2. Expression of reactive stroma genes in DRS cell lines

A. RT-PCR of reactive stroma genes upregulated in human prostate cancer reactive stroma in DRS cell lines 19I, 19B and 33B. LNCaP was included as a positive control for FoxA1 since this gene was only weakly expressed in a single DRS cell line. The “X” indicates a skipped well in this gel. GAPDH was used as a positive control for mRNA amount and reverse transcription. B. Quantitative RT-PCR of selected reactive sroma genes in DRS cell lines. β-actin was used for copy number normalization. Expression is shown relative to 19I cells (100%). Mean +/− standard deviation of triplicate determinations of biological triplicates are shown. Significant differences in expression relative to 19I cells by t-test are indicated by asterisks (p=.04 Gli2 33B; p<.02 COMP; all others p<.001)

Figure 3. Tumorigenesis in a 28-day DRS model is inhibited by knockdown of Wnt10B or c-Kit

LNCaP cells were injected subcutaneously in nude mice with Matrigel and 19I DRS prostate stromal cells with shRNAs targeting Wnt10B (A) or c-Kit (B) or vector controls. Gene knockdown was confirmed by Q-RT-PCR prior to injection. After 28 days the number of palpable tumors was scored and palpable tumors were excised and weighed. All values are relative to vector controls and are expressed as mean +/− SEM, n=18 injections in a single experiment. Significant differences relative to 19I vector control cells are indicated by asterisks. For gene expression: p<.01, t-test. For tumor weight: p<.003, t-test. For tumor incidence: p=.01, Fisher exact test.

Figure 4. Inhibition of tumorigenesis by reactive stroma gene knockdown in a 10-day DRS model

LNCaP cells were injected subcutaneously in nude mice with Matrigel and 19I DRS prostate stromal cells with shRNAs targeting Wnt10B, COMP, Gli2, Bmi1 or vector controls. A. Gene expression prior to injection by quantitative-RT-PCR. B. Tumor weight after 10 days. All values are relative to controls and are expressed as mean +/− SEM. Results represent two separate experiments with 12 injections in each experiment. Significant differences relative to control cells are indicated by asterisks. For gene expression: p<.001, t-test. For tumor weight: p<.001, t-test. C. Histology of 10 day DRS tumors from control and shBmi1 tumors. Residual matrigel is present (arrowheads) as are islands of LNCaP cells (arrows) and stromal cells and blood vessels (double arrows). Note the smaller and more sparse clusters LNCaP cells and decreased blood vessels in the shBmi1 tumors. D. Immunohistochemistry with anti-Bmi1 antibody. LNCaP cells (arrows) are positive as are a subset of stromal cells (half shaded arrows). Staining of stromal cells was visibly decreased in tumors with shBmi1 stromal cells and this was confirmed by image analysis. All images 200X magnification.

Figure 5. Biological impact of stromal gene knockdown in DRS tumors

Tumors from the 10-day DRS experiment with gene knockdown of the indicated stromal gene (or vector controls) were analyzed for: (A) number of LNCaP cells; (B) proliferation of LNCaP cells, by Ki67 IHC; (C) apoptosis in LNCaP cells, by TUNEL and (D) angiogenesis, by anti-CD-31 IHC. Values were determined using image analysis as described in Materials and Methods. All values are expressed as mean +/− SEM. Significant differences relative to tumors with control cells are indicated by asterisks and is all cases p<.005 by t-test.

Figure 6. Decreased expression of mesenchymal stem cell associated genes in prostate stromal cells with knockdown of Wnt10B

Individual Q-RT-PCR reactions were carried out for four mesenchymal stem cell related genes to confirm the results of the Q-RT-PCR array. Expression relative to vector controls is shown. All values are means of triplicates. Standard deviations were less than 2% and thus are not shown since they are not visible on the graph. Significant differences relative to control cells by t-test are indicated by asterisks (ABCB1, p<.001; OCT4, p=.024; BDNF, p<.001; THY1, p=.01).