Phenotypic characterization of telomerase-immortalized primary non-malignant and malignant tumor-derived human prostate epithelial cell lines

In vitro human prostate cell culture models are critical for clarifying the mechanism of prostate cancer progression and for testing preventive and therapeutic agents. Cell lines ideal for the study of human primary prostate tumors would be those derived from spontaneously immortalized tumor cells; unfortunately, explanted primary prostate cells survive only short-term in culture, and rarely immortalize spontaneously. Therefore, we recently have generated five immortal human prostate epithelial cell cultures derived from both the benign and malignant tissues of prostate cancer patients with telomerase, a gene that prevents cellular senescence. Examination of these cell lines for their morphologies and proliferative capacities, their abilities to grow in low serum, to respond to androgen stimulation, to grow above the agar layer, to form tumors in SCID mice, suggests that they may serve as valid, useful tools for the elucidation of early events in prostate tumorigenesis. Furthermore, the chromosome alterations observed in these immortalized cell lines expressing aspects of the malignant phenotypes imply that these cell lines accurately recapitulate the genetic composition of primary tumors. These novel in vitro models may offer unique models for the study of prostate carcinogenesis and also provide the means for testing both chemopreventive and chemotherapeutic agents.     

Tumor models for prostate cancer exemplified by fibroblast growth factor 8-induced tumorigenesis and tumor progression

Prostate cancer is a very common malignancy among Western males. Although most tumors are indolent and grow slowly, some grow and metastasize aggressively. Because prostate cancer growth is usually androgen-dependent, androgen ablation offers a therapeutic option to treat post-resection tumor recurrence or primarily metastasized prostate cancer. However, patients often relapse after the primary response to androgen ablation therapy, and there is no effective cure for cases of castration-resistant prostate cancer (CRPC). The mechanisms of tumor growth in CRPC are poorly understood. Although the androgen receptors (ARs) remain functional in CRPC, other mechanisms are clearly activated (e.g., disturbed growth factor signaling). Results from our laboratory and others have shown that dysregulation of fibroblast growth factor (FGF) signaling, including FGF receptor 1 (FGFR1) activation and FGF8b overexpression, has an important role in prostate cancer growth and progression. Several experimental models have been developed for prostate tumorigenesis and various stages of tumor progression. These models include genetically engineered mice and rats, as well as induced tumors and xenografts in immunodeficient mice. The latter was created using parental and genetically modified cell lines. All of these models greatly helped to elucidate the roles of different genes in prostate carcinogenesis and tumor progression. Recently, patient-derived xenografts have been studied for possible use in testing individual, specific responses of tumor tissue to different treatment options. Feasible and functional CRPC models for drug responsiveness analysis and the development of effective therapies targeting the FGF signaling pathway and other pathways in prostate cancer are being actively investigated.     

Stemness markers characterize IGR-CaP1, a new cell line derived from primary epithelial prostate cancer

Deciphering molecular pathways involved in the early steps of prostate oncogenesis requires both in vitro and in vivo models derived from human primary tumors. However the few recognized models of human prostate epithelial cancer originate from metastases. To date, very few models are proposed from primary tumors and immortalizing normal human prostate cells does not recapitulate the natural history of the disease. By culturing human prostate primary tumor cells onto human epithelial extra-cellular matrix, we successfully selected a new prostate cancer cell line, IGR-CaP1, and clonally-derived subclones. IGR-CaP1 cells, that harbor a tetraploid karyotype, high telomerase activity and mutated TP53, rapidly induced subcutaneous xenografts in nude mice. Furthermore, IGR-CaP1 cell lines, all exhibiting negativity for the androgen receptor and PSA, express the specific prostate markers alpha-methylacyl-CoA racemase and a low level of the prostate-specific membrane antigen PSMA, along with the prostate basal epithelial markers CK5 and CK14. More importantly, these clones express high CD44, CD133, and CXCR4 levels associated with high expression of α2β1-integrin and Oct4 which are reported to be prostate cancer stemness markers. RT-PCR data also revealed high activation of the Sonic Hedgehog signalling pathway in these cells. Additionally, the IGR-CaP1 cells possess a 3D sphere-forming ability and a renewal capacity by maintaining their CSC potential after xenografting in mice. As a result, the hormone-independent IGR-CaP1 cellular clones exhibit the original features of both basal prostate tissue and cancer stemness. Tumorigenic IGR-CaP1 clones constitute invaluable human models for studying prostate cancer progression and drug assessment in vitro as well as in animals specifically for developing new therapeutic approaches targeting prostate cancer stem cells.     

The use of genetically engineered mouse models of prostate cancer for nutrition and cancer chemoprevention research

The ability to modify the expression of specific genes in the mouse through genetic engineering technologies allows for the generation of previously unavailable models for prostate cancer prevention research. Although animal models have existed for some time for the study of prostate cancer prevention (primarily in the rat), it is uncertain if the mechanisms that drive prostate carcinogenesis in these models are relevant to those in human prostate cancer. Cell culture studies are of limited usefulness because the conditions are inherently artificial. Factors such as relevant physiologic concentrations and metabolism of putative chemoprevention compounds are difficult to model in an in vitro system. These studies also preclude the types of interactions known to occur between multiple cell types in vivo. In addition, all prostate cancer cell lines are already highly progressed and are not representative of the type of cells to which most preventive strategies would be targeted. Due to the advent of genetically engineered mouse (GEM) models, we now have models of prostate cancer that are dependent on molecular mechanisms already implicated in human prostate carcinogenesis. With these models we can perform a variety of experiments that could previously only be done in cell culture or in prostate cancer cell line xenografts. The currently available GEM models of prostate cancer have been extensively reviewed therefore, this review will focus on the types of models available and their usefulness for various types of preclinical studies relevant to prostate cancer prevention.     

Caveolin-1, a metastasis-related gene that promotes cell survival in prostate cancer

Metastasis represents the ultimate target in cancer therapy as this complex biological process is the direct cause of mortality for a variety of human malignancies. The current high level of mortality from prostate cancer results in large part from the inexorable growth of overt or occult metastasis present at the time of diagnosis. Currently, there are no curative therapies for metastatic prostate cancer. To better understand the metastatic phenotype in prostate cancer, we developed a strategy to identify mRNAs that are expressed differentially in cell lines derived from primary versus metastatic mouse prostate cancer using differential display-PCR. In using this system a number of metastasis-related sequences were identified including a cDNA that encodes caveolin-1. Caveolin-1 was found to be overexpressed not only in metastatic mouse prostate cancer, but also in human metastatic disease. Recent studies have indicated that suppression of caveolin-1 expression induces androgen sensitivity in high caveolin-1, androgen-insensitive mouse prostate cancer cells derived from metastases. Conversely, overexpression of caveolin-1 leads to androgen insensitivity in low caveolin, androgen-sensitive mouse prostate cancer cells. Caveolin-1, therefore, is both a metastasis-related gene as well as a candidate androgen resistance gene for prostate cancer in man. Interestingly, recent studies also point to a potential role for caveolin-1 in the resistance of various malignancies to multiple antineoplastic agents. The linkage of caveolin-1 expression with the androgen-resistant phenotype in prostate cancer and the multidrug resistance phenotype in various solid tumors establishes a novel paradigm for understanding these clinically important and now potentially related processes in malignant progression.     

Molecular Validation of PACE4 as a Target in Prostate Cancer

Prostate cancer remains the single most prevalent cancer in men. Standard therapies are still limited and include androgen ablation that initially causes tumor regression. However, tumor cells eventually relapse and develop into a hormone-refractory prostate cancer. One of the current challenges in this disease is to define new therapeutic targets, which have been virtually unchanged in the past 30 years. Recent studies have suggested that the family of enzymes known as the proprotein convertases (PCs) is involved in various types of cancers and their progression. The present study examined PC expression in prostate cancer and validates one PC, namely PACE4, as a target. The evidence includes the observed high expression of PACE4 in all different clinical stages of human prostate tumor tissues. Gene silencing studies targeting PACE4 in the DU145 prostate cancer cell line produced cells (cell line 4-2) with slower proliferation rates, reduced clonogenic activity, and inability to grow as xenografts in nude mice. Gene expression and proteomic profiling of the 4-2 cell line reveals an increased expression of known cancer-related genes (e.g., GJA1, CD44, IGFBP6) that are downregulated in prostate cancer. Similarly, cancer genes whose expression is decreased in the 4-2 cell line were upregulated in prostate cancer (e.g., MUC1, IL6). The direct role of PACE4 in prostate cancer is most likely through the upregulated processing of growth factors or through the aberrant processing of growth factors leading to sustained cancer progression, suggesting that PACE4 holds a central role in prostate cancer.     

Hepsin Paradox Reveals Unexpected Complexity of Metastatic Process

The existing models of cancer progression assume that a linear sequence of geneticand epigenetic events occurs during this process. In this representation every new event(either loss of a tumor-suppressor, or activation of a proto-oncogene) makes cells even moremalignant. The result is a “super” cell that can form metastases at the distant sites.Metastatic cells are believed to carry all genetic and epigenetic characteristics that arenecessary for metastasis formation. Recently, we have shown that cell-surface proteasehepsin causes disorganization of the basement membrane and promotes prostate cancerprogression and metastasis. In human prostate cancer hepsin is upregulated in theprecancerous lesions and this upregulation is maintained in the primary tumors. Remarkablyand completely unexpected for a metastasis-promoting gene, hepsin is expressed at lowlevels in metastatic lesions and the message is completely absent in metastasis-derivedprostate cancer cell lines. These results demonstrate that genes that play an important role inmetastatic process may exercise their role only at the specific fragments of cancerprogression pathway (for example, during initial invasion and tissue disorganization in theprimary organ) and may have no role in metastatic lesions. Future treatment of cancerpatients may rely heavily on monitoring of tumor progression, as treatment efficient inattenuation of initial tumor progression may be inefficient or even adverse at the advancestages of disease.     

Evaluating Baculovirus as a Vector for Human Prostate Cancer Gene Therapy

Gene therapy represents an attractive strategy for the non-invasive treatment of prostate cancer, where current clinical interventions show limited efficacy. Here, we evaluate the use of the insect virus, baculovirus (BV), as a novel vector for human prostate cancer gene therapy. Since prostate tumours represent a heterogeneous environment, a therapeutic approach that achieves long-term regression must be capable of targeting multiple transformed cell populations. Furthermore, discrimination in the targeting of malignant compared to non-malignant cells would have value in minimising side effects. We employed a number of prostate cancer models to analyse the potential for BV to achieve these goals. In vitro, both traditional prostate cell lines as well as primary epithelial or stromal cells derived from patient prostate biopsies, in two- or three-dimensional cultures, were used. We also evaluated BV in vivo in murine prostate cancer xenograft models. BV was capable of preferentially transducing invasive malignant prostate cancer cell lines compared to early stage cancers and non-malignant samples, a restriction that was not a function of nuclear import. Of more clinical relevance, primary patient-derived prostate cancer cells were also efficiently transduced by BV, with robust rates observed in epithelial cells of basal phenotype, which expressed BV-encoded transgenes faster than epithelial cells of a more differentiated, luminal phenotype. Maximum transduction capacity was observed in stromal cells. BV was able to penetrate through three-dimensional structures, including in vitro spheroids and in vivo orthotopic xenografts. BV vectors containing a nitroreductase transgene in a gene-directed enzyme pro-drug therapy approach were capable of efficiently killing malignant prostate targets following administration of the pro-drug, CB1954. Thus, BV is capable of transducing a large proportion of prostate cell types within a heterogeneous 3-D prostate tumour, can facilitate cell death using a pro-drug approach, and shows promise as a vector for the treatment of prostate cancer.     

Bub1 up-regulation and hyperphosphorylation promote malignant transformation in SV40 tag-induced transgenic mouse models

Rodents do not naturally develop prostate cancer. Currently, most widely used genetically engineered mouse prostate cancer models use SV40 T/tag oncogene. To understand the mechanism underlying prostate cancer development in transgenic and knock-in SV40 Tag mouse models, we did cDNA microarray analyses, comparing gene expression profiles of prostate cancer tissues from early-, late-, and advance-stage androgen-independent prostate cancers. Of the 67 genes that were up-regulated by > or = 10-fold, 40 are known to be required for chromosome stability. In particular, the spindle checkpoint component Bub1 was persistently up-regulated from early to advanced androgen-independent prostate cancer lesions. Significantly, Bub1, which is required for accurate chromosome segregation during mitosis, has recently been reported to bind SV40 Tag. Consistent with a spindle checkpoint defect, flow cytometry experiments indicate that advanced androgen-independent prostate cancer tumors exhibit aneuploidy, along with up-regulation of levels of both Bub1 mRNA and Bub1 protein or hyperphosphorylation. Importantly, up-regulation and hyperphosphorylation of Bub1 were also observed in established human prostate cancer cell lines and in clinical studies. Furthermore, analysis of human prostate cancer lines showed impaired spindle checkpoint function and endoreduplication following exposure to spindle toxins. Small interfering RNA-mediated repression of Bub1 in the human prostate cancer line PC-3 restrained cell proliferation, an effect mimicked by inhibition of mitogen-activated protein kinase, an upstream activator of Bub1. Thus, by perturbing Bub1 function, our observations suggest a new mechanism whereby the SV40 Tag oncoprotein promotes chromosomal instability and aneuploidy in transgenic mouse prostate cancer models. Whereas the exact details of this mechanism remain unclear, our novel findings raise the possibility of exploiting Bub1 as a new therapeutic target in the treatment of prostate cancer, the most common cancer in adult men in North America.     

miR-888 is an expressed prostatic secretions-derived microRNA that promotes prostate cell growth and migration

microRNAs (miRNAs) are a growing class of small non-coding RNAs that exhibit widespread dysregulation in prostate cancer. We profiled miRNA expression in syngeneic human prostate cancer cell lines that differed in their metastatic potential in order to determine their role in aggressive prostate cancer. miR-888 was the most differentially expressed miRNA observed in human metastatic PC3-ML cells relative to non-invasive PC3-N cells, and its levels were higher in primary prostate tumors from cancer patients, particularly those with seminal vesicle invasion. We also examined a novel miRNA-based biomarker source called expressed prostatic secretions in urine (EPS urine) for miR-888 expression and found that its levels were preferentially elevated in prostate cancer patients with high-grade disease. These expression studies indicated a correlation for miR-888 in disease progression. We next tested how miR-888 regulated cancer-related pathways in vitro using human prostate cancer cell lines. Overexpression of miR-888 increased proliferation and migration, and conversely inhibition of miR-888 activity blocked these processes. miR-888 also increased colony formation in PC3-N and LNCaP cells, supporting an oncogenic role for this miRNA in the prostate. Our data indicates that miR-888 functions to promote prostate cancer progression and can suppress protein levels of the tumor suppressor genes RBL1 and SMAD4. This miRNA holds promise as a diagnostic tool using an innovative prostatic fluid source as well as a therapeutic target for aggressive prostate cancer.     

Prostate cancer progression and surrounding microenvironment

Bidirectional cellular interactions between prostate cancer and prostate or bone stroma are needed for local tumor growth and distant metastasis. The genetics of cancer cells is affected by the host microenvironment and, reciprocally, permanent gene expression changes occur in the stroma surrounding epithelial cancer cells. The immune-mediated micromilieu also affects the progression of prostate cancer; the role of the immune system in controlling the growth of prostate cancer cells is complex, with immune escape mechanisms prevailing over effective antitumor response. Moreover, tumor stem cell models to explain the origin and progression of prostate cancer require appropriate environmental conditions. On the basis of a review of the literature, this article aims to outline the recent advances in the elucidation of the molecular mechanisms underlying the interactions between prostate cancer and its microenvironment.     

MicroRNA-1 is a candidate tumor suppressor and prognostic marker in human prostate cancer

We previously reported that miR-1 is among the most consistently down-regulated miRs in primary human prostate tumors. In this follow-up study, we further corroborated this finding in an independent data set and made the novel observation that miR-1 expression is further reduced in distant metastasis and is a candidate predictor of disease recurrence. Moreover, we performed in vitro experiments to explore the tumor suppressor function of miR-1. Cell-based assays showed that miR-1 is epigenetically silenced in human prostate cancer. Overexpression of miR-1 in these cells led to growth inhibition and down-regulation of genes in pathways regulating cell cycle progression, mitosis, DNA replication/repair and actin dynamics. This observation was further corroborated with protein expression analysis and 3'-UTR-based reporter assays, indicating that genes in these pathways are either direct or indirect targets of miR-1. A gene set enrichment analysis revealed that the miR-1-mediated tumor suppressor effects are globally similar to those of histone deacetylase inhibitors. Lastly, we obtained preliminary evidence that miR-1 alters the cellular organization of F-actin and inhibits tumor cell invasion and filipodia formation. In conclusion, our findings indicate that miR-1 acts as a tumor suppressor in prostate cancer by influencing multiple cancer-related processes and by inhibiting cell proliferation and motility.     

Xanthohumol Impairs Human Prostate Cancer Cell Growth and Invasion and Diminishes the Incidence and Progression of Advanced Tumors in TRAMP Mice

Despite recent advances in understanding the biological basis of prostate cancer, management of the disease, especially in the phase resistant to androgen ablation, remains a significant challenge. The long latency and high incidence of prostate carcinogenesis provides the opportunity to intervene with chemoprevention to prevent or eradicate prostate malignancies. In this study, we have used human hormone-resistant prostate cancer cells, DU145 and PC3, as an in vitro model to assess the efficacy of xanthohumol (XN) against cell growth, motility and invasion. We observed that treatment of prostate cancer cells with low micromolar doses of XN inhibits proliferation and modulates focal adhesion kinase (FAK) and AKT phosphorylation leading to reduced cell migration and invasion. Oxidative stress by increased production of reactive oxygen species (ROS) was associated with these effects. Transgenic adenocarcinoma of the mouse prostate (TRAMP) transgenic mice were used as an in vivo model of prostate adenocarcinoma. Oral gavage of XN, three times per week, beginning at 4 wks of age, induced a decrease in the average weight of the urogenital (UG) tract, delayed advanced tumor progression and inhibited the growth of poorly differentiated prostate carcinoma. The ability of XN to inhibit prostate cancer in vitro and in vivo suggests that XN may be a novel agent for the management of prostate cancer.