ADORA2A-driven proline synthesis triggers epigenetic reprogramming in neuroendocrine prostate and lung cancers.

Cell lineage plasticity is one of the major causes for the failure of targeted therapies in various cancers. However, the driver and actionable drug targets in promoting cancer cell lineage plasticity are scarcely identified. Here, we found that a G protein-coupled receptor, ADORA2A, is specifically upregulated during neuroendocrine differentiation, a common form of lineage plasticity in prostate cancer and lung cancer following targeted therapies. Activation of the ADORA2A signaling rewires the proline metabolism via an ERK/MYC/PYCR cascade. Increased proline synthesis promotes deacetylases SIRT6/7-mediated deacetylation of histone H3 at lysine 27 (H3K27), and thereby biases a global transcriptional output toward a neuroendocrine lineage profile. Ablation of Adora2a in genetically engineered mouse models inhibits the development and progression of neuroendocrine prostate and lung cancers, and, intriguingly, prevents the adenocarcinoma-to-neuroendocrine phenotypic transition. Importantly, pharmacological blockade of ADORA2A profoundly represses neuroendocrine prostate and lung cancer growth in vivo. Therefore, we believe that ADORA2A can be used as a promising therapeutic target to govern the epigenetic reprogramming in neuroendocrine malignancies.

IL-1ß Is an Androgen-Responsive Target in Macrophages for Immunotherapy of Prostate Cancer.

Great attention is paid to the role of androgen receptor (AR) as a central transcriptional factor in driving the growth of prostate cancer (PCa) epithelial cells. However, the understanding of the role of androgen in PCa-infiltrated immune cells and the impact of androgen deprivation therapy (ADT), the first-line treatment for advanced PCa, on the PCa immune microenvironment remains limited. On the other hand, immune checkpoint blockade has revolutionized the treatment of certain cancer types, but fails to achieve any benefit in advanced PCa, due to an immune suppressive environment. In this study, it is reported that AR signaling pathway is evidently activated in tumor-associated macrophages (TAMs) of PCa both in mice and humans. AR acts as a transcriptional repressor for IL1B in TAMs. ADT releases the restraint of AR on IL1B and therefore leads to an excessive expression and secretion of IL-1ß in TAMs. IL-1ß induces myeloid-derived suppressor cells (MDSCs) accumulation that inhibits the activation of cytotoxic T cells, leading to the immune suppressive microenvironment. Critically, anti-IL-1ß antibody coupled with ADT and the immune checkpoint inhibitor anti-PD-1 antibody exerts a stronger anticancer effect on PCa following castration. Together, IL-1ß is an important androgen-responsive immunotherapeutic target for advanced PCa.

Numb/Parkin-directed mitochondrial fitness governs cancer cell fate via metabolic regulation of histone lactylation.

Cell plasticity and neuroendocrine differentiation in prostate and lung adenocarcinomas are one of the major reasons for therapeutic resistance to targeted therapy. Whether and how metabolic changes contribute to this adenocarcinoma-to-neuroendocrine cell fate transition remains largely unclear. Here we show that neuroendocrine prostate or lung cancer cells possess mostly fragmented mitochondria with low membrane potential and rely on glycolysis for energy metabolism. We further show an important role of the cell fate determinant Numb in mitochondrial quality control via binding to Parkin and facilitating Parkin-mediated mitophagy. Deficiency in the Numb/Parkin pathway in prostate or lung adenocarcinomas causes a metabolic reprogramming featured with a significant increase in production of lactate acid, which subsequently leads to an upregulation of histone lactylation and transcription of neuroendocrine-associated genes. Collectively, the Numb/Parkin-directed mitochondrial fitness is a key metabolic switch and a promising therapeutic target on cancer cell plasticity through the regulation of histone lactylation.

Extracellular vesicles produced by human-induced pluripotent stem cell-derived endothelial cells can prevent arterial stenosis in mice via autophagy regulation.

Intravascular transplantation of human-induced pluripotent stem cells (hiPSCs) demonstrated a significant therapeutic effect in the treatment of restenosis by the paracrine function of extracellular vesicles (EVs). However, the risk of tumorigenicity and poor cell survival limits its clinical applications. In this study, we for the first time applied a highly efficient and robust three-dimensional (3D) protocol for hiPSC differentiation into endothelial cells (ECs) with subsequent isolation of EVs from the derived hiPSC-EC (ECs differentiated from hiPSCs), and validated their therapeutic effect in intimal hyperplasia (IH) models. We found that intravenously (iv) injected EVs could accumulate on the carotid artery endothelium and significantly alleviate the intimal thickening induced by the carotid artery ligation. To elucidate the mechanism of this endothelial protection, we performed miRNA expression profiling and found out that among the most conserved endothelial miRNAs, miR-126 was the most abundant in hiPSC-EC-produced EVs (hiPSC-EC-EV). MiR-126 depletion from hiPSC-EC-EV can hinder its protective effect on human umbilical vein endothelial cells (HUVECs) in an inflammatory process. A variety of functional in vitro studies revealed that miR-126 was able to prevent endothelial apoptosis after inflammatory stimulation, as well as promote EC migration and tube formation through autophagy upregulation. The latter was supported by in vivo studies demonstrating that treatment with hiPSC-EC-EV can upregulate autophagy in mouse carotid artery ECs, thereby preventing IH and modulating vascular homeostasis via remodeling of the vascular intima. Our findings suggest a regulatory mechanism for the therapeutic effect on arterial restenosis by autophagy regulation, and provide a potential strategy for clinical treatment of the disease.

Zeb1 sustains hematopoietic stem cell functions by suppressing mitofusin-2-mediated mitochondrial fusion.

Metabolic status is essential in maintaining normal functions of hematopoietic stem cells (HSCs). However, how the dynamic of the mitochondrion, as a central organelle in metabolism, is molecularly regulated to orchestrate metabolism and HSC stemness remains to be elucidated. Here, we focus on the role of Zeb1, a well-characterized epithelial-to-mesenchymal transition (EMT) inducer which has been demonstrated to confer stem-cell-like characteristics in multiple cancer types in stemness regulation of HSCs. Using a Zeb1-tdTomato reporter mouse model, we find that Zeb1+Lin-Sca-1+c-Kit+ cells (Zeb1+-LSKs) represent a subset of functional long-term HSCs. Zeb1+LSKs exhibit a reduced reactive oxygen species (ROS) level, low mitochondrial mass, low mitochondrial membrane potential (MMP), and particularly small, round fragmented mitochondria. Of note, ectopic expression of Zeb1 leads to a fragmented mitochondrial morphology with a low mitochondrial metabolic status in EML cells. In addition, Zeb1-knockout (Zeb1-KO) LSKs from fetal liver display an exhausted stem-cell activity. Zeb1 deficiency results in elongated and tubulated mitochondria with increased mitochondrial mass, elevated MMP, and higher ROS production. Mechanistically, Zeb1 acts as a transcriptional suppressor on the key mitochondrial-fusion protein Mitofusin-2 (encoded by Mfn2). We highlight an important role of Zeb1 in the regulation of mitochondrial morphology in HSC and the metabolic control of HSC stemness by repressing Mfn2-mediated mitochondrial fusion.

Gremlin1 is a therapeutically targetable FGFR1 ligand that regulates lineage plasticity and castration resistance in prostate cancer.

Among the greatest hurdles in clinical management of prostate cancer (PCa) are the progression to lethal castration-resistant prostate cancer (CRPC) and the lack of suitable targeted therapies for advanced disease. Here we identify Gremlin1 as a ligand for fibroblast growth factor receptor 1 (FGFR1), which promotes lineage plasticity and drives castration resistance. Importantly, we generate a specific anti-Gremlin1 therapeutic antibody and demonstrate synergistic effect with androgen deprivation therapy (ADT) in CRPC. GREM1 transcription is suppressed by androgen receptor (AR) and released following ADT. We show that Gremlin1 binds to FGFR1 and activates downstream MAPK signaling. Gremlin1 interacts with FGFR1 differently to its canonical ligand FGF1, as revealed through protein structure docking and mutagenesis experiments. Altogether, our data indicate Gremlin1 as a promising candidate therapeutic target for CRPC.

The Cell-Isolation Capsules with Rod-Like Channels Ensure the Survival and Response of Cancer Cells to Their Microenvironment.

Current macrocapsules with semipermeable but immunoprotective polymeric membranes are attractive devices to achieve the purpose of immunoisolation, however, their ability to allow diffusion of essential nutrients and oxygen is limited, which leads to a low survival rate of encapsulated cells. Here, a novel method is reported by taking advantage of thermotropic liquid crystals, sodium laurylsulfonate (SDS) liquid crystals (LCs), and rod-like crystal fragments (LCFs) to develop engineered alginate hydrogels with rod-like channels. This cell-isolation capsule with an engineered alginate hydrogel-wall allows small molecules, large molecules, and bacteria to diffuse out from the capsules freely but immobilizes the encapsulated cells inside and prevents cells in the microenvironment from moving in. The encapsulated cells show a high survival rate with isolation of host immune cells and long-term growth with adequate nutrients and oxygen supply. In addition, by sharing and responding to the normal molecular and vesicular microenvironment (NMV microenvironment), encapsulated cancer cells display a transition from tumorous phenotypes to ductal features of normal epithelial cells. Thus, this device will be potentially useful for clinical application in cell therapy by secreting molecules and for establishment of patient-derived xenograft (PDX) models that are often difficult to achieve for certain types of tumors, such as prostate cancer.

Single-cell RNA sequencing reveals the epithelial cell heterogeneity and invasive subpopulation in human bladder cancer.

Bladder cancer represents a highly heterogeneous disease characterized by distinct histological, molecular and clinical phenotypes, and a detailed analysis of tumor cell invasion and crosstalks within bladder tumor cells has not been determined. Here, we applied droplet-based single-cell RNA sequencing (scRNA-seq) to acquire transcriptional profiles of 36 619 single cells isolated from seven patients. Single cell transcriptional profiles matched well with the pathological basal/luminal subtypes. Notably, in T1 tumors diagnosed as luminal subtype, basal cells displayed characteristics of epithelial-mesenchymal transition (EMT) and mainly located at the tumor-stromal interface as well as micrometastases in the lamina propria. In one T3 tumor, muscle-invasive tumor showed significantly higher expression of cancer stem cell markers SOX9 and SOX2 than the primary tumor. We additionally analyzed communications between tumor cells and demonstrated its relevance to basal/luminal phenotypes. Overall, our single-cell study provides a deeper insight into the tumor cell heterogeneity associated with bladder cancer progression.

Stepwise Induction of Inner Ear Hair Cells From Mouse Embryonic Fibroblasts via Mesenchymal- to-Epithelial Transition and Formation of Otic Epithelial Cells.

Although embryonic stem cells or induced pluripotent stem cells are able to differentiate into inner ear hair cells (HCs), they have drawbacks limiting their clinical application, including a potential risk of tumourigenicity. Direct reprogramming of fibroblasts to inner ear HCs could offer an alternative solution to this problem. Here, we present a stepwise guidance protocol to induce mouse embryonic fibroblasts to differentiate into inner ear HC-like cells (HCLs) via mesenchymal-to-epithelial transition and then acquisition of otic sensory epithelial cell traits by overexpression of three key transcription factors. These induced HCLs express multiple HC-specific proteins, display protrusions reminiscent of ciliary bundle structures, respond to voltage stimulation, form functional mechanotransduction channels, and exhibit a transcriptional profile of HC signature. Together, our work provides a new method to produce functional HCLs in vitro, which may have important implications for studies of HC development, drug discovery, and cell replacement therapy for hearing loss.

A novel mouse model for liver metastasis of prostate cancer reveals dynamic tumour-immune cell communication.

OBJECTIVES: In contrast to extensive studies on bone metastasis in advanced prostate cancer (PCa), liver metastasis has been under-researched so far. In order to decipher molecular and cellular mechanisms underpinning liver metastasis of advanced PCa, we develop a rapid and immune sufficient mouse model for liver metastasis of PCa via orthotopic injection of organoids from PbCre+ ; rb1f/f ;p53f/f mice. MATERIALS AND METHODS: PbCre+ ;rb1f/f ;p53f/f and PbCre+ ;ptenf/f ;p53f/f mice were used to generate PCa organoid cultures in vitro. Immune sufficient liver metastasis models were established via orthotopic transplantation of organoids into the prostate of C57BL/6 mice. Immunofluorescent and immunohistochemical staining were performed to characterize the lineage profile in primary tumour and organoid-derived tumour (ODT). The growth of niche-labelling reporter infected ODT can be visualized by bioluminescent imaging system. Immune cells that communicated with tumour cells in the liver metastatic niche were determined by flow cytometry. RESULTS: A PCa liver metastasis model with full penetrance is established in immune-intact mouse. This model reconstitutes the histological and lineage features of original tumours and reveals dynamic tumour-immune cell communication in liver metastatic foci. Our results suggest that a lack of CD8+ T cell and an enrichment of CD163+ M2-like macrophage as well as PD1+ CD4+ T cell contribute to an immuno-suppressive microenvironment of PCa liver metastasis. CONCLUSIONS: Our model can be served as a reliable tool for analysis of the molecular pathogenesis and tumour-immune cell crosstalk in liver metastasis of PCa, and might be used as a valuable in vivo model for therapy development.

Single-cell analysis supports a luminal-neuroendocrine transdifferentiation in human prostate cancer.

Neuroendocrine prostate cancer is one of the most aggressive subtypes of prostate tumor. Although much progress has been made in understanding the development of neuroendocrine prostate cancer, the cellular architecture associated with neuroendocrine differentiation in human prostate cancer remain incompletely understood. Here, we use single-cell RNA sequencing to profile the transcriptomes of 21,292 cells from needle biopsies of 6 castration-resistant prostate cancers. Our analyses reveal that all neuroendocrine tumor cells display a luminal-like epithelial phenotype. In particular, lineage trajectory analysis suggests that focal neuroendocrine differentiation exclusively originate from luminal-like malignant cells rather than basal compartment. Further tissue microarray analysis validates the generality of the luminal phenotype of neuroendocrine cells. Moreover, we uncover neuroendocrine differentiation-associated gene signatures that may help us to further explore other intrinsic molecular mechanisms deriving neuroendocrine prostate cancer. In summary, our single-cell study provides direct evidence into the cellular states underlying neuroendocrine transdifferentiation in human prostate cancer.

Blockade of ß-Catenin-Induced CCL28 Suppresses Gastric Cancer Progression via Inhibition of Treg Cell Infiltration.

Dysregulation of Wnt/ß-catenin signaling is frequently observed in human gastric cancer. Elucidation of the tumor immune microenvironment is essential for understanding tumorigenesis and for the development of immunotherapeutic strategies. However, it remains unclear how ß-catenin signaling regulates the tumor immune microenvironment in the stomach. Here, we identify CCL28 as a direct transcriptional target gene of ß-catenin/T-cell factor (TCF). Protein levels of ß-catenin and CCL28 positively correlated in human gastric adenocarcinoma. ß-Catenin-activated CCL28 recruited regulatory T (Treg) cells in a transwell migration assay. In a clinically relevant mouse gastric cancer model established by Helicobacter (H.) felis infection and N-methyl-N-nitrosourea (MNU) treatment, inhibition of ß-catenin/TCF activity by a pharmacologic inhibitor iCRT14 suppressed CCL28 expression and Treg cell infiltration in the stomach. Moreover, an anti-CCL28 antibody attenuated Treg cell infiltration and tumor progression in H. felis/MNU mouse models. Diphtheria toxin-induced Treg cell ablation restrained gastric cancer progression in H. felis/MNU-treated DEREG (Foxp3-DTR) mice, clarifying the tumor-promoting role of Treg cells. Thus, the ß-catenin-CCL28-Treg cell axis may serve as an important mechanism for immunosuppression of the stomach tumor microenvironment. Our findings reveal an immunoregulatory role of ß-catenin signaling in stomach tumors and highlight the therapeutic potential of CCL28 blockade for the treatment of gastric cancer. SIGNIFICANCE: These findings demonstrate an immunosuppressive role of tumor-intrinsic ß-catenin signaling and the therapeutic potential of CCL28 blockade in gastric cancer.

Identification of a Zeb1 expressing basal stem cell subpopulation in the prostate.

The basal cell compartment in many epithelial tissues is generally believed to serve as an important pool of stem cells. However, basal cells are heterogenous and the stem cell subpopulation within basal cells is not well elucidated. Here we uncover that the core epithelial-to-mesenchymal transition (EMT) inducer Zeb1 is expressed in a prostate basal cell subpopulation. The Zeb1+ prostate epithelial cells are multipotent prostate basal stem cells (PBSCs) that can self-renew and generate functional prostatic glandular structures at the single-cell level. Genetic ablation studies reveal an indispensable role for Zeb1 in prostate basal cell development. Utilizing unbiased single-cell transcriptomic analysis of over 9000 mouse prostate basal cells, we confirm the existence of the Zeb1+ basal cell subset. Moreover, Zeb1+ epithelial cells can be detected in mouse and human prostate tumors. Identification of the PBSC and its transcriptome profile is crucial to advance our understanding of prostate development and tumorigenesis.

The histone methyltransferase Setd2 is indispensable for V(D)J recombination.

The diverse repertoire of T cell receptors (TCR) and immunoglobulins is generated through the somatic rearrangement of respective V, D and J gene segments, termed V(D)J recombination, during early T or B cell development. However, epigenetic regulation of V(D)J recombination is still not fully understood. Here we show that the deficiency of Setd2, a histone methyltransferase that catalyzes lysine 36 trimethylation on histone 3 (H3K36me3) in mice, causes a severe developmental block of thymocytes at the CD4-CD8- DN3 stage. While H3K36me3 is normally enriched at the TCRß locus, Setd2 deficiency reduces TCRß H3K36me3 and suppresses TCRß V(D)J rearrangement by impairing RAG1 binding to TCRß loci and the DNA double-strand break repair. Similarly, Setd2 ablation also impairs immunoglobulin V(D)J rearrangement to induce B cell development block at the pro-B stage. Lastly, SETD2 is frequently mutated in patients with primary immunodeficiency. Our study thus demonstrates that Setd2 is required for optimal V(D)J recombination and normal lymphocyte development.

Downregulation of the histone methyltransferase SETD2 promotes imatinib resistance in chronic myeloid leukaemia cells.

OBJECTIVES: Epigenetic modifiers were important players in the development of haematological malignancies and sensitivity to therapy. Mutations of SET domain-containing 2 (SETD2), a methyltransferase that catalyses the trimethylation of histone 3 on lysine 36 (H3K36me3), were found in various myeloid malignancies. However, the detailed mechanisms through which SETD2 confers chronic myeloid leukaemia progression and resistance to therapy targeting on BCR-ABL remain unclear. MATERIALS AND METHODS: The level of SETD2 in imatinib-sensitive and imatinib-resistant chronic myeloid leukaemia (CML) cells was examined by immunoblotting and quantitative real-time PCR. We analysed CD34+ CD38- leukaemic stem cells by flow cytometry and colony formation assays upon SETD2 knockdown or overexpression. The impact of SETD2 expression alterations or small-molecule inhibitor JIB-04 targeting H3K36me3 loss on imatinib sensitivity was assessed by IC50, cell apoptosis and proliferation assays. Finally, RNA sequencing and ChIP-quantitative PCR were performed to verify putative downstream targets. RESULTS: SETD2 was found to act as a tumour suppressor in CML. The novel oncogenic targets MYCN and ERG were shown to be the direct downstream targets of SETD2, where their overexpression induced by SETD2 knockdown caused imatinib insensitivity and leukaemic stem cell enrichment in CML cell lines. Treatment with JIB-04, an inhibitor that restores H3K36me3 levels through blockade of its demethylation, successfully improved the cell imatinib sensitivity and enhanced the chemotherapeutic effect. CONCLUSIONS: Our study not only emphasizes the regulatory mechanism of SETD2 in CML, but also provides promising therapeutic strategies for overcoming the imatinib resistance in patients with CML.

Aphthous ulcer drug inhibits prostate tumor metastasis by targeting IKKɛ/TBK1/NF-κB signaling.

Tumor metastasis is the major cause of death for prostate cancer (PCa) patients. However, the treatment options for metastatic PCa are very limited. Epithelial-mesenchymal transition (EMT) has been reported to be an indispensable step for tumor metastasis and is suggested to associate with acquisition of cancer stem cell (CSC) attributes. We propose that small-molecule compounds that can reverse EMT or induce mesenchymal-epithelial transition (MET) of PCa cells may serve as drug candidates for anti-metastasis therapy. Methods: The promoters of CDH1 and VIM genes were sub-cloned to drive the expression of firefly and renilla luciferase reporter in a lentiviral vector. Mesenchymal-like PCa cells were infected with the luciferase reporter lentivirus and subjected to drug screening from a 1274 approved small-molecule drug library for the identification of agents to reverse EMT. The dosage-dependent effect of candidate compounds was confirmed by luciferase reporter assay and immunoblotting. Wound-healing assay, sphere formation, transwell migration assay, and in vivo intracardiac and orthotopic tumor xenograft experiments were used to evaluate the mobility, metastasis and tumor initiating capacity of PCa cells upon treatment. Possible downstream signaling pathways affected by the candidate compound treatment were analyzed by RNA sequencing and immunoblotting. Results: Drug screening identified Amlexanox, a drug used for recurrent aphthous ulcers, as a strong agent to reverse EMT. Amlexanox induced significant suppression of cell mobility, invasion, serial sphere formation and in vivo metastasis and tumor initiating capacity of PCa cells. Amlexanox treatment led to downregulation of the IKK-ɛ/ TBK1/ NF-κB signaling pathway. The effect of Amlexanox on EMT reversion and cell mobility inhibition can be mimicked by other IKK-ɛ/TBK1 inhibitors and rescued by reconstitution of dominant active NF-κB. Conclusions: Amlexanox can sufficiently suppress PCa metastasis by reversing EMT through downregulating the IKK-ɛ/TBK1/NF-κB signaling axis.

E-cadherin bridges cell polarity and spindle orientation to ensure prostate epithelial integrity and prevent carcinogenesis in vivo.

Cell polarity and correct mitotic spindle positioning are essential for the maintenance of a proper prostate epithelial architecture, and disruption of the two biological features occurs at early stages in prostate tumorigenesis. However, whether and how these two epithelial attributes are connected in vivo is largely unknown. We herein report that conditional genetic deletion of E-cadherin, a key component of adherens junctions, in a mouse model results in loss of prostate luminal cell polarity and randomization of spindle orientations. Critically, E-cadherin ablation causes prostatic hyperplasia which progresses to invasive adenocarcinoma. Mechanistically, E-cadherin and the spindle positioning determinant LGN interacts with the PDZ domain of cell polarity protein SCRIB and form a ternary protein complex to bridge cell polarity and cell division orientation. These findings provide a novel mechanism by which E-cadherin acts an anchor to maintain prostate epithelial integrity and to prevent carcinogenesis in vivo.

Inactivation of STAT3 Signaling Impairs Hair Cell Differentiation in the Developing Mouse Cochlea.

Although STAT3 signaling is demonstrated to regulate sensory cell differentiation and regeneration in the zebrafish, its exact role is still unclear in mammalian cochleae. Here, we report that STAT3 and its activated form are specifically expressed in hair cells during mouse cochlear development. Importantly, conditional cochlear deletion of Stat3 leads to an inhibition on hair cell differentiation in mice in vivo and in vitro. By cell fate analysis, inactivation of STAT3 signaling shifts the cell division modes from asymmetric to symmetric divisions from supporting cells. Moreover, inhibition of Notch signaling stimulates STAT3 phosphorylation, and inactivation of STAT3 signaling attenuates production of supernumerary hair cells induced by a Notch pathway inhibitor. Our findings highlight an important role of the STAT3 signaling during mouse cochlear hair cell differentiation and may have clinical implications for the recovery of hair cell loss-induced hearing impairment.

Numb-/low Enriches a Castration-Resistant Prostate Cancer Cell Subpopulation Associated with Enhanced Notch and Hedgehog Signaling.

Purpose: To elucidate the role and molecular mechanism of Numb in prostate cancer and the functional contribution of Numb-/low prostate cancer cells in castration resistance.Experimental Design: The expression of Numb was assessed using multiple Oncomine datasets and prostate cancer tissues from both humans and mice. The biological effects of the overexpression and knockdown of Numb in human prostate cancer cell lines were investigated in vitro and in vivo In addition, we developed a reliable approach to distinguish between prostate cancer cell populations with a high or low endogenous expression of Numb protein using a Numb promoter-based lentiviral reporter system. The difference between Numb-/low and Numbhigh prostate cancer cells in the response to androgen-deprivation therapy (ADT) was then tested. The likely downstream factors of Numb were analyzed using luciferase reporter assays, immunoblotting, and quantitative real-time PCR.Results: We show here that Numb was downregulated and negatively correlated with prostate cancer advancement. Functionally, Numb played an inhibitory role in xenograft prostate tumor growth and castration-resistant prostate cancer development by suppressing Notch and Hedgehog signaling. Using a Numb promoter-based lentiviral reporter system, we were able to distinguish Numb-/low prostate cancer cells from Numbhigh cells. Numb-/low prostate cancer cells were smaller and quiescent, preferentially expressed Notch and Hedgehog downstream and stem-cell-associated genes, and associated with a greater resistance to ADT. The inhibition of the Notch and Hedgehog signaling pathways significantly increased apoptosis in Numb-/low cells in response to ADT.Conclusions: Numb-/low enriches a castration-resistant prostate cancer cell subpopulation that is associated with unregulated Notch and Hedgehog signaling. Clin Cancer Res; 23(21); 6744-56. ©2017 AACR.

WNT/ß-Catenin Directs Self-Renewal Symmetric Cell Division of hTERThigh Prostate Cancer Stem Cells.

Cancer stem-like cells (CSC) drive cancer progression and recurrence. Self-renewal expansion of CSC is achieved through symmetric cell division, yet how external stimuli affect intracellular regulatory programs of CSC division modes and stemness remains obscure. Here, we report that the hTERThigh prostate cancer cells exhibit CSC properties, including a stem cell-associated gene expression signature, long-term tumor-propagating capacity and epithelial-to-mesenchymal transition. In promoting the self-renewal symmetric division of hTERThigh prostate cancer cells, WNT3a dramatically decreased the ratio of hTERThigh prostate cancer cells undergoing asymmetric division. Increased WNT/ß-catenin signal activation was also detected in hTERThigh prostate cancer cells. hTERT-mediated CSC properties were at least partially dependent on ß-catenin. These findings provide novel cellular and molecular mechanisms for the self-renewal of CSC orchestrated by tumor microenvironmental stimuli and intracellular signals. Cancer Res; 77(9); 2534-47. ©2017 AACR.