SUMMARY Prostate cancer (PCa) is heterogeneous and contains both differentiated and undifferentiated tumor cells, but the relative functional contribution of these two cell populations remains unclear. Here we report distinct molecular, cellular, and tumor-propagating properties of PCa cells that express high (PSA+) and low (PSA−/lo) levels of the differentiation marker PSA. PSA−/lo PCa cells are quiescent and refractory to stresses including androgen deprivation, exhibit high clonogenic potential, and possess long-term tumor-propagating capacity. They preferentially express stem cell genes and can undergo asymmetric cell division generating PSA+ cells. Importantly, PSA−/lo PCa cells can initiate robust tumor development and resist androgen ablation in castrated hosts, and harbor highly tumorigenic castration-resistant PCa cells that can be prospectively enriched using ALDH+CD44+α2β1+ phenotype. In contrast, PSA+ PCa cells possess more limited tumor-propagating capacity, undergo symmetric division and are sensitive to castration. Together, our study suggests PSA−/lo cells may represent a critical source of castration-resistant PCa cells.

Human cancers are heterogeneous containing stem-like cancer cells operationally defined as cancer stem cells (CSCs) that possess great tumor-initiating and long-term tumor-propagating properties. In this study, we systematically dissect the phenotypic, functional and tumorigenic heterogeneity in human prostate cancer (PCa) using xenograft models and >70 patient tumor samples. In the first part, we further investigate the PSA−/lo PCa cell population, which we have recently shown to harbor self-renewing long-term tumor-propagating cells and present several novel findings. We show that discordant AR and PSA expression in both untreated and castration-resistant PCa (CRPC) results in AR+PSA+, AR+PSA−, AR−PSA−, and AR−PSA+ subtypes of PCa cells that manifest differential sensitivities to therapeutics. We further demonstrate that castration leads to a great enrichment of PSA−/lo PCa cells in both xenograft tumors and CRPC samples and systemic androgen levels dynamically regulate the relative abundance of PSA+ versus PSA−/lo PCa cells that impacts the kinetics of tumor growth. We also present evidence that the PSA−/lo PCa cells possess distinct epigenetic profiles. As the PSA−/lo PCa cell population is heterogeneous, in the second part, we employ two PSA− (Du145 and PC3) and two PSA+ (LAPC9 and LAPC4) PCa models as well as patient tumor cells to further dissect the clonogenic and tumorigenic subsets. We report that different PCa models possess distinct tumorigenic subpopulations that both commonly and uniquely express important signaling pathways that could represent therapeutic targets. Our results have important implications in understanding PCa cell heterogeneity, response to clinical therapeutics, and cellular mechanisms underlying CRPC.

Background Lung cancer treatment has become increasingly dependent upon invasive biopsies to profile tumors for personalized therapy. Recently, tumor expression of PD-L1 has gained interest as a potential predictor of response to immunotherapy. Circulating biomarkers present an opportunity for tumor profiling without the risks of invasive procedures. We characterized PD-L1 expression within populations of nucleated cells in the peripheral blood of lung cancer patients in hopes of expanding the role of liquid biopsy in this setting. Methods Peripheral blood samples from a multi-institutional prospective study of patients with clinical diagnosis of lung cancer were subjected to cytomorphometric and immunohistochemical evaluation using single-cell, automated slide-based, digital pathology. PD-L1 expression was determined by immunofluorescence. Results PD-L1 expression was detected within peripheral circulating cells associated with malignancy (CCAM) in 26/112(23%) non-small cell lung cancer patients. Two distinct populations of nucleated, non-hematolymphoid, PD-L1 expressing cells were identified; cytokeratin positive (CK+, PD-L1+, CD45−) and cytokeratin negative (CK−, PD-L1+, CD45−) cells, both with cytomorphometric features (size, nuclear to cytoplasm ratio) consistent with tumor cells. Patients with >1.1 PD-L1(+) cell/mL (n=14/112) experienced worse overall survival than patients with ≤1.1 PD-L1(+) cell/ml (2-yearOS:31.2% vs 78.8%, p=0.00159). In a Cox model adjusting for stage, high PD-L1(+) cell burden remained a significant predictor of mortality (HR=3.85, 95%CI:1.64–9.09, p=0.002). Conclusions PD-L1 expression is detectable in two distinct cell populations in the peripheral blood of lung cancer patients and is associated with worse survival.

15-Lipoxygenase 2 (15-LOX2), a lipid-peroxidizing enzyme, is mainly expressed in the luminal compartment of the normal human prostate and often decreased or lost in prostate cancer. Previous studies from our lab implicate 15-LOX2 as a functional tumor suppressor. To better understand the biological role of 15-LOX2 in vivo, we established prostate-specific 15-LOX2 transgenic mice using the ARR2PB promoter. Unexpectedly, transgenic expression of 15-LOX2 or 15-LOX2sv-b, a splice variant that lacks the arachidonic acid metabolizing activity, resulted in age-dependent prostatic hyperplasia and enlargement of the prostate. Prostatic hyperplasia induced by both 15-LOX2 and 15-LOX2sv-b was associated with an increase in luminal and Ki-67+ cells; however, 15-LOX2-transgenic prostates also showed a prominent increase in basal cells. Microarray analysis revealed distinct gene expression profiles that could help explain the prostate phenotypes. Strikingly, 15-LOX2, but not 15-LOX2sv-b, transgenic prostate showed upregulation of several well-known stem/progenitor cell molecules including Sca-1, Trop2, p63, Nkx3.1 and Psca. Prostatic hyperplasia caused by both 15-LOX2 and 15-LOX2sv-b did not progress to prostatic intraprostate neoplasia (PIN) or carcinoma and, mechanistically, prostate lobes (especially those of the 15-LOX2 mice) showed a dramatic increase in senescent cells as revealed by increased SA-βgal, p27Kip1 and HP1γ staining. Collectively, our results suggest that 15-LOX2 expression in mouse prostate leads to hyperplasia and also induces cell senescence, which may, in turn, function as a barrier to tumor development.

Normal human prostate (NHP) epithelial cells undergo senescence in vitro and in vivo, but the underlying molecular mechanisms remain obscure. Here we show that the senescence of primary NHP cells, which are immunophenotyped as intermediate basal-like cells expressing progenitor cell markers CD44, ␣2␤1, p63, hTERT, and CK5/CK18, involves loss of telomerase expression, up-regulation of p16, and activation of p53. Using genetically defined manipulations of these three signaling pathways, we show that p16 is the primary determinant of the NHP cell proliferative capacity and that hTERT is required for unlimited proliferative life span. Hence, suppression of p16 significantly extends NHP cell life span, but both p16 inhibition and hTERT are required to immortalize NHP cells. Importantly, immortalized NHP cells retain expression of most progenitor markers, demonstrate gene expression profiles characteristic of proliferating progenitor cells, and possess multilineage differentiation potential generating functional prostatic glands. Our studies shed important light on the molecular mechanisms regulating the proliferative life span of NHP progenitor cells.The prostatic glands contain neuroendocrine (NE) 7 cells and two epithelial cells: 1) luminal cells expressing cytokeratin 8 (CK8) and CK18, androgen receptor (AR), prostate-specific antigen (PSA), prostatic acid phosphatase, CD26, CD57, and 15-lipoxygenase 2 (15-LOX2) and 2) basal cells expressing CK5/CK14, CD44, CD104 (integrin ␤4), Bcl-2, p63, telomerase, and glutathione S-transferase-(1-3). It has been proposed that a common stem/progenitor cell may generate both basal and luminal cells (4). Alternatively, basal cells may function as progenitors to luminal cells (5, 6).The adult rodent prostate possesses regenerative stem cells (SCs) (6 -8). Whether adult human prostate contains definitive SCs is less certain, although there exists strong evidence that the basal cell layer harbors regenerative cells (1, 6, 9 -12), and several candidate populations of human prostate stem/progenitor cells, preferentially localized in the basal layer, have been reported. These include the cells that preferentially express CD44 (13), ␣2␤1 (i.e. ␣2␤1 hi ) (14), or CD133 (15, 16) and the side population (17), whose phenotype is mediated by multidrug resistance family proteins, such as MDR-1 and ABCG2 (18). Interestingly, the ABCG2 ϩ cells in the benign prostate constitute Ͻ1% of total basal cell population and share essentially the same transcriptome as the side population cells (19 * This work was supported, in whole or in part, by National Institutes of Health Grants R01-AG023374, R01-ES015888, and R21-ES015893-01A1. This work was also supported by American Cancer Society Grant RSG MGO-105961, Department of Defense Grants W81XWH-07-1-0616 and PC073751, the Prostate Cancer Foundation, the Elsa Pardee Foundation (to D. G. T.), and two Center Grants, CCSG-5 P30 CA166672 and ES07784. The costs of publication of this article were defrayed in part by the payment of page charges. This article m...