Hsueh-Ping Chao

In order to exploit the specific vulnerabilities of tumors, it is urgent to identify the basis of associated defects in genome maintenance. One unsolved problem is the mechanism of inhibition of processing of DNA double-strand break repair by REV7 and its influence on DNA repair pathways. We searched for REV7-associated proteins in human cells and found FAM35A, a protein of previously unknown function. By analyzing the FAM35A sequence we discovered that FAM35A has an unstructured N-terminal region and a C-terminal region harboring three OB-fold domains similar to single-stranded binding protein RPA. Knockdown of FAM35A caused sensitivity to DNA damaging agents, and FAM35A re-localized in damaged cell nuclei. In a BRCA1 mutant cell line, however, depletion of FAM35A increased resistance to camptothecin, suggesting that FAM35A participates in processing of DNA ends to allow more efficient DNA repair. Moreover, we found FAM35A absent in one widely used BRCA1-mutant cancer cell line (HCC1937) with anomalous resistance to PARP inhibitors. A survey of FAM35A alterations in cancer revealed that the gene is altered at the highest frequency in prostate cancers (up to 13%) and significantly less expressed in metastatic cases. The results reveal a new DNA repair factor with promise as a therapeutically relevant cancer marker.

MicroRNAs play important roles in regulating tumour development, progression and metastasis. Here we show that one of the miR-200 family members, miR-141, is under-expressed in several prostate cancer (PCa) stem/progenitor cell populations in both xenograft and primary patient tumours. Enforced expression of miR-141 in CD44⁺ and bulk PCa cells inhibits cancer stem cell properties including holoclone and sphere formation, as well as invasion, and suppresses tumour regeneration and metastasis. Moreover, miR-141 expression enforces a strong epithelial phenotype with a partial loss of mesenchymal phenotype. Whole-genome RNA sequencing uncovers novel miR-141-regulated molecular targets in PCa cells including the Rho GTPase family members (for example, CDC42, CDC42EP3, RAC1 and ARPC5) and stem cell molecules CD44 and EZH2, all of which are validated as direct and functionally relevant targets of miR-141. Our results suggest that miR-141 employs multiple mechanisms to obstruct tumour growth and metastasis.

Elucidating the cell of origin of cancer has great significance in stratifying patients into appropriate treatment groups and for developing novel targeted therapies. Early studies demonstrate that only stem-like basal cells in the normal human prostate (NHP) can function as the cell of origin for prostate cancer (PCa). Here, we show that the organoids derived from bulk NHP luminal cells can also be tumorigenically transformed. We further show that the WIT medium, which is used to culture human mammary epithelial progenitor cells, when combined with the ROCK inhibitor, can readily propagate a population of progenitor-like cells from the primary NHP luminal cell isolates. Such functionally defined luminal progenitors can be transformed by distinct sets of genetic perturbations (i.e., AR+AKT/ERG or c-MYC+PTEN knockout) to form tumor glands. Genome-wide RNA-Seq analysis of freshly purified unperturbed human benign prostatic basal and luminal cells and culture-expanded lineage-specific stem/progenitor populations reveals that the luminal progenitors possess a distinct gene expression profile that is greatly enriched in advanced, castration-resistant, and metastatic PCa, and it associates with poor patient survival. The ability of the simple two-dimensional culture system reported herein to greatly enrich NHP progenitor-like cells should facilitate biological and biochemical studies as well as high-throughput screening in these cells and in progenitor-like PCa cells.

Purpose: We have shown that the phenotypically undifferentiated (PSA−/lo) prostate cancer cell population harbors long-term self-renewing cancer stem cells (CSC) that resist castration, and a subset of the cells within the PSA−/lo population bearing the ALDHhiCD44+α2β1+ phenotype (Triple Marker+/TM+) is capable of robustly initiating xenograft tumors in castrated mice. The goal of the current project is to further characterize the biologic properties of TM+ prostate cancer cell population, particularly in the context of initiating and propagating castration-resistant prostate cancer (CRPC). Experimental Design: The in vivo CSC activities were measured by limiting-dilution serial tumor transplantation assays in both androgen-dependent and androgen-independent prostate cancer xenograft models. In vitro clonal, clonogenic, and sphere-formation assays were conducted in cells purified from xenograft and patient tumors. qPCR, Western blot, lentiviral-mediated gene knockdown, and human microRNA arrays were performed for mechanistic studies. Results: By focusing on the LAPC9 model, we show that the TM+ cells are CSCs with both tumor-initiating and tumor-propagating abilities for CRPC. Moreover, primary patient samples have TM+ cells, which possess CSC activities in “castrated” culture conditions. Mechanistically, we find that (i) the phenotypic markers are causally involved in CRPC development; (ii) the TM+ cells preferentially express castration resistance and stem cell–associated molecules that regulate their CSC characteristics; and (iii) the TM+ cells possess distinct microRNA expression profiles and miR-499-5p functions as an oncomir. Conclusions: Our results define the TM+ prostate cancer cells as a population of preexistent stem-like cancer cells that can both mediate and propagate CRPC and highlight the TM+ cell population as a therapeutic target. Clin Cancer Res; 22(17); 4505–16. ©2016 AACR.

The prostate gland mainly contains basal and luminal cells constructed as a pseudostratified epithelium. Annotation of prostate epithelial transcriptomes provides a foundation for discoveries that can impact disease understanding and treatment. Here we describe a genome-wide transcriptome analysis of human benign prostatic basal and luminal epithelial populations using deep RNA sequencing. Through molecular and biological characterizations, we show that the differential gene-expression profiles account for their distinct functional properties. Strikingly, basal cells preferentially express gene categories associated with stem cells, neurogenesis and ribosomal RNA (rRNA) biogenesis. Consistent with this profile, basal cells functionally exhibit intrinsic stem-like and neurogenic properties with enhanced rRNA transcription activity. Of clinical relevance, the basal cell gene-expression profile is enriched in advanced, anaplastic, castration-resistant and metastatic prostate cancers. Therefore, we link the cell-type-specific gene signatures to aggressive subtypes of prostate cancer and identify gene signatures associated with adverse clinical features.

Longitudinal tracking of subpopulation dynamics and molecular changes during LNCaP cell castration and identification of inhibitors that could target the PSA−/lo castration-resistant cells

Systematic dissection of phenotypic, functional, and tumorigenic heterogeneity of human prostate cancer cells Systematic dissection of phenotypic, functional, and tumorigenic heterogeneity of human prostate cancer cells

The homeobox domain transcription factor NANOG, a key regulator of embryonic development and cellular reprogramming, has been reported to be broadly expressed in human cancers. Functional studies have provided strong evidence that NANOG possesses protumorigenic attributes. In addition to promoting self-renewal and long-term proliferative potential of stem-like cancer cells, NANOG-mediated oncogenic reprogramming may underlie clinical manifestations of malignant disease. In this review, we examine the molecular origin, expression, biological activities, and mechanisms of action of NANOG in various malignancies. We also consider clinical implications such as correlations between NANOG expression and cancer prognosis and/or response to therapy. We surmise that NANOG potentiates the molecular circuitry of tumorigenesis, and thus may represent a novel therapeutic target or biomarker for the diagnosis, prognosis, and treatment outcome of cancer. Finally, we present critical pending questions relating NANOG to cancer stem cells and tumor development. Stem Cells 2015;33:2381–2390

BACKGROUND: GP.Mur (Mi.III) is a glycophorin B‐A‐B hybrid sialoglycoprotein expressing several potent immunogens, including Mi^a, Mur, and Hil. GP.Mur is considered one of the most important red blood cell (RBC) phenotypes in blood banking in Southeast Asia. However, there are no antibodies commercially available for the screening of GP.Mur RBCs. STUDY DESIGN AND METHODS: To develop a direct blood polymerase chain reaction (PCR) approach for the screening of GP.Mur cells, we first confirmed the genomic sequence differences among four GP.Mur and three Mi(a–) samples by sequencing their GYP.Mur and GYPB genes. With these data, we designed PCR primers that best discriminate GYPB and GYP.Mur. Our primer design also allows the detection of other Hil+ glycophorin variants. We also constructed two plasmids—pGBi2i3 and pMiIIIi2i3—which serve as the negative and positive control DNA, respectively, for the PCR procedure. Additionally, we designed a control PCR to be run side by side with the typing PCR. RESULTS: Because of the high specificity of our primers, we found it unnecessary to extract DNA from blood samples for PCR. We have tested this PCR method on 379 fresh and frozen blood samples. The results were further validated by serology and DNA sequencing and were shown to be completely accurate in our hand. We also found that the rapid genotyping method—high‐resolution melting—can be a timesaving alternative for DNA sequencing. CONCLUSION: This direct blood PCR approach for determination of GP.Mur and related Hil+ phenotypes is reliable and economical and is expected to be useful for blood banking in Southeast Asia.