Characterizing the contribution of stem/progenitor cells to tumorigenesis in the Pten-/-TP53-/- prostate cancer model.

Loss of PTEN is one of the most common mutations in prostate cancer, and loss of wild-type TP53 is associated with prostate cancer progression and castrate resistance. Modeling prostate cancer in the mouse has shown that while Pten deletion in prostate epithelial cells leads to adenocarcinoma, combined loss of Pten and TP53 results in rapidly developing disease with greater tumor burden and early death. TP53 contributes significantly to the regulation of stem cell self-renewal, and we hypothesized that loss of Pten/TP53 would result in measurable changes in prostate cancer stem/progenitor cell properties. Clonogenic assays that isolate progenitor function in primary prostate epithelial cells were used to measure self-renewal, differentiation, and tumorigenic potential. Pten/TP53 null as compared with wild-type protospheres showed increased self-renewal activity and modified lineage commitment. Orthotopic transplantation of Pten/TP53 null cells derived from protospheres produced invasive Prostatic Intraepithelial Neoplasia (PIN)/adenocarcinoma, recapitulating the pathology seen in primary tumors. Pten/TP53 null progenitors relative to wild type also demonstrated increased dependence on the AKT/mammalian target of rapamycin complex 1 (mTORC1) and androgen receptor (AR) pathways for clonogenic and tumorigenic growth. These data demonstrate roles for Pten/TP53 in prostate epithelial stem/progenitor cell function, and moreover, as seen in patients with castrate-resistant prostate cancer, suggest for the involvement of an AR-dependent axis in the clonogenic expansion of prostate cancer stem cells.

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.

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.

Molecular pathway for thymoquinone-induced cell-cycle arrest and apoptosis in neoplastic keratinocytes.

Thymoquinone (TQ), the most abundant constituent in black seed, was shown to possess potent chemopreventive activities against DMBA-initiated TPA-promoted skin tumors in mice. Despite the potential interest in TQ as a skin antineoplastic agent, its mechanism of action has not been examined yet. Using primary mouse keratinocytes, papilloma (SP-1) and spindle (I7) carcinoma cells, we studied the cellular and molecular events involved in TQ's antineoplastic activity. We show that non-cytotoxic concentrations of TQ reduce the proliferation of neoplastic keratinocytes by 50%. The sensitivity of cells to TQ treatment appears to be stage dependent such that papilloma cells are twice as sensitive to the growth inhibitory effects of TQ as the spindle cancer cells. TQ treatment of SP-1 cells induced G0/G1 cell-cycle arrest, which correlated with sharp increases in the expression of the cyclin-dependent kinase inhibitor p16 and a decrease in cyclin D1 protein expression. TQ-induced growth inhibition in I7 cells by inducing G2/M cell-cycle arrest, which was associated with an increase in the expression of the tumor suppressor protein p53 and a decrease in cyclin B1 protein. At longer times of incubation, TQ induced apoptosis in both cell lines by remarkably increasing the ratio of Bax/Bcl-2 protein expression and decreasing Bcl-xL protein. The apoptotic effects of TQ were more pronounced in SP-1 than in I7 cells. Collectively, these findings support a potential role for TQ as a chemopreventive agent, particularly at the early stages of skin tumorigenesis.