Oral pimonidazole unveils clinicopathologic and epigenetic features of hypoxic tumour aggressiveness in localized prostate cancer.

BACKGROUND: Tumor hypoxia is associated with prostate cancer (PCa) treatment resistance and poor prognosis. Pimonidazole (PIMO) is an investigational hypoxia probe used in clinical trials. A better understanding of the clinical significance and molecular alterations underpinning PIMO-labeled tumor hypoxia is needed for future clinical application. Here, we investigated the clinical significance and molecular alterations underpinning PIMO-labeled tumor hypoxia in patients with localized PCa, in order to apply PIMO as a prognostic tool and to identify potential biomarkers for future clinical translation. METHODS: A total of 39 patients with localized PCa were recruited and administered oral PIMO before undergoing radical prostatectomy (RadP). Immunohistochemical staining for PIMO was performed on 37 prostatectomy specimens with staining patterns evaluated and clinical association analyzed. Whole genome bisulfite sequencing was performed using laser-capture of microdissected specimen sections comparing PIMO positive and negative tumor areas. A hypoxia related methylation molecular signature was generated by integrating the differentially methylated regions with previously established RNA-seq datasets. RESULTS: Three PIMO staining patterns were distinguished: diffuse, focal, and comedo-like. The comedo-like staining pattern was more commonly associated with adverse pathology. PIMO-defined hypoxia intensity was positively correlated with advanced pathologic stage, tumor invasion, and cribriform and intraductal carcinoma morphology. The generated DNA methylation signature was found to be a robust hypoxia biomarker, which could risk-stratify PCa patients across multiple clinical datasets, as well as be applicable in other cancer types. CONCLUSIONS: Oral PIMO unveiled clinicopathologic features of disease aggressiveness in localized PCa. The generated DNA methylation signature is a novel and robust hypoxia biomarker that has the potential for future clinical translation.

N6-Methyladenosine Reader YTHDF1 Promotes ARHGEF2 Translation and RhoA Signaling in Colorectal Cancer.

BACKGROUND & AIMS: N6-methyladenosine (m6A) governs the fate of RNAs through m6A readers. Colorectal cancer (CRC) exhibits aberrant m6A modifications and expression of m6A regulators. However, how m6A readers interpret oncogenic m6A methylome to promote malignant transformation remains to be illustrated. METHODS: YTH N6-methyladenosine RNA binding protein 1 (Ythdf1) knockout mouse was generated to determine the effect of Ythdf1 in CRC tumorigenesis in vivo. Multiomic analysis of RNA-sequencing, m6A methylated RNA immunoprecipitation sequencing, YTHDF1 RNA immunoprecipitation sequencing, and proteomics were performed to unravel targets of YTHDF1 in CRC. The therapeutic potential of targeting YTHDF1-m6A-Rho/Rac guanine nucleotide exchange factor 2 (ARHGEF2) was evaluated using small interfering RNA (siRNA) encapsulated by lipid nanoparticles (LNP). RESULTS: DNA copy number gain of YTHDF1 is a frequent event in CRC and contributes to its overexpression. High expression of YTHDF1 is significantly associated with metastatic gene signature in patient tumors. Ythdf1 knockout in mice dampened tumor growth in an inflammatory CRC model. YTHDF1 promotes cell growth in CRC cell lines and primary organoids and lung and liver metastasis in vivo. Integrative multiomics analysis identified RhoA activator ARHGEF2 as a key downstream target of YTHDF1. YTHDF1 binds to m6A sites of ARHGEF2 messenger RNA, resulting in enhanced translation of ARHGEF2. Ectopic expression of ARHGEF2 restored impaired RhoA signaling, cell growth, and metastatic ability both in vitro and in vivo caused by YTHDF1 loss, verifying that ARHGEF2 is a key target of YTHDF1. Finally, ARHGEF2 siRNA delivered by LNP significantly suppressed tumor growth and metastasis in vivo. CONCLUSIONS: We identify a novel oncogenic epitranscriptome axis of YTHDF1-m6A-ARHGEF2, which regulates CRC tumorigenesis and metastasis. siRNA-delivering LNP drug validated the therapeutic potential of targeting this axis in CRC.

GRB10 sustains AR activity by interacting with PP2A in prostate cancer cells.

Prostate cancer (PCa) progression is driven by androgen receptor (AR) signaling. Unfortunately, androgen-deprivation therapy and the use of even more potent AR pathway inhibitors (ARPIs) cannot bring about a cure. ARPI resistance (ie, castration-resistant PCa, CRPC) will inevitably develop. Previously, we demonstrated that GRB10 is an AR transcriptionally repressed gene that functionally contributes to CRPC development and ARPI resistance. GRB10 expression is elevated prior to CRPC development in our patient-derived xenograft models and is significantly upregulated in clinical CRPC samples. Here, we analyzed transcriptomic data from GRB10 knockdown in PCa cells and found that AR signaling is downregulated. While the mRNA expression of AR target genes decreased upon GRB10 knockdown, AR expression was not affected at the mRNA or protein level. We further found that phosphorylation of AR serine 81 (S81), which is critical for AR transcriptional activity, is decreased by GRB10 knockdown and increased by its overexpression. Luciferase assay using GRB10-knockdown cells also indicate reduced AR activity. Immunoprecipitation coupled with mass spectrometry revealed an interaction between GRB10 and the PP2A complex, which is a known phosphatase of AR. Further validations and analyses showed that GRB10 binds to the PP2Ac catalytic subunit with its PH domain. Mechanistically, GRB10 knockdown increased PP2Ac protein stability, which in turn decreased AR S81 phosphorylation and reduced AR activity. Our findings indicate a reciprocal feedback between GRB10 and AR signaling, implying the importance of GRB10 in PCa progression.

A synopsis of prostate organoid methodologies, applications, and limitations.

BACKGROUND: Current in vitro modeling systems do not fully reflect the biologic and clinical diversity of prostate cancer (PCa). Organoids are 3D in vitro cell cultures that recapitulate disease heterogeneity, retain prostate gland architecture, and mirror parental tumor characteristics. METHODS: To make better use of organoid models in the PCa research field, we provide a review of cutting-edge prostate organoid methodologies, applications, and limitations. RESULTS: We summarize methodologies for the establishment of benign prostate and PCa organoids and describe some of the model's practical applications and challenges. We highlight the patient-derived xenograft (PDX)-organoid interface model, which may allow for the generation of organoids from primary and rare PCa subtypes. Finally, we discuss potential future utilizations of PCa organoids in the realms of drug development and precision oncology. CONCLUSIONS AND FUTURE DIRECTIONS: Organoids represent a quasi in vivo modeling system that can be easily amenable to genetic modification and functional studies. As such, organoids may serve as an intermediate preclinical model for studying PCa. Future directions may include the refinement of culturing conditions to increase drug response fidelity in PCa organoids. The PDX-organoid interface model may enable the future establishment of primary and rare subtype PCa organoid lines.

CINP is a novel cofactor of KLF5 required for its role in the promotion of cell proliferation, survival and tumor growth.

Krüppel-like factor 5 (KLF5) both suppresses and promotes tumor growth depending on cellular context. The mechanisms underlying tumor promotion could be targetable for therapy. Although a number of transcriptional targets of KLF5 have been identified and implicated in KLF5-mediated tumor growth, how KLF5 regulates these genes remains to be addressed. Here we performed coimmunoprecipitation (co-IP) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the TSU-Pr1 bladder cancer cell line, in which KLF5 is shown to promote tumor growth, to identify KLF5-interacting nuclear proteins that are necessary for KLF5's tumor promoting function. LC-MS/MS revealed 122 potential KLF5 binding proteins in the nuclear proteins precipitated by the KLF5 antibody, and the top nine candidates included AHNAK, TFAM, HSDL2, HNRNPC, CINP, IST1, FBL, PABPC1 and SNRNP40. SRB assays of these nine proteins indicated that silencing CINP had the most potent inhibitory effect on cell growth in KLF5-expressing cells but did not affect parental TSU-Pr1 cells. Further analyses not only confirmed the physical interaction between KLF5 and CINP, also demonstrated that knockdown of CINP attenuated the effects of KLF5 on cell cycle progression, apoptosis and tumorigenesis. Silencing CINP also attenuated the effect of KLF5 on the expression of a number of genes and signaling pathways, including cell cycle regulator Cyclin D1 and apoptosis-related Caspase 7. These results suggest that CINP is a cofactor of KLF5 that is crucial for the promotion of tumor growth, and that the KLF5-CINP interaction could be a novel therapeutic target for inhibiting KLF5-promoted tumor growth.

Treatment with docetaxel in combination with Aneustat leads to potent inhibition of metastasis in a patient-derived xenograft model of advanced prostate cancer.

BACKGROUND: Docetaxel used for first-line treatment of advanced prostate cancer (PCa) is only marginally effective. We previously showed, using the LTL-313H subrenal capsule patient-derived metastatic PCa xenograft model, that docetaxel combined with Aneustat (OMN54), a multivalent plant-derived therapeutic, led to marked synergistic tumour growth inhibition. Here, we investigated the effect of docetaxel+Aneustat on metastasis. METHODS: C4-2 cells were incubated with docetaxel, Aneustat and docetaxel+Aneustat to assess effects on cell migration. The LTL-313H model, similarly treated, was analysed for effects on lung micro-metastasis and kidney invasion. The LTL-313H gene expression profile was compared with profiles of PCa patients (obtained from Oncomine) and subjected to IPA to determine involvement of cancer driver genes. RESULTS: Docetaxel+Aneustat markedly inhibited C4-2 cell migration and LTL-313H lung micro-metastasis/kidney invasion. Oncomine analysis indicated that treatment with docetaxel+Aneustat was associated with improved patient outcome. The drug combination markedly downregulated expression of cancer driver genes such as FOXM1 (and FOXM1-target genes). FOXM1 overexpression reduced the anti-metastatic activity of docetaxel+Aneustat. CONCLUSIONS: Docetaxel+Aneustat can inhibit PCa tissue invasion and metastasis. This activity appears to be based on reduced expression of cancer driver genes such as FOXM1. Use of docetaxel+Aneustat may provide a new, more effective regimen for therapy of metastatic PCa.

KLF5 inhibits angiogenesis in PTEN-deficient prostate cancer by attenuating AKT activation and subsequent HIF1α accumulation.

BACKGROUND: KLF5 is a basic transcriptional factor that regulates multiple physiopathological processes. Our recent study showed that deletion of Klf5 in mouse prostate promotes tumorigenesis initiated by the deletion of Pten. While molecular characterization of Klf5-null tumors suggested that angiogenesis was partially responsible for tumor promotion, the precise function and mechanism of KLF5 deletion in prostate tumor angiogenesis remain unclear. RESULTS: Applying histological staining to Pten-null mouse prostates, we observed that deletion of Klf5 significantly increased the number of microvessels, accompanied by the upregulation of multiple angiogenesis-related genes based on microarray analysis with MetaCore software. In human umbilical vein endothelial cells (HuVECs), tube formation and migration, both of which are indicators of angiogenic activities, were decreased by conditioned media from PC-3 and DU 145 human prostate cancer cells with KLF5 overexpression, but increased by media from cells with KLF5 knockdown. HIF1α, a key angiogenesis inducer, was upregulated by KLF5 loss at the protein but not the mRNA level in both mouse tissues and human cell lines, as determined by immunohistochemical staining, real-time RT-PCR and Western blotting. Consistently, KLF5 loss also upregulated VEGF and PDGF, two pro-angiogenic mediators of HIF1α function, as analyzed by immunohistochemical staining in mouse tissues and ELISA in conditioned media. Mechanistically, AKT activity, which caused the accumulation of HIF1α, was increased by KLF5 knockout or knockdown but decreased by KLF5 overexpression. PI3K/AKT inhibitors consistently abolished the effects of KLF5 knockdown on angiogenic activity, HIF1α accumulation, and VEGF and PDGF expression. CONCLUSION: KLF5 loss enhances tumor angiogenesis by attenuating PI3K/AKT signaling and subsequent accumulation of HIF1α in PTEN deficient prostate tumors.

Interruption of KLF5 acetylation converts its function from tumor suppressor to tumor promoter in prostate cancer cells.

KLF5 possesses both tumor suppressing and tumor promoting activities, though the mechanism controlling these opposing functions is unknown. In cultured noncancerous epithelial cells, KLF5 converts from proproliferative to antiproliferative activity upon TGFß-induced acetylation, which sequentially alters the KLF5 transcriptional complex and the expression of genes such as p15 and MYC. In this study, we tested whether the acetylation status of KLF5 also determines its opposing functions in tumorigenesis using the PC-3 and DU 145 prostate cancer cell lines, whose proliferation is inhibited by TGFß. KLF5 inhibited the proliferation of these cancer cells, and the inhibition was dependent on KLF5 acetylation. MYC and p15 showed the same patterns of expression change found in noncancerous cells. In nude mice, KLF5 also suppressed tumor growth in an acetylation-dependent manner. Furthermore, deacetylation switched KLF5 to tumor promoting activity, and blocking TGFß signaling attenuated the tumor suppressor activity of KLF5. RNA sequencing and comprehensive data analysis suggest that multiple molecules, including RELA, p53, CREB1, MYC, JUN, ER, AR and SP1, mediate the opposing functions of AcKLF5 and unAcKLF5. These results provide novel insights into the mechanism by which KLF5 switches from antitumorigenic to protumorigenic function and also suggest the roles of AcKLF5 and unAcKLF5, respectively, in the tumor suppressing and tumor promoting functions of TGFß.

In vivo CRISPR screens identify a dual function of MEN1 in regulating tumor-microenvironment interactions.

Functional genomic screens in two-dimensional cell culture models are limited in identifying therapeutic targets that influence the tumor microenvironment. By comparing targeted CRISPR-Cas9 screens in a two-dimensional culture with xenografts derived from the same cell line, we identified MEN1 as the top hit that confers differential dropout effects in vitro and in vivo. MEN1 knockout in multiple solid cancer types does not impact cell proliferation in vitro but significantly promotes or inhibits tumor growth in immunodeficient or immunocompetent mice, respectively. Mechanistically, MEN1 knockout redistributes MLL1 chromatin occupancy, increasing H3K4me3 at repetitive genomic regions, activating double-stranded RNA expression and increasing neutrophil and CD8+ T cell infiltration in immunodeficient and immunocompetent mice, respectively. Pharmacological inhibition of the menin-MLL interaction reduces tumor growth in a CD8+ T cell-dependent manner. These findings reveal tumor microenvironment-dependent oncogenic and tumor-suppressive functions of MEN1 and provide a rationale for targeting MEN1 in solid cancers.

Modeling Androgen Deprivation Therapy-Induced Prostate Cancer Dormancy and Its Clinical Implications.

Treatment-induced tumor dormancy is a state in cancer progression where residual disease is present but remains asymptomatic. Dormant cancer cells are treatment-resistant and responsible for cancer recurrence and metastasis. Prostate cancer treated with androgen-deprivation therapy (ADT) often enters a dormant state. ADT-induced prostate cancer dormancy remains poorly understood due to the challenge in acquiring clinical dormant prostate cancer cells and the lack of representative models. In this study, we aimed to develop clinically relevant models for studying ADT-induced prostate cancer dormancy. Dormant prostate cancer models were established by castrating mice bearing patient-derived xenografts (PDX) of hormonal naïve or sensitive prostate cancer. Dormancy status and tumor relapse were monitored and evaluated. Paired pre- and postcastration (dormant) PDX tissues were subjected to morphologic and transcriptome profiling analyses. As a result, we established eleven ADT-induced dormant prostate cancer models that closely mimicked the clinical courses of ADT-treated prostate cancer. We identified two ADT-induced dormancy subtypes that differed in morphology, gene expression, and relapse rates. We discovered transcriptomic differences in precastration PDXs that predisposed the dormancy response to ADT. We further developed a dormancy subtype-based, predisposed gene signature that was significantly associated with ADT response in hormonal naïve prostate cancer and clinical outcome in castration-resistant prostate cancer treated with ADT or androgen-receptor pathway inhibitors. IMPLICATIONS: We have established highly clinically relevant PDXs of ADT-induced dormant prostate cancer and identified two dormancy subtypes, leading to the development of a novel predicative gene signature that allows robust risk stratification of patients with prostate cancer to ADT or androgen-receptor pathway inhibitors.

Molecular events in neuroendocrine prostate cancer development.

Neuroendocrine prostate cancer (NEPC) is a lethal subtype of prostate cancer. NEPC arises de novo only rarely; the disease predominantly develops from adenocarcinoma in response to drug-induced androgen receptor signalling inhibition, although the mechanisms behind this transdifferentiation are a subject of debate. The survival of patients with NEPC is poor, and few effective treatment options are available. To improve clinical outcomes, understanding of the biology and molecular mechanisms regulating NEPC development is crucial. Various NEPC molecular drivers make temporal contributions during NEPC development, and despite the limited treatment options available, several novel targeted therapeutics are currently under research.

ZRSR2 overexpression is a frequent and early event in castration-resistant prostate cancer development.

BACKGROUND: Androgen deprivation therapy (ADT) remains the leading systemic therapy for locally advanced and metastatic prostate cancers (PCa). While a majority of PCa patients initially respond to ADT, the durability of response is variable and most patients will eventually develop incurable castration-resistant prostate cancer (CRPC). Our research objective is to identify potential early driver genes responsible for CRPC development. METHODS: We have developed a unique panel of hormone-naïve PCa (HNPC) patient-derived xenograft (PDX) models at the Living Tumor Laboratory. The PDXs provide a unique platform for driver gene discovery as they allow for the analysis of differentially expressed genes via transcriptomic profiling at various time points after mouse host castration. In the present study, we focused on genes with expression changes shortly after castration but before CRPC has fully developed. These are likely to be potential early drivers of CRPC development. Such genes were further validated for their clinical relevance using data from PCa patient databases. ZRSR2 was identified as a top gene candidate and selected for further functional studies. RESULTS: ZRSR2 is significantly upregulated in our PDX models during the early phases of CRPC development after mouse host castration and remains consistently high in fully developed CRPC PDX models. Moreover, high ZRSR2 expression is also observed in clinical CRPC samples. Importantly, elevated ZRSR2 in PCa samples is correlated with poor patient treatment outcomes. ZRSR2 knockdown reduced PCa cell proliferation and delayed cell cycle progression at least partially through inhibition of the Cyclin D1 (CCND1) pathway. CONCLUSION: Using our unique HNPC PDX models that develop into CRPC after host castration, we identified ZRSR2 as a potential early driver of CRPC development.

The evolutionarily conserved long non-coding RNA LINC00261 drives neuroendocrine prostate cancer proliferation and metastasis via distinct nuclear and cytoplasmic mechanisms.

Metastatic neuroendocrine prostate cancer (NEPC) is a highly aggressive disease, whose incidence is rising. Long noncoding RNAs (lncRNAs) represent a large family of disease- and tissue-specific transcripts, most of which are still functionally uncharacterized. Thus, we set out to identify the highly conserved lncRNAs that play a central role in NEPC pathogenesis. To this end, we performed transcriptomic analyses of donor-matched patient-derived xenograft models (PDXs) with immunohistologic features of prostate adenocarcinoma (AR+ /PSA+ ) or NEPC (AR- /SYN+ /CHGA+ ) and through differential expression analyses identified lncRNAs that were upregulated upon neuroendocrine transdifferentiation. These genes were prioritized for functional assessment based on the level of conservation in vertebrates. Here, LINC00261 emerged as the top gene with over 3229-fold upregulation in NEPC. Consistently, LINC00261 expression was significantly upregulated in NEPC specimens in multiple patient cohorts. Knockdown of LINC00261 in PC-3 cells dramatically attenuated its proliferative and metastatic abilities, which are explained by parallel downregulation of CBX2 and FOXA2 through distinct molecular mechanisms. In the cell cytoplasm, LINC00261 binds to and sequesters miR-8485 from targeting the CBX2 mRNA, while inside the nucleus, LINC00261 functions as a transcriptional scaffold to induce SMAD-driven expression of the FOXA2 gene. For the first time, these results demonstrate hyperactivation of the LINC00261-CBX2-FOXA2 axes in NEPC to drive proliferation and metastasis, and that LINC00261 may be utilized as a therapeutic target and a biomarker for this incurable disease.

KLF5 Is Crucial for Androgen-AR Signaling to Transactivate Genes and Promote Cell Proliferation in Prostate Cancer Cells.

Androgen/androgen receptor (AR) signaling drives both the normal prostate development and prostatic carcinogenesis, and patients with advanced prostate cancer often develop resistance to androgen deprivation therapy. The transcription factor Krüppel-like factor 5 (KLF5) also regulates both normal and cancerous development of the prostate. In this study, we tested whether and how KLF5 plays a role in the function of AR signaling in prostate cancer cells. We found that KLF5 is upregulated by androgen depending on AR in LNCaP and C4-2B cells. Silencing KLF5, in turn, reduced AR transcriptional activity and inhibited androgen-induced cell proliferation and tumor growth in vitro and in vivo. Mechanistically, KLF5 occupied the promoter of AR, and silencing KLF5 repressed AR transcription. In addition, KLF5 and AR physically interacted with each other to regulate the expression of multiple genes (e.g., MYC, CCND1 and PSA) to promote cell proliferation. These findings indicate that, while transcriptionally upregulated by AR signaling, KLF5 also regulates the expression and transcriptional activity of AR in androgen-sensitive prostate cancer cells. The KLF5-AR interaction could provide a therapeutic opportunity for the treatment of prostate cancer.

Conditionally Reprogrammed Cells from Patient-Derived Xenograft to Model Neuroendocrine Prostate Cancer Development.

Neuroendocrine prostate cancer (NEPC) is a lethal subtype of prostate cancer. It develops mainly via NE transdifferentiation of prostate adenocarcinoma in response to androgen receptor (AR)-inhibition therapy. The study of NEPC development has been hampered by a lack of clinically relevant models. We previously established a unique and first-in-field patient-derived xenograft (PDX) model of adenocarcinoma (LTL331)-to-NEPC (LTL331R) transdifferentiation. In this study, we applied conditional reprogramming (CR) culture to establish a LTL331 PDX-derived cancer cell line named LTL331_CR_Cell. These cells retain the same genomic mutations as the LTL331 parental tumor. They can be continuously propagated in vitro and can be genetically manipulated. Androgen deprivation treatment on LTL331_CR_Cells had no effect on cell proliferation. Transcriptomic analyses comparing the LTL331_CR_Cell to its parental tumor revealed a profound downregulation of the androgen response pathway and an upregulation of stem and basal cell marker genes. The transcriptome of LTL331_CR_Cells partially resembles that of post-castrated LTL331 xenografts in mice. Notably, when grafted under the renal capsules of male NOD/SCID mice, LTL331_CR_Cells spontaneously gave rise to NEPC tumors. This is evidenced by the histological expression of the NE marker CD56 and the loss of adenocarcinoma markers such as PSA. Transcriptomic analyses of the newly developed NEPC tumors further demonstrate marked enrichment of NEPC signature genes and loss of AR signaling genes. This study provides a novel research tool derived from a unique PDX model. It allows for the investigation of mechanisms underlying NEPC development by enabling gene manipulations ex vivo and subsequent functional evaluations in vivo.

Klf5 acetylation regulates luminal differentiation of basal progenitors in prostate development and regeneration.

Prostate development depends on balanced cell proliferation and differentiation, and acetylated KLF5 is known to alter epithelial proliferation. It remains elusive whether post-translational modifications of transcription factors can differentially determine adult stem/progenitor cell fate. Here we report that, in human and mouse prostates, Klf5 is expressed in both basal and luminal cells, with basal cells preferentially expressing acetylated Klf5. Functionally, Klf5 is indispensable for maintaining basal progenitors, their luminal differentiation, and the proliferation of their basal and luminal progenies. Acetylated Klf5 is also essential for basal progenitors' maintenance and proper luminal differentiation, as deacetylation of Klf5 causes excess basal-to-luminal differentiation; attenuates androgen-mediated organoid organization; and retards postnatal prostate development. In basal progenitor-derived luminal cells, Klf5 deacetylation increases their proliferation and attenuates their survival and regeneration following castration and subsequent androgen restoration. Mechanistically, Klf5 deacetylation activates Notch signaling. Klf5 and its acetylation thus contribute to postnatal prostate development and regeneration by controlling basal progenitor cell fate.

ONECUT2 is a driver of neuroendocrine prostate cancer.

Neuroendocrine prostate cancer (NEPC), a lethal form of the disease, is characterized by loss of androgen receptor (AR) signaling during neuroendocrine transdifferentiation, which results in resistance to AR-targeted therapy. Clinically, genomically and epigenetically, NEPC resembles other types of poorly differentiated neuroendocrine tumors (NETs). Through pan-NET analyses, we identified ONECUT2 as a candidate master transcriptional regulator of poorly differentiated NETs. ONECUT2 ectopic expression in prostate adenocarcinoma synergizes with hypoxia to suppress androgen signaling and induce neuroendocrine plasticity. ONEUCT2 drives tumor aggressiveness in NEPC, partially through regulating hypoxia signaling and tumor hypoxia. Specifically, ONECUT2 activates SMAD3, which regulates hypoxia signaling through modulating HIF1α chromatin-binding, leading NEPC to exhibit higher degrees of hypoxia compared to prostate adenocarcinomas. Treatment with hypoxia-activated prodrug TH-302 potently reduces NEPC tumor growth. Collectively, these results highlight the synergy between ONECUT2 and hypoxia in driving NEPC, and emphasize the potential of hypoxia-directed therapy for NEPC patients.

Targeting MCT4 to reduce lactic acid secretion and glycolysis for treatment of neuroendocrine prostate cancer.

Development of neuroendocrine prostate cancer (NEPC) is emerging as a major problem in clinical management of advanced prostate cancer (PCa). As increasingly potent androgen receptor (AR)-targeting antiandrogens are more widely used, PCa transdifferentiation into AR-independent NEPC as a mechanism of treatment resistance becomes more common and precarious, since NEPC is a lethal PCa subtype urgently requiring effective therapy. Reprogrammed glucose metabolism of cancers, that is elevated aerobic glycolysis involving increased lactic acid production/secretion, plays a key role in multiple cancer-promoting processes and has been implicated in therapeutics development. Here, we examined NEPC glucose metabolism using our unique panel of patient-derived xenograft PCa models and patient tumors. By calculating metabolic pathway scores using gene expression data, we found that elevated glycolysis coupled to increased lactic acid production/secretion is an important metabolic feature of NEPC. Specific inhibition of expression of MCT4 (a plasma membrane lactic acid transporter) by antisense oligonucleotides led to reduced lactic acid secretion as well as reduced glucose metabolism and NEPC cell proliferation. Taken together, our results indicate that elevated glycolysis coupled to excessive MCT4-mediated lactic acid secretion is clinically relevant and functionally important to NEPC. Inhibition of MCT4 expression appears to be a promising therapeutic strategy for NEPC.

Heterochromatin Protein 1α Mediates Development and Aggressiveness of Neuroendocrine Prostate Cancer.

Neuroendocrine prostate cancer (NEPC) is a lethal subtype of prostate cancer arising mostly from adenocarcinoma via neuroendocrine transdifferentiation following androgen deprivation therapy. Mechanisms contributing to both NEPC development and its aggressiveness remain elusive. In light of the fact that hyperchromatic nuclei are a distinguishing histopathologic feature of NEPC, we utilized transcriptomic analyses of our patient-derived xenograft (PDX) models, multiple clinical cohorts, and genetically engineered mouse models to identify 36 heterochromatin-related genes that are significantly enriched in NEPC. Longitudinal analysis using our unique, first-in-field PDX model of adenocarcinoma-to-NEPC transdifferentiation revealed that, among those 36 heterochromatin-related genes, heterochromatin protein 1α (HP1α) expression increased early and steadily during NEPC development and remained elevated in the developed NEPC tumor. Its elevated expression was further confirmed in multiple PDX and clinical NEPC samples. HP1α knockdown in the NCI-H660 NEPC cell line inhibited proliferation, ablated colony formation, and induced apoptotic cell death, ultimately leading to tumor growth arrest. Its ectopic expression significantly promoted NE transdifferentiation in adenocarcinoma cells subjected to androgen deprivation treatment. Mechanistically, HP1α reduced expression of androgen receptor and RE1 silencing transcription factor and enriched the repressive trimethylated histone H3 at Lys9 mark on their respective gene promoters. These observations indicate a novel mechanism underlying NEPC development mediated by abnormally expressed heterochromatin genes, with HP1α as an early functional mediator and a potential therapeutic target for NEPC prevention and management.Significance: Heterochromatin proteins play a fundamental role in NEPC, illuminating new therapeutic targets for this aggressive disease. Cancer Res; 78(10); 2691-704. ©2018 AACR.