We are interested in understanding the general principles governing cancer stem cell development. The lab currently focuses on elucidating the cellular basis and molecular regulation of prostate cancer (PCa) cell heterogeneity. Our research, encompassing four inter-connected parts (see below), has been supported by extramural grants from the NCI, DOD, CPRIT, and many other funding agencies. The ultimate goal of our research is to identify novel therapeutics that specifically targets the undifferentiated prostate cancer stem cells (PCSCs) and to utilize such novel therapeutics as adjuvant therapy for PCa patients to prevent recurrence and metastasis. The general principles uncovered in studying PCa, and the novel therapeutic targets and therapeutics identified, are applicable to many other cancers. 

I.Cell(s)-of-origin of prostate cancer

We are taking two complimentary approaches to studying the cell(s)-of-origin of PCa. The first approach employs developing novel animal models. The mouse prostate, although not developing spontaneous malignancies, has been widely used to study prostate and PCa biology. At least four putative mouse prostate epithelial stem cells have been reported (Laffin & Tang, Cell Stem Cell, 2010). Among the most pressing questions are what is the interrelationship between these subpopulations of stem/progenitor cells and how we can use the mouse models to understand the involvement of stem cells vs. their progeny in PCa initiation, propagation, progression, metastasis, and therapy resistance. One common property shared by virtually all somatic stem cells is their quiescence or dormancy (Rycaj and Tang, Cancer Res, 2015). Taking advantage of this biological property, we have established a novel, tet-off, bigenic mouse model that allows us, for the first time, to purify out LIVE slow-cycling (i.e., GFP-retaining) mouse prostatic epithelial cells. Preliminary evidence indicates that the long-term label-retaining prostatic epithelial cells have very primitive stem cell properties and possess great regenerative capacity. Not only will this novel model help clarify the interrelationship between the reported prostate stem cell populations but also we could cross our animals with PCa-prone mouse models (such as the Hi-Myc and Pten-/- models) to address the important question of whether dormant cancer cells are CSCs and to perform co-clinical trials on novel therapeutics we are developing.

In the second approach, we utilize freshly purified human normal/benign prostate luminal (CD49f-Trop2+) and basal (CD49fhiTrop2+) epithelial cells to perform genome-wide RNA-Seq analysis, which has revealed distinct gene expression profiles in the two cell populations: basal cells are enriched in genes associated with development & stem cells, EMT/cell movement & motitlity, proneural and neuronal development, and MYC-regulated ribosome biogenesis whereas luminal cells preferentially express genes involved in steroid hormone synthesis and metabolism. Importantly, the basal/stem cell gene expression profile is linked to aggressive and anaplastic PCa variants as well as castration-resistant PCa (CRPC) (Nat Commun, 2016).  Of significance, we have developed a novel culture system that allows, for the first time, the propagation of luminal progenitor cells. We further demonstrate that the human prostate luminal progenitor cells, just like basal cells, can be tumorigenically transformed.

Altogether, our studies of the cell(s)-of origin for PCa are expected to generate critical information to help stratify cancer patients and develop personalized therapies.

II.Systematic efforts in dissecting the cellular heterogeneity of PCSCs

Tremendous cellular heterogeneity contributes greatly to the ultimate treatment failure, tumor relapse, and distant metastasis. Using multiple xenograft models and >220 primary prostate tumors, we have provided the direct experimental evidence that human PCa cells are not all equal with respect to their tumor-regenerating and tumor-propagating activities. Rather, there exist small subsets of PCa cells endowed with much enhanced tumor-initiating and stem cell properties (Cancer Res, 2005; Oncogene 2006; Cancer Res., 2007; Cancer Res, 2008). Recently, using novel lentiviral reporters, we have prospectively purified the differentiated (PSA+) and undifferentiated (PSA-/lo) PCa cells to show that the PSA-/lo cell population harbors cells that can undergo asymmetric cell division and possess long-term tumor-propagating activity in androgen-proficient mice and remarkable tumor-generating capacity in castrated hosts. Furthermore, the PSA-/lo PCa cells are highly enriched in stem cell associated genes (Cell Stem Cell, 2012). Moreover, the PSA-/lo PCa cell population possesses distinct epigenetic landscape and is heterogeneous harboring many tumorigenic  subsets (Oncotarget, 2015) including highly castration-resistant TM+ subpopulation (Clin Cancer Res, 2016). We have recently performed and are currently analyzing whole-genome RNA-seq and ChIP-Seq (for H3K4me1, H3K4me3, and H3K27me3 marks) in PSA+ and PSA-/lo PCa cells. The same global strategy is being employed to study the plasticity in and dedifferentiation of PSA+ PCa cells upon persistent castration. This line of research is expected to uncover novel epigenetic regulators that govern PCSC development and identify novel epigenetic targets for PCSCs. Our studies in human PCa have clear-cut implications in understanding cellular heterogeneity and plasticity in other tumor systems (Cell Res, 2012).

By establishing an innovative experimental system, we have uncovered a prominent microenvironment reprogrammed stem cell associated metastasis gene signature that can predict the metastatic propensity of patient prostate tumors. A novel signaling pathway is also uncovered in which HOXB9 directly regulates two key downstream molecules, i.e., osteopontin and CD44 that mediate the enhanced metastasis of human PCa cells. Importantly, we have zeroed in on a potent subpopulation of metastatic PCSCs that harbor virtually all the metastatic capacity.

III.Molecular regulators of PCSCs

We recently identified a retrotransposed gene, NanogP8, to be expressed in all somatic cancer cell types examined. NanogP8 is a homolog to Nanog1, the latter of which is expressed exclusively in ES cells. We have demonstrated that NanogP8 is required for the tumorigenicity of somatic cancer cells via regulating cancer stem cell (CSC) properties (Stem Cells, 2009). Conversely, inducible expression of NanogP8 is sufficient to reprogram bulk cancer cells to CSCs and to promote castration-resistant PCa regeneration (Oncogene, 2011). This line of research suggests that somatic cancer cells reactivate NanogP8 to positively regulate CSC properties (Stem Cells, 2015). Our ongoing whole-genome ChIP-Seq analysis indicates that NanogP8 in PCa cells binds to genomic loci both similar to and different from Nanog1. We have also identified a novel NanogP8 interacting protein complex that regulates CSC activity.

We have performed a miRNA expression library screening in several highly purified PCa stem/progenitor cell populations. The results show that, remarkably, only 4 miRNAs (miR-34a, let-7, miR-106a, and miR-141) are commonly under-expressed and 2 miRNAs (miR-301 and miR-452) commonly overexpressed in all PCSC subpopulations. By focusing on miR-34a, we have shown that it inhibits many PCSC properties as well as PCa metastasis by directly suppressing CD44 (Nature Med, 2011). The follow-up study shows that let-7 also negatively regulates PCa cell tumorigenicity but through different mechanisms (Cancer Res, 2012). Very excitingly, miR-141, one of the tumor-suppressive miR-200 family member, is most dramatically downregulated in tumorigenic subsets purified from patient prostate tumors. By whole-genome RNA-Seq analysis, we have uncovered that miR-141 inhibits PCa metastasis by targeting a cohort of genes in the RhoGTPase family. Our recent work demonstrates that miR-128, which is often downregulated or lost in PCa and metastasis, also exhibits tumor-suppressive functions against the PCSCs by targeting BMI-1 (Cancer Res, 2014). Finally, miR-199a-3p, which is dramatically under-expressed in the CD44+ PCSCs, has also been found to suppress PCSC properties and PCa growth (Cancer Res, 2016). Our studies are consistent with the emerging theme that multiple tumor-suppressive miRNAs coordinately and distinctively regulate CSC properties (Cancer Res, 2011). The lab continues to focus on miRNA and recently, lncRNA, regulation of tumorigenic PCa cells.

IV.Therapeutic targeting of castration-resistant and metastasis-prone PCSCs

Several lines of our research are providing critical information on developing novel PCSC-targeting therapeutics. 1) Tumor-suppressive miRNAs identified above, e.g., miR-34a, let-7, miR-128, and miR-141 that preferentially target PCa stem/progenitor cells are being exploited as novel replacement therapeutics against PCa recurrence and metastasis. 2) Strategies inhibiting positive regulators of PCSCs such as NanogP8 and Bmi-1 are being developed to target the self-renewal machinery of PCSCs. These novel strategies and therapeutics target the ‘stemness’ of the cancer cells. 3) With our ability to obtain increasingly enriched PCSC subpopulations, we have started a phage-display library screening in attempt to identify peptides that can specifically home in on the PSA-/lo (and other populations of) PCSCs. By far we have identified two peptides that have demonstrated preferential binding to PSA-/lo PCa cells and we are currently testing their utilities as therapeutics. 4) We have observed that the PSA+ and PSA-/lo undergo dynamic changes resulting in the homogeneous population of PSA-/lo cells upon persistent castration. Using these cells, we have performed drug screenings including targeted drug screening using individual compounds/agents, the FDA 1,200 drug library, and the 740 human kinome inhibitor libraries (Oncotarget, 2016). We are also planning high-throughput chemical library and RNAi library screening. Our ultimate goal is to identify novel chemicals and molecular therapeutics that can target undifferentiated and drug-resistant PCSCs.