Adaptive (TINT) Changes in the Tumor Bearing Organ Are Related to Prostate Tumor Size and Aggressiveness

In order to grow, tumors need to induce supportive alterations in the tumor-bearing organ, by us named tumor instructed normal tissue (TINT) changes. We now examined if the nature and magnitude of these responses were related to tumor size and aggressiveness. Three different Dunning rat prostate tumor cells were implanted into the prostate of immune-competent rats; 1) fast growing and metastatic MatLyLu tumor cells 2) fast growing and poorly metastatic AT-1 tumor cells, and 3) slow growing and non-metastatic G tumor cells. All tumor types induced increases in macrophage, mast cell and vascular densities and in vascular cell-proliferation in the tumor-bearing prostate lobe compared to controls. These increases occurred in parallel with tumor growth. The most pronounced and rapid responses were seen in the prostate tissue surrounding MatLyLu tumors. They were, also when small, particularly effective in attracting macrophages and stimulating growth of not only micro-vessels but also small arteries and veins compared to the less aggressive AT-1 and G tumors. The nature and magnitude of tumor-induced changes in the tumor-bearing organ are related to tumor size but also to tumor aggressiveness. These findings, supported by previous observation in patient samples, suggest that one additional way to evaluate prostate tumor aggressiveness could be to monitor its effect on adjacent tissues.     

Characterization of a novel human breast cancer associated gene (BCA3) encoding an alternatively spliced proline-rich protein

As part of an integrated study of breast cancer gene expression, partial cDNAs were cloned from normal and tumor breast cells by subtractive-hybridization and differential display cloning. The DNA sequence for one of these breast cancer associated genes was used to construct the larger 1319 bp BCA3 cDNA sequence using ESTs without assigned names or functions. High-level BCA3 mRNA expression was found in breast and prostate tumor cell lines whereas normal breast and prostate tissues have low-level expression. Further analysis revealed possible functional domains and alternative splicing of BCA3 that we confirmed by RT-PCR analysis. Immunohistochemistry revealed that the protein is expressed in breast tumor cells in vivo, and not in surrounding stromal tissue.     

BRG1 is a prognostic indicator and a potential therapeutic target for prostate cancer

Brahma-related gene 1 (BRG1) is one of two mutually exclusive ATPases that function as the catalytic subunit of human SWItch/Sucrose NonFermentable (SWI/SNF) chromatin remodeling enzymes. BRG1 has been identified as a tumor suppressor in some cancer types but has been shown to be expressed at elevated levels, relative to normal tissue, in other cancers. Using TCGA (The Cancer Genome Atlas) prostate cancer database, we determined that BRG1 mRNA and protein expression is elevated in prostate tumors relative to normal prostate tissue. Only 3 of 491 (0.6%) sequenced tumors showed amplification of the locus or mutation in the protein coding sequence, arguing against the idea that elevated expression due to amplification or expression of a mutant BRG1 protein is associated with prostate cancer. Kaplan-Meier survival curves showed that BRG1 expression in prostate tumors inversely correlated with survival. However, BRG1 expression did not correlate with Gleason score/International Society of Urological Pathology (ISUP) Grade Group, indicating it is an independent predictor of tumor progression/patient outcome. To experimentally assess BRG1 as a possible therapeutic target, we treated prostate cancer cells with a biologic inhibitor called ADAADi (active DNA-dependent ATPase A Domain inhibitor) that targets the activity of the SNF2 family of ATPases in biochemical assays but showed specificity for BRG1 in prior tissue culture experiments. The inhibitor decreased prostate cancer cell proliferation and induced apoptosis. When directly injected into xenografts established by injection of prostate cancer cells in mouse flanks, the inhibitor decreased tumor growth and increased survival. These results indicate the efficacy of pursuing BRG1 as both an indicator of patient outcome and as a therapeutic target.     

Contact stimulation of prostate cancer cell migration: the role of gap junctional coupling and migration stimulated by heterotypic cell-to-cell contacts in determination of the metastatic phenotype of Dunning rat prostate cancer cells

BACKGROUND INFORMATION: Motile activity of tumour cells is regarded as a critical factor determining their metastatic potential. We have shown previously that contrary to the majority of normal cells, homotypic contacts between some tumour cells, among them low metastatic (AT-2) and highly metastatic (MAT-LyLu) rat prostate cancer cells, increase the speed of their movements. The aim of the present study was to determine the effect of heterotypic cell-to-cell contacts on the migration of rat prostate cancer cells differing in metastatic potential, and to correlate it with the intensity of homo- and heterologous gap junctional coupling. RESULTS: MAT-LyLu and AT-2 cells moving on the surface of fibroblasts displayed significantly greater cell displacement than those moving on plastic substrata. This effect correlated with the polarization (contact guidance) and increased speed of cell movements. However, in contrast with the migration on plastic substrata, where MAT-LyLu cells displayed considerably higher motility than AT-2 cells, no differences between both cell lines were observed on the surface of fibroblasts. On the other hand, in contrast with AT-2, Mat-LyLu cells displayed extensive homologous coupling mediated by connexin43 and were able to couple with normal fibroblasts. CONCLUSION: Heterotypic contacts between migrating prostatic cancer cells and normal fibroblasts can strongly stimulate their migration during invasion; however, this effect does not correlate with the gap junctional coupling between cancer cells and normal fibroblasts.     

Beyond comparing means: the usefulness of analyzing interindividual variation in gene expression for identifying genes associated with cancer development

Identifying genes associated with cancer development is typically accomplished by comparing mean expression values in normal and tumor tissues, which identifies differentially expressed (DE) genes. Interindividual variation (IV) in gene expression is indirectly included in DE gene identification because given the same absolute differences in means, genes with lower variance tend to have lower p-values. We explored the direct use of IV in gene expression to identify candidate genes associated with cancer development. We focused on prostate (PCa) and lung (LC) cancers and compared IV in the expression level of genes shown to be cancer related with that in all other genes in the human genome. Compared with all those other genes, cancer-related genes tended to have greater IV in normal tissues and a greater increase in IV during the transition from normal to tumorous tissue. Genes without significantly different mean expression values between tumor and normal tissues but with greater IV in tumor than in normal tissue (note: the DE-based approach completely ignores those genes) had stronger associations with clinically important features like Gleason score in PCa or tumor histology in LC than all other genes were. Our results suggest that analyzing IV in gene expression level is useful in identifying novel candidate genes associated with cancer development.     

Global methylation analysis identifies PITX2 as an upstream regulator of the androgen receptor and IGF-I receptor genes in prostate cancer

The insulin-like growth factor-I (IGF-IR) and androgen (AR) receptors are important players in prostate cancer. Functional interactions between the IGF-I and androgen signaling pathways have crucial roles in the progression of prostate cancer from early to advanced stages. DNA methylation is a major epigenetic alteration affecting gene expression. Hypermethylation of tumor suppressor promoters is a frequent event in human cancer, leading to inactivation and repression of specific genes. The aim of the present study was to identify the entire set of methylated genes ("methylome") in a cellular model that replicates prostate cancer progression. The methylation profiles of the P69 (early stage, benign) and M12 (advanced stage, metastatic) prostate cancer cell lines were established by treating cells with the demethylating agent 5-aza-2'-deoxycytidine (5-Aza) followed by DNA microarray analysis. Comparative genome-wide methylation analyses of 5-Aza-treated versus untreated cells identified 297 genes overexpressed in P69 and 191 genes overexpressed in M12 cells. 102 genes were upregulated in both benign and metastatic cell lines. In addition, our analyses identified the PITX2 gene as a master regulator upstream of the AR and IGF-IR genes. The PITX2 promoter was semi-methylated in P69 cells but fully methylated (i. e., silenced) in M12 cells. Epigenetic regulation of PITX2 during the course of the disease may lead to orchestrated control of the AR and IGF signaling pathways. In summary, our results provide new insights into the epigenetic changes associated with progression of prostate cancer from an organ confined, androgen-sensitive disorder to an aggressive, androgen-insensitive disease.     

Loss of mir-146a function in hormone-refractory prostate cancer

The pattern of microRNA (miRNA) expression is associated with the degree of tumor cell differentiation in human prostate cancer. MiRNAs bind complementarily to either oncogenes or tumor suppressor genes, which are consequently silenced, resulting in alterations of tumorigenecity. We have detected eight down-regulated and three up-regulated known miRNAs in androgen-independent human prostate cancer cells compared to those in androgen-dependent cells, using miRNA microarray analyses. These identified miRNAs showed the same expression patterns in hormone-refractory prostate carcinomas (HRPC) compared to androgen-sensitive noncancerous prostate epithelium as determined by fluorescent in situ hybridization assays in human prostate cancer tissue arrays. One of the eight down-regulated miRNAs, mir-146a, was selected and constitutively expressed to examine its effects on suppression of prostate cancer transformation from androgen-dependent to -independent cells as determined by in vitro tumorigenecity assays. Transfection of mir-146a, which perpetually express the miRNA, suppressed >82% of the expression of the targeted protein-coding gene, ROCK1, in androgen-independent PC3 cells, consequently markedly reducing cell proliferation, invasion, and metastasis to human bone marrow endothelial cell monolayers. Given that ROCK1 is one of the key kinases for the activation of hyaluronan (HA)-mediated HRPC transformation in vivo and in PC3 cells, mir-146a may function as a tumor-suppressor gene in modulating HA/ROCK1-mediated tumorigenecity in androgen-dependent prostate cancer.     

Analysis of the 10q11 cancer risk locus implicates MSMB and NCOA4 in human prostate tumorigenesis

Genome-wide association studies (GWAS) have established a variant, rs10993994, on chromosome 10q11 as being associated with prostate cancer risk. Since the variant is located outside of a protein-coding region, the target genes driving tumorigenesis are not readily apparent. Two genes nearest to this variant, MSMB and NCOA4, are strong candidates for mediating the effects of rs109939934. In a cohort of 180 individuals, we demonstrate that the rs10993994 risk allele is associated with decreased expression of two MSMB isoforms in histologically normal and malignant prostate tissue. In addition, the risk allele is associated with increased expression of five NCOA4 isoforms in histologically normal prostate tissue only. No consistent association with either gene is observed in breast or colon tissue. In conjunction with these findings, suppression of MSMB expression or NCOA4 overexpression promotes anchorage-independent growth of prostate epithelial cells, but not growth of breast epithelial cells. These data suggest that germline variation at chromosome 10q11 contributes to prostate cancer risk by influencing expression of at least two genes. More broadly, the findings demonstrate that disease risk alleles may influence multiple genes, and associations between genotype and expression may only be observed in the context of specific tissue and disease states.     

Expression and function of the leucine zipper protein Par-4 in apoptosis.

The prostate apoptosis response-4 (par-4) gene was identified by differential screening for genes that are upregulated when prostate cancer cells are induced to undergo apoptosis. The par-4 gene is induced by apoptotic signals but not by growth-arresting, necrotic, or growth-stimulatory signals. The deduced amino acid sequence of par-4 predicts a protein with a leucine zipper domain at its carboxy terminus. We have recently shown that the Par-4 protein binds, via its leucine zipper domain, to the zinc finger domain of Wilms' tumor protein WT1 (R. W. Johnstone et al., Mol. Cell. Biol. 16:6945-6956, 1996). In experiments aimed at determining the functional role of par-4 in apoptosis, an antisense par-4 oligomer abrogated par-4 expression and activator-driven apoptosis in rat prostate cancer cell line AT-3, suggesting that par-4 is required for apoptosis in these cells. Consistent with a functional role for par-4 in apoptosis, ectopic overexpression of par-4 in prostate cancer cell line PC-3 and melanoma cell line A375-C6 conferred supersensitivity to apoptotic stimuli. Transfection studies with deletion mutants of Par-4 revealed that full-length Par-4, but not mutants that lacked the leucine zipper domain of Par-4, conferred enhanced sensitivity to apoptotic stimuli. Most importantly, ectopic coexpression of the leucine zipper domain of Par-4 inhibited the ability of Par-4 to enhance apoptosis. Finally, ectopic expression of WT1 attenuated apoptosis, and coexpression of Par-4 but not a leucine zipperless mutant of Par-4 rescued the cells from the antiapoptotic effect of WT1. These findings suggest that the leucine zipper domain is required for the Par-4 protein to function in apoptosis.     

Expression of SPANX proteins in normal prostatic tissue and in prostate cancer

The sperm protein associated with the nucleus in the X chromosome (SPANX) gene family encodes for proteins that are not only expressed in germ cells, but also in a number of tumors. In addition, SPANX genes map in an interval of the X chromosome (namely, Xq27), which has been found to be associated with familial prostate cancer by linkage analysis. The aim of this study was therefore to evaluate SPANX protein expression in normal prostate tissues and in prostate carcinoma. For this purpose, formalin-fixed and paraffin-embedded sections obtained from 15 normal (at autopsy) donors and 12 men with prostate cancer were analyzed by immunohistochemistry. About 40% of both normal and tumor prostate samples resulted SPANX positive. Signals were exclusively within the nucleus in normal prostate cells, whereas both nuclear and cytoplasmic positivity was observed in tumor cells. In conclusion, these findings showed that SPANX genes are expressed in both normal and tumor prostate gland, but the latter showed a peculiar cytoplasmic staining positivity. This suggests a possible association between SPANX over expression and prostate cancer development. Additional studies are needed to corroborate this hypothesis.Key words: cancer, prostate, immunohistochemistry, SPANX gene     

Effects of androgen receptor and androgen on gene expression in prostate stromal fibroblasts and paracrine signaling to prostate cancer cells

The androgen receptor (AR) is expressed in a subset of prostate stromal cells and functional stromal cell AR is required for normal prostate developmental and influences the growth of prostate tumors. Although we are broadly aware of the specifics of the genomic actions of AR in prostate cancer cells, relatively little is known regarding the gene targets of functional AR in prostate stromal cells. Here, we describe a novel human prostate stromal cell model that enabled us to study the effects of AR on gene expression in these cells. The model involves a genetically manipulated variant of immortalized human WPMY-1 prostate stromal cells that overexpresses wildtype AR (WPMY-AR) at a level comparable to LNCaP cells and is responsive to dihydrotestosterone (DHT) stimulation. Use of WPMY-AR cells for gene expression profiling showed that the presence of AR, even in the absence of DHT, significantly altered the gene expression pattern of the cells compared to control (WPMY-Vec) cells. Treatment of WPMY-AR cells, but not WPMY-Vec control cells, with DHT resulted in further changes that affected the expression of 141 genes by 2-fold or greater compared to vehicle treated WPMY-AR cells. Remarkably, DHT significantly downregulated more genes than were upregulated but many of these changes reversed the initial effects of AR overexpression alone on individual genes. The genes most highly effected by DHT treatment were categorized based upon their role in cancer pathways or in cell signaling pathways (transforming growth factor-β, Wnt, Hedgehog and MAP Kinase) thought to be involved in stromal-epithelial crosstalk during prostate or prostate cancer development. DHT treatment of WPMY-AR cells was also sufficient to alter their paracrine potential for prostate cancer cells as conditioned medium from DHT-treated WPMY-AR significantly increased growth of LNCaP cells compared to DHT-treated WPMY-Vec cell conditioned medium.     

Expression of heat shock proteins and heat shock protein messenger ribonucleic acid in human prostate carcinoma in vitro and in tumors in vivo

Heat shock proteins (HSPs) are thought to play a role in the development of cancer and to modulate tumor response to cytotoxic therapy. In this study, we have examined the expression of hsf and HSP genes in normal human prostate epithelial cells and a range of prostate carcinoma cell lines derived from human tumors. We have observed elevated expressions of HSF1, HSP60, and HSP70 in the aggressively malignant cell lines PC-3, DU-145, and CA-HPV-10. Elevated HSP expression in cancer cell lines appeared to be regulated at the post-messenger ribonucleic acid (mRNA) levels, as indicated by gene chip microarray studies, which indicated little difference in heat shock factor (HSF) or HSP mRNA expression between the normal and malignant prostate cell lines. When we compared the expression patterns of constitutive HSP genes between PC-3 prostate carcinoma cells growing as monolayers in vitro and as tumor xenografts growing in nude mice in vivo, we found a marked reduction in expression of a wide spectrum of the HSPs in PC-3 tumors. This decreased HSP expression pattern in tumors may underlie the increased sensitivity to heat shock of PC-3 tumors. However, the induction by heat shock of HSP genes was not markedly altered by growth in the tumor microenvironment, and HSP40, HSP70, and HSP110 were expressed abundantly after stress in each growth condition. Our experiments indicate therefore that HSF and HSP levels are elevated in the more highly malignant prostate carcinoma cells and also show the dominant nature of the heat shock-induced gene expression, leading to abundant HSP induction in vitro or in vivo.     

The microRNA miR-34a inhibits prostate cancer stem cells and metastasis by directly repressing CD44

Cancer stem cells (CSCs), or tumor-initiating cells, are involved in tumor progression and metastasis. MicroRNAs (miRNAs) regulate both normal stem cells and CSCs, and dysregulation of miRNAs has been implicated in tumorigenesis. CSCs in many tumors--including cancers of the breast, pancreas, head and neck, colon, small intestine, liver, stomach, bladder and ovary--have been identified using the adhesion molecule CD44, either individually or in combination with other marker(s). Prostate CSCs with enhanced clonogenic and tumor-initiating and metastatic capacities are enriched in the CD44(+) cell population, but whether miRNAs regulate CD44(+) prostate cancer cells and prostate cancer metastasis remains unclear. Here we show, through expression analysis, that miR-34a, a p53 target, was underexpressed in CD44(+) prostate cancer cells purified from xenograft and primary tumors. Enforced expression of miR-34a in bulk or purified CD44(+) prostate cancer cells inhibited clonogenic expansion, tumor regeneration, and metastasis. In contrast, expression of miR-34a antagomirs in CD44(-) prostate cancer cells promoted tumor development and metastasis. Systemically delivered miR-34a inhibited prostate cancer metastasis and extended survival of tumor-bearing mice. We identified and validated CD44 as a direct and functional target of miR-34a and found that CD44 knockdown phenocopied miR-34a overexpression in inhibiting prostate cancer regeneration and metastasis. Our study shows that miR-34a is a key negative regulator of CD44(+) prostate cancer cells and establishes a strong rationale for developing miR-34a as a novel therapeutic agent against prostate CSCs.