Prostate cancer-derived holoclones: a novel and effective model for evaluating cancer stemness.

Prostate cancer accounts for approximately 13.5% of all newly diagnosed male cancer cases. Significant clinical burdens remain in terms of ineffective prognostication, with overtreatment of insignificant disease. Additionally, the pathobiology underlying disease heterogeneity remains poorly understood. As the role of cancer stem cells in the perpetuation of aggressive carcinoma is being substantiated by experimental evidence, it is crucially important to understand the molecular mechanisms, which regulate key features of cancer stem cells. We investigated two methods for in vitro cultivation of putative prostate cancer stem cells based on 'high-salt agar' and 'monoclonal cultivation'. Data demonstrated 'monoclonal cultivation' as the superior method. We demonstrated that 'holoclones' expressed canonical stem markers, retained the exclusive ability to generate poorly differentiated tumours in NOD/SCID mice and possessed a unique mRNA-miRNA gene signature. miRNA:Target interactions analysis visualised potentially critical regulatory networks, which are dysregulated in prostate cancer holoclones. The characterisation of this tumorigenic population lays the groundwork for this model to be used in the identification of proteomic or small non-coding RNA therapeutic targets for the eradication of this critical cellular population. This is significant, as it provides a potential route to limit development of aggressive disease and thus improve survival rates.

EGR1 regulates angiogenic and osteoclastogenic factors in prostate cancer and promotes metastasis.

Early growth response-1 (EGR1) is a transcription factor correlated with prostate cancer (PC) progression in a variety of contexts. For example, EGR1 levels increase in response to suppressed androgen receptor signaling or loss of the tumor suppressor, PTEN. EGR1 has been shown to regulate genes influencing proliferation, apoptosis, immune cell activation, and matrix degradation, among others. Despite this, the impact of EGR1 on PC metastatic colonization is unclear. We demonstrate using a PC model (DU145/RasB1) of bone and brain metastasis that EGR1 expression regulates angiogenic and osteoclastogenic properties of metastases. We have shown previously that FN14 (TNFRSF12A) and downstream NF-κB signaling is required for metastasis in this model. Here we demonstrate that FN14 ligation also leads to NF-κB-independent, MEK-dependent EGR1 expression. EGR1-depletion in DU145/RasB1 cells reduced both the number and size of metastases but did not affect primary tumor growth. Decreased EGR1 expression led to reduced blood vessel density in brain and bone metastases as well as decreased osteolytic bone lesion area and reduced numbers of osteoclasts at the bone-tumor interface. TWEAK (TNFSF12) induced several EGR1-dependent angiogenic and osteoclastogenic factors (e.g., PDGFA, TGFB1, SPP1, IL6, IL8, and TGFA, among others). Consistent with this, in clinical samples of PC, the level of several genes encoding angiogenic/osteoclastogenic pathway effectors correlated with EGR1 levels. Thus, we show here that EGR1 has a direct effect on prostate cancer metastases. EGR1 regulates angiogenic and osteoclastogenic factors, informing the underlying signaling networks that impact autonomous and microenvironmental mechanisms of cancer metastases.

Identification of Different Classes of Luminal Progenitor Cells within Prostate Tumors.

Primary prostate cancer almost always has a luminal phenotype. However, little is known about the stem/progenitor properties of transformed cells within tumors. Using the aggressive Pten/Tp53-null mouse model of prostate cancer, we show that two classes of luminal progenitors exist within a tumor. Not only did tumors contain previously described multipotent progenitors, but also a major population of committed luminal progenitors. Luminal cells, sorted directly from tumors or grown as organoids, initiated tumors of adenocarcinoma or multilineage histological phenotypes, which is consistent with luminal and multipotent differentiation potentials, respectively. Moreover, using organoids we show that the ability of luminal-committed progenitors to self-renew is a tumor-specific property, absent in benign luminal cells. Finally, a significant fraction of luminal progenitors survived in vivo castration. In all, these data reveal two luminal tumor populations with different stem/progenitor cell capacities, providing insight into prostate cancer cells that initiate tumors and can influence treatment response.

Loss of Androgen-Regulated MicroRNA 1 Activates SRC and Promotes Prostate Cancer Bone Metastasis.

Bone metastasis is the hallmark of progressive and castration-resistant prostate cancers. MicroRNA 1 (miR-1) levels are decreased in clinical samples of primary prostate cancer and further reduced in metastases. SRC has been implicated as a critical factor in bone metastasis, and here we show that SRC is a direct target of miR-1. In prostate cancer patient samples, miR-1 levels are inversely correlated with SRC expression and a SRC-dependent gene signature. Ectopic miR-1 expression inhibited extracellular signal-regulated kinase (ERK) signaling and bone metastasis in a xenograft model. In contrast, SRC overexpression was sufficient to reconstitute bone metastasis and ERK signaling in cells expressing high levels of miR-1. Androgen receptor (AR) activity, defined by an AR output signature, is low in a portion of castration-resistant prostate cancer. We show that AR binds to the miR-1-2 regulatory region and regulates miR-1 transcription. Patients with low miR-1 levels displayed correlated low canonical AR gene signatures. Our data support the existence of an AR-miR-1-SRC regulatory network. We propose that loss of miR-1 is one mechanistic link between low canonical AR output and SRC-promoted metastatic phenotypes.

TMPRSS2- driven ERG expression in vivo increases self-renewal and maintains expression in a castration resistant subpopulation.

Genomic rearrangements commonly occur in many types of cancers and often initiate or alter the progression of disease. Here we describe an in vivo mouse model that recapitulates the most frequent rearrangement in prostate cancer, the fusion of the promoter region of TMPRSS2 with the coding region of the transcription factor, ERG. A recombinant bacterial artificial chromosome including an extended TMPRSS2 promoter driving genomic ERG was constructed and used for transgenesis in mice. TMPRSS2-ERG expression was evaluated in tissue sections and FACS-fractionated prostate cell populations. In addition to the anticipated expression in luminal cells, TMPRSS2-ERG was similarly expressed in the Sca-1(hi)/EpCAM(+) basal/progenitor fraction, where expanded numbers of clonogenic self-renewing progenitors were found, as assayed by in vitro sphere formation. These clonogenic cells increased intrinsic self renewal in subsequent generations. In addition, ERG dependent self-renewal and invasion in vitro was demonstrated in prostate cell lines derived from the model. Clinical studies have suggested that the TMPRSS2-ERG translocation occurs early in prostate cancer development. In the model described here, the presence of the TMPRSS2-ERG fusion alone was not transforming but synergized with heterozygous Pten deletion to promote PIN. Taken together, these data suggest that one function of TMPRSS2-ERG is the expansion of self-renewing cells, which may serve as targets for subsequent mutations. Primary prostate epithelial cells demonstrated increased post transcriptional turnover of ERG compared to the TMPRSS2-ERG positive VCaP cell line, originally isolated from a prostate cancer metastasis. Finally, we determined that TMPRSS2-ERG expression occurred in both castration-sensitive and resistant prostate epithelial subpopulations, suggesting the existence of androgen-independent mechanisms of TMPRSS2 expression in prostate epithelium.

Analysis of gene expression in non-regressed and regressed bovine corpus luteum tissue using a customized ovarian cDNA array.

The lifespan of the bovine corpus luteum (CL) is an important factor in the control of normal ovarian cyclicity and the establishment and maintenance of pregnancy. There is increasing evidence that CL lifespan is regulated by alternative expression of genes that promote or inhibit luteolysis. To gain further insights into these events a 434 character ovarian cDNA array comprising genes attributed to key aspects of CL function including more than 100 anonymous expressed sequence tags (ESTs) was constructed and screened with alpha(33)P dATP labeled RNA isolated from non-regressed (n=6) and regressed (n=6) CL tissue. Significance analysis of microarrays (SAM) identified 15 genes that changed expression 1.7-fold or more with a false discovery rate of <5%. The differentially expressed genes encoded enzymes involved in steroid biosynthesis and oxygen radical metabolism and proteins involved in extracellular matrix remodeling, apoptosis and cell structure. Results for five of the differentially expressed genes including matrix gla protein and collagen alpha1(I) (extracellular matrix), glutathione-S-transferase alpha I (oxygen metabolism), clusterin (apoptosis) and scavenger receptor BI (steroid biosynthesis) were confirmed by Northern blot analysis and found to be significantly different (P<0.01) between non-regressed and regressed CL tissue. Collectively this study identified genes with recognized roles in CL regression, genes with potential roles in this process and genes whose function have yet to be defined in this event.

Analysis of gene expression in the bovine corpus luteum through generation and characterisation of 960 ESTs.

To gain new insights into gene identity and gene expression in the bovine corpus luteum (CL) a directionally cloned CL cDNA library was constructed, screened with a total CL cDNA probe and clones representing abundant and rare mRNA transcripts isolated. The 5'-terminal DNA sequence of 960 cDNA clones, composed of 192 abundant and 768 rare mRNA transcripts was determined and clustered into 351 non-redundant expressed sequence tag (EST) groups. Bioinformatic analysis revealed that 309 (88%) of the ESTs showed significant homology to existing sequences in the protein and nucleotide public databases. Several previously unidentified bovine genes encoding proteins associated with key aspects of CL function including extracellular matrix remodelling, lipid metabolism/steroid biosynthesis and apoptosis, were identified. Forty-two (12%) of the ESTs showed homology with human or with other uncharacterised ESTs, some of these were abundantly expressed and may therefore play an important role in primary CL function. Tissue-specificity and temporal CL gene expression of selected clones previously unidentified in bovine CL tissue was also examined. The most interesting finds indicated that mRNA encoding squalene epoxidase was constitutively expressed in CL tissue throughout the oestrous cycle and 7-fold down-regulated (P < 0.05) in late luteal tissue, concomitant with the disappearance of systemic progesterone, suggesting that de novo cholesterol biosynthesis plays an important role in steroidogenesis. The mRNA encoding the growth factor, insulin-like growth factor-binding protein-related protein 1 (IGFBP-rP1), remained constant during the oestrous cycle and was 1.8-fold up-regulated (P < 0.05) in late luteal tissue implying a role in CL regression.

Postmortem stability of RNA isolated from bovine reproductive tissues.

Molecular biology is being increasingly used to address the complex problem of bovine infertility. One common concern shared by many of these studies is the postmortem delay in obtaining reproductive tissues and the effect this may have on RNA dependent studies. To address this concern, bovine ovarian, oviduct and uterine tissue samples, collected over intervals ranging from 0 to 96 h postmortem to freeze storage, were analysed to determine the potential effects on RNA quantity and quality. The analysis showed that total RNA yields were not changed significantly by postmortem interval up to 96 h while 28S ribosomal RNA remained intact up to 24 h postmortem. Specific messenger RNA transcripts encoding beta-actin, GAPDH and transforming growth factor-beta were detected in all tissues up to 96 h postmortem using reverse transcriptase-polymerase chain reaction and Northern analysis indicated no detectable mRNA degradation up to 24 h postmortem. Finally, using poly(A)(+) mRNA isolated from ovarian tissues frozen 2 h postmortem, we constructed corpus luteum and ovarian cortex cDNA libraries containing 7.65x10(4) and 1.9x10(6) primary transformants with average cDNA lengths of 2.3 and 1.6 kb respectively. Taken together, these data show that a postmortem delay of up to 24 h does not significantly affect the yield or quality of RNA prepared from bovine reproductive tissues.