Reprogramming of the FOXA1 cistrome in treatment-emergent neuroendocrine prostate cancer.

Lineage plasticity, the ability of a cell to alter its identity, is an increasingly common mechanism of adaptive resistance to targeted therapy in cancer. An archetypal example is the development of neuroendocrine prostate cancer (NEPC) after treatment of prostate adenocarcinoma (PRAD) with inhibitors of androgen signaling. NEPC is an aggressive variant of prostate cancer that aberrantly expresses genes characteristic of neuroendocrine (NE) tissues and no longer depends on androgens. Here, we investigate the epigenomic basis of this resistance mechanism by profiling histone modifications in NEPC and PRAD patient-derived xenografts (PDXs) using chromatin immunoprecipitation and sequencing (ChIP-seq). We identify a vast network of cis-regulatory elements (N~15,000) that are recurrently activated in NEPC. The FOXA1 transcription factor (TF), which pioneers androgen receptor (AR) chromatin binding in the prostate epithelium, is reprogrammed to NE-specific regulatory elements in NEPC. Despite loss of dependence upon AR, NEPC maintains FOXA1 expression and requires FOXA1 for proliferation and expression of NE lineage-defining genes. Ectopic expression of the NE lineage TFs ASCL1 and NKX2-1 in PRAD cells reprograms FOXA1 to bind to NE regulatory elements and induces enhancer activity as evidenced by histone modifications at these sites. Our data establish the importance of FOXA1 in NEPC and provide a principled approach to identifying cancer dependencies through epigenomic profiling.

Combined TP53 and RB1 Loss Promotes Prostate Cancer Resistance to a Spectrum of Therapeutics and Confers Vulnerability to Replication Stress.

Prostate cancers (PCs) with loss of the potent tumor suppressors TP53 and RB1 exhibit poor outcomes. TP53 and RB1 also influence cell plasticity and are frequently lost in PCs with neuroendocrine (NE) differentiation. Therapeutic strategies that address these aggressive variant PCs are urgently needed. Using deep genomic profiling of 410 metastatic biopsies, we determine the relationships between combined TP53 and RB1 loss and PC phenotypes. Notably, 40% of TP53/RB1-deficient tumors are classified as AR-active adenocarcinomas, indicating that NE differentiation is not an obligate consequence of TP53/RB1 inactivation. A gene expression signature reflecting TP53/RB1 loss is associated with diminished responses to AR antagonists and reduced survival. These tumors exhibit high proliferation rates and evidence of elevated DNA repair processes. While tumor cells lacking TP53/RB1 are highly resistant to all single-agent therapeutics tested, the combination of PARP and ATR inhibition is found to produce significant responses, reflecting a clinically exploitable vulnerability resulting from replication stress.

Identification of Therapeutic Vulnerabilities in Small-cell Neuroendocrine Prostate Cancer.

PURPOSE: Small-cell neuroendocrine prostate cancer (SCNPC) exhibits an aggressive clinical course and incidence rates seem to be increasing following resistance to potent androgen receptor (AR) antagonists. Currently, treatment options are limited and few model systems are available to identify new approaches for treatment. We sought to evaluate commonalities between SCNPC and other aggressive neuroendocrine carcinomas to identify therapeutic targets. EXPERIMENTAL DESIGN: We generated whole transcriptome RNA-sequencing data from AR-active prostate cancers (ARPCs) and SCNPCs from tumors collected at rapid autopsy and two other neuroendocrine carcinomas, Merkel cell carcinoma (MCC), and small-cell lung cancer. We performed cross-tumor comparisons to identify conserved patterns of expression of druggable targets. We tested inhibitors to highly upregulated drug targets in a panel of prostate cancer cell lines and in vivo patient-derived xenograft (PDX) models. RESULTS: We identified BCL2 as highly upregulated in SCNPC compared with ARPC. Inhibitors targeting BCL2 induced apoptotic cell death in SCNPC cell lines at nanomolar concentrations while ARPC cell lines were resistant. Treatment with the BCL2 inhibitor navitoclax leads to a reduction of growth of SCNPC PDX tumors in vivo, whereas ARPC PDX models were more resistant. We identified Wee1 as a second druggable target upregulated in SCNPC. Treatment with the combination of navitoclax and the Wee1 inhibitor AZD-1775 repressed the growth of SCNPC PDX resistant to single-agent BCL2 inhibitors. CONCLUSIONS: The combination of BCL2 and Wee1 inhibition presents a novel therapeutic strategy for the treatment of SCNPC.

Molecular determinants of response to high-dose androgen therapy in prostate cancer.

Clinical trials of high-dose androgen (HDA) therapy for prostate cancer (PC) have shown promising efficacy but are limited by lack of criteria to identify likely responders. To elucidate factors that govern the growth-repressive effects of HDAs, we applied an unbiased integrative approach using genetic screens and transcriptional profiling of PC cells with or without demonstrated phenotypic sensitivity to androgen-mediated growth repression. Through this comprehensive analysis, we identified genetic events and related signaling networks that determine the response to both HDA and androgen withdrawal. We applied these findings to develop a gene signature that may serve as an early indicator of treatment response and identify men with tumors that are amenable to HDA therapy.

Testosterone accumulation in prostate cancer cells is enhanced by facilitated diffusion.

BACKGROUND: Testosterone is a driver of prostate cancer (PC) growth via ligand-mediated activation of the androgen receptor (AR). Tumors that have escaped systemic androgen deprivation, castration-resistant prostate cancers (CRPC), have measurable intratumoral levels of testosterone, suggesting that a resistance mechanism still depends on androgen-simulated growth. However, AR activation requires an optimal intracellular concentration of androgens, a situation challenged by low circulating testosterone concentrations. Notably, PC cells may optimize their androgen levels by regulating the expression of steroid metabolism enzymes that convert androgen precursors into androgens. Here we propose that testosterone entry into the cell could be another control point. METHODS: To determine whether testosterone enters cells via a transporter, we performed in vitro 3 H-testosterone uptake assays in androgen-dependent LNCaP and androgen and AR-independent PC3 cells. To determine if the uptake mechanism depended on a concentration gradient, we modified UGT2B17 levels in LNCaP cells and measured androgen levels by liquid-liquid extraction-mass spectrometry. We also analyzed CRPC metastases for expression of AKR1C3 to determine whether this enzyme that converts adrenal androgens to testosterone was present in the tumor stroma (microenvironment) in addition to its expression in the tumor epithelium. RESULTS: Testosterone uptake followed a concentration gradient but unlike in passive diffusion, was saturable and temperature-dependent, thus suggesting facilitated transport. Suppression of UGT2B17 to abrogate a testosterone gradient reduced testosterone transport while overexpression of the enzyme enhanced it. The facilitated transport suggests a paracrine route of testosterone uptake for maintaining optimal intracellular levels. We found that AKR1C3 was expressed in the tumor microenvironment of CRPC metastases in addition to epithelial cells and the pattern of relative abundance of the enzyme in epithelium vs stroma varied substantially between the metastatic sites. CONCLUSIONS: Our findings suggest that in addition to testosterone transport and metabolism by tumor epithelium, testosterone could also be produced by components of the tumor microenvironment. Facilitated testosterone uptake by tumor cells supports a cell nonautonomous mechanism for testosterone signaling in CRPC.

Molecular profiling stratifies diverse phenotypes of treatment-refractory metastatic castration-resistant prostate cancer.

Metastatic castration-resistant prostate cancer (mCRPC) is a heterogeneous disease with diverse drivers of disease progression and mechanisms of therapeutic resistance. We conducted deep phenotypic characterization of CRPC metastases and patient-derived xenograft (PDX) lines using whole genome RNA sequencing, gene set enrichment analysis and immunohistochemistry. Our analyses revealed five mCRPC phenotypes based on the expression of well-characterized androgen receptor (AR) or neuroendocrine (NE) genes: (i) AR-high tumors (ARPC), (ii) AR-low tumors (ARLPC), (iii) amphicrine tumors composed of cells co-expressing AR and NE genes (AMPC), (iv) double-negative tumors (i.e. AR-/NE-; DNPC) and (v) tumors with small cell or NE gene expression without AR activity (SCNPC). RE1-silencing transcription factor (REST) activity, which suppresses NE gene expression, was lost in AMPC and SCNPC PDX models. However, knockdown of REST in cell lines revealed that attenuated REST activity drives the AMPC phenotype but is not sufficient for SCNPC conversion. We also identified a subtype of DNPC tumors with squamous differentiation and generated an encompassing 26-gene transcriptional signature that distinguished the five mCRPC phenotypes. Together, our data highlight the central role of AR and REST in classifying treatment-resistant mCRPC phenotypes. These molecular classifications could potentially guide future therapeutic studies and clinical trial design.

Characterization of lymphatic malformations using primary cells and tissue transcriptomes.

Lymphatic malformations (LMs) are disfiguring congenital anomalies characterized by aberrant growth of lymphatic vessels. They are broadly categorized histopathologically as macrocystic and microcystic. Although sclerotherapy has shown some success in the treatment of macrocystic malformations, there has been less progress with developing treatment strategies for microcystic malformations. In this study, we characterized lymphatic endothelial cells isolated from lymphatic and lymphaticovenous malformations. When compared to cells from normal lymphatic vessels, we found that the primary cultured malformed cells are morphologically different and also exhibited differences in binding, proliferation, migration and tube formation. Transcriptome analysis identified several genes whose expression was substantially higher in malformed compared to normal lymphatic endothelium, including DIRAS3 and FOXF1. Further analysis of LM tissue samples revealed distinguishing gene expression patterns that could pave the way to understanding the molecular pathogenesis of LMs. Based on gene expression signatures, we propose a new hypothesis that the subtype of localized LMs could be formed because of disruptions in lymph node development.

Resolvins suppress tumor growth and enhance cancer therapy.

Cancer therapy reduces tumor burden by killing tumor cells, yet it simultaneously creates tumor cell debris that may stimulate inflammation and tumor growth. Thus, conventional cancer therapy is inherently a double-edged sword. In this study, we show that tumor cells killed by chemotherapy or targeted therapy ("tumor cell debris") stimulate primary tumor growth when coinjected with a subthreshold (nontumorigenic) inoculum of tumor cells by triggering macrophage proinflammatory cytokine release after phosphatidylserine exposure. Debris-stimulated tumors were inhibited by antiinflammatory and proresolving lipid autacoids, namely resolvin D1 (RvD1), RvD2, or RvE1. These mediators specifically inhibit debris-stimulated cancer progression by enhancing clearance of debris via macrophage phagocytosis in multiple tumor types. Resolvins counterregulate the release of cytokines/chemokines, including TNFα, IL-6, IL-8, CCL4, and CCL5, by human macrophages stimulated with cell debris. These results demonstrate that enhancing endogenous clearance of tumor cell debris is a new therapeutic target that may complement cytotoxic cancer therapies.

Androgen Receptor Pathway-Independent Prostate Cancer Is Sustained through FGF Signaling.

Androgen receptor (AR) signaling is a distinctive feature of prostate carcinoma (PC) and represents the major therapeutic target for treating metastatic prostate cancer (mPC). Though highly effective, AR antagonism can produce tumors that bypass a functional requirement for AR, often through neuroendocrine (NE) transdifferentiation. Through the molecular assessment of mPCs over two decades, we find a phenotypic shift has occurred in mPC with the emergence of an AR-null NE-null phenotype. These "double-negative" PCs are notable for elevated FGF and MAPK pathway activity, which can bypass AR dependence. Pharmacological inhibitors of MAPK or FGFR repressed the growth of double-negative PCs in vitro and in vivo. Our results indicate that FGF/MAPK blockade may be particularly efficacious against mPCs with an AR-null phenotype.

Association of Tissue Abiraterone Levels and SLCO Genotype with Intraprostatic Steroids and Pathologic Response in Men with High-Risk Localized Prostate Cancer.

Purpose: Germline variation in solute carrier organic anion (SLCO) genes influences cellular steroid uptake and is associated with prostate cancer outcomes. We hypothesized that, due to its steroidal structure, the CYP17A inhibitor abiraterone may undergo transport by SLCO-encoded transporters and that SLCO gene variation may influence intracellular abiraterone levels and outcomes.Experimental Design: Steroid and abiraterone levels were measured in serum and tissue from 58 men with localized prostate cancer in a clinical trial of LHRH agonist plus abiraterone acetate plus prednisone for 24 weeks prior to prostatectomy. Germline DNA was genotyped for 13 SNPs in six SLCO genes.Results: Abiraterone levels spanned a broad range (serum median 28 ng/mL, 108 nmol/L; tissue median 77 ng/mL, 271 nmol/L) and were correlated (r = 0.355, P = 0.001). Levels correlated positively with steroids upstream of CYP17A (pregnenolone, progesterone), and inversely with steroids downstream of CYP17A (DHEA, AED, testosterone). Serum PSA and tumor volumes were higher in men with undetectable versus detectable tissue abiraterone at prostatectomy (median 0.10 vs. 0.03 ng/dL, P = 0.02; 1.28 vs. 0.44 cc, P = 0.09, respectively). SNPs in SLCO2B1 associated with significant differences in tissue abiraterone (rs1789693, P = 0.0008; rs12422149, P = 0.03) and higher rates of minimal residual disease (tumor volume < 0.5 cc; rs1789693, 67% vs. 27%, P = 0.009; rs1077858, 46% vs. 0%, P = 0.03). LNCaP cells expressing SLCO2B1 showed two- to fourfold higher abiraterone levels compared with vector controls (P < 0.05).Conclusions: Intraprostatic abiraterone levels and genetic variation in SLCO genes are associated with pathologic responses in high-risk localized prostate cancer. Variation in SLCO genes may serve as predictors of response to abiraterone treatment. Clin Cancer Res; 23(16); 4592-601. ©2017 AACR.

Long non-coding RNA: A newly deciphered "code" in prostate cancer.

As one of the most frequently diagnosed cancers in males, the development and progression of prostate cancer remains an open area of research. The role of lncRNAs in prostate cancer is an emerging field of study. In this review, we summarize what is currently known about lncRNAs in prostate cancer while focusing on a few key lncRNAs. PCA3 was the first lncRNA identified in prostate cancer and has been shown to be expressed in a majority of prostate cancer cases. It may act in both an androgen dependent and independent fashion and has clinical utility as a biomarker. Other lncRNAs are known to interact directly with the androgen receptor pathway including PlncRNA-1, HOTAIR, PRNCR1 and PCGEM1. Additionally, lncRNAs have been shown to interfere with tumor suppressors, DNA break repair, transcription and alternate RNA splicing. While only in its infancy, an understanding of the role of lncRNAs in prostate cancer development should present ample opportunities for the discovery of new cancer biomarkers and therapeutic targets.

Epoxyeicosanoids promote organ and tissue regeneration.

Epoxyeicosatrienoic acids (EETs), lipid mediators produced by cytochrome P450 epoxygenases, regulate inflammation, angiogenesis, and vascular tone. Despite pleiotropic effects on cells, the role of these epoxyeicosanoids in normal organ and tissue regeneration remains unknown. EETs are produced predominantly in the endothelium. Normal organ and tissue regeneration require an active paracrine role of the microvascular endothelium, which in turn depends on angiogenic growth factors. Thus, we hypothesize that endothelial cells stimulate organ and tissue regeneration via production of bioactive EETs. To determine whether endothelial-derived EETs affect physiologic tissue growth in vivo, we used genetic and pharmacological tools to manipulate endogenous EET levels. We show that endothelial-derived EETs play a critical role in accelerating tissue growth in vivo, including liver regeneration, kidney compensatory growth, lung compensatory growth, wound healing, corneal neovascularization, and retinal vascularization. Administration of synthetic EETs recapitulated these results, whereas lowering EET levels, either genetically or pharmacologically, delayed tissue regeneration, demonstrating that pharmacological modulation of EETs can affect normal organ and tissue growth. We also show that soluble epoxide hydrolase inhibitors, which elevate endogenous EET levels, promote liver and lung regeneration. Thus, our observations indicate a central role for EETs in organ and tissue regeneration and their contribution to tissue homeostasis.

Epoxyeicosanoids stimulate multiorgan metastasis and tumor dormancy escape in mice.

Epoxyeicosatrienoic acids (EETs) are small molecules produced by cytochrome P450 epoxygenases. They are lipid mediators that act as autocrine or paracrine factors to regulate inflammation and vascular tone. As a result, drugs that raise EET levels are in clinical trials for the treatment of hypertension and many other diseases. However, despite their pleiotropic effects on cells, little is known about the role of these epoxyeicosanoids in cancer. Here, using genetic and pharmacological manipulation of endogenous EET levels, we demonstrate that EETs are critical for primary tumor growth and metastasis in a variety of mouse models of cancer. Remarkably, we found that EETs stimulated extensive multiorgan metastasis and escape from tumor dormancy in several tumor models. This systemic metastasis was not caused by excessive primary tumor growth but depended on endothelium-derived EETs at the site of metastasis. Administration of synthetic EETs recapitulated these results, while EET antagonists suppressed tumor growth and metastasis, demonstrating in vivo that pharmacological modulation of EETs can affect cancer growth. Furthermore, inhibitors of soluble epoxide hydrolase (sEH), the enzyme that metabolizes EETs, elevated endogenous EET levels and promoted primary tumor growth and metastasis. Thus, our data indicate a central role for EETs in tumorigenesis, offering a mechanistic link between lipid signaling and cancer and emphasizing the critical importance of considering possible effects of EET-modulating drugs on cancer.

Cytochrome P450-derived eicosanoids: the neglected pathway in cancer.

Endogenously produced lipid autacoids are locally acting small molecule mediators that play a central role in the regulation of inflammation and tissue homeostasis. A well-studied group of autacoids are the products of arachidonic acid metabolism, among which the prostaglandins and leukotrienes are the best known. They are generated by two pathways controlled by the enzyme systems cyclooxygenase and lipoxygenase, respectively. However, arachidonic acid is also substrate for a third enzymatic pathway, the cytochrome P450 (CYP) system. This third eicosanoid pathway consists of two main branches: ω-hydroxylases convert arachidonic acid to hydroxyeicosatetraenoic acids (HETEs) and epoxygenases convert it to epoxyeicosatrienoic acids (EETs). This third CYP pathway was originally studied in conjunction with inflammatory and cardiovascular disease. Arachidonic acid and its metabolites have recently stimulated great interest in cancer biology; but, unlike prostaglandins and leukotrienes the link between cytochome P450 metabolites and cancer has received little attention. In this review, the emerging role in cancer of cytochrome P450 metabolites, notably 20-HETE and EETs, are discussed.

Inhibition of tumor angiogenesis by oral etoposide.

The chemotherapeutic agent etoposide is a topoisomerase II inhibitor widely used for cancer therapy. Low-dose oral etoposide, administered at close regular intervals, has potent anti-tumor activity in patients who are refractory to intravenous etoposide; however, the mechanism remains unclear. Since endothelial cells may be more sensitive than tumor cells to chemotherapy agents, we determined the effects of etoposide alone and in combination with oral cyclooxygenase-2 inhibitors and peroxisome-proliferator activated receptor γ ligands on angiogenesis and tumor growth in xenograft tumor models. Optimal anti-angiogenic (metronomic) and anti-tumor doses of etoposide on angiogenesis, primary tumor growth and metastasis were established alone and in combination therapy. Etoposide inhibited endothelial and tumor cell proliferation, decreased vascular endothelial growth factor (VEGF) production by tumor cells and suppressed endothelial tube formation at non-cytotoxic concentrations. In our in vivo studies, oral etoposide inhibited fibroblast growth factor 2 and VEGF-induced corneal neovascularization, VEGF-induced vascular permeability and increased levels of the endogenous angiogenesis inhibitor endostatin in mice. In addition, etoposide inhibited Lewis lung carcinoma (LLC) and human glioblastoma (U87) primary tumor growth as well as spontaneous lung metastasis in a LLC resection model. Furthermore, etoposide had synergistic anti-tumor activity in combination with celecoxib and rosiglitazone, which are also oral anti-angiogenic and anti-tumor agents. Etoposide inhibits angiogenesis in vitro and in vivo by indirect and direct mechanisms of action. Combining etoposide with celecoxib and rosiglitazone increases its efficacy and merits further investigation in future clinical trials to determine the potential usefulness of etoposide in combinatory anti-angiogenic chemotherapy.

Effects of poly-N-acetyl glucosamine (pGlcNAc) patch on wound healing in db/db mouse.

BACKGROUND: Poly-N-acetyl glucosamine (pGlcNAc) nanofiber-based materials, produced by a marine microalga, have been characterized as effective hemostatic agents. In this study, we hypothesized that a pGlcNAc fiber patch may enhance wound healing in the db/db mouse. METHODS: pGlcNAc patches were applied on 1-cm, full-thickness, skin wounds in the db/db mouse model. Wounds (n = 15 per group) were dressed with a pGlcNAc nanofiber patch for 1 hour, 24 hours, or left untreated. After the application time, patches were removed and wounds were allowed to heal spontaneously. The rate of wound closure was evaluated by digital analysis of unclosed wound area as a function of time. At day 10, wounds (n = 7 per group) were harvested and quantified with immunohistochemical markers of proliferation (Ki-67) and vascularization (platelet endothelial cell adhesion molecule). RESULTS: Wounds dressed with pGlcNAc patches for 1 hour closed faster than control wounds, reaching 90% closure in 16.6 days, 9 days faster than untreated wounds. Granulation tissue showed higher levels of proliferation and vascularization after 1-hour treatment than the 24-hour and left-untreated groups. Foreign body reaction to the material was not noted in applications up to 24 hours. DISCUSSION: In addition to its hemostatic properties, the pGlcNAc material also appears to accelerate wound closure in healing-impaired genetically diabetic mice. This material, with its combination of hemostatic and wound healing properties, has the potential to be effective agent for the treatment of complicated wounds.

Sucrose octasulfate regulates fibroblast growth factor-2 binding, transport, and activity: potential for regulation of tumor growth.

The antithrombotic activity of heparin has largely been credited with the success found in some cancer treatment by heparin. There are, however, many potent growth factors involved in tumor and blood vessel growth that bind to heparin with high affinity and their regulation by heparin may play a role in heparin's efficacy. We therefore chose to study the activity of a heparin analog, sucrose octasulfate (SOS), which has been similarly shown to interact with heparin-binding growth factors. Using mouse melanoma and lung carcinoma models, we demonstrate in vivo inhibition of tumor growth by SOS. SOS, however, showed little effect in coagulation assays indicating that this activity was not a primary mechanism of action for this molecule. Studies were then performed to assess the effect of SOS on basic fibroblast growth factor (FGF-2) activity, a growth factor which promotes tumor and blood vessel growth and is produced by B16 melanoma cells. SOS potently inhibited FGF-2 binding to endothelial cells and stripped pre-bound FGF-2 from cells. SOS also regulated FGF-2 stimulated proliferation. Further, SOS facilitated FGF-2 diffusion through Descemet's membrane, a heparan sulfate-rich basement membrane from the cornea, suggesting a possible role in FGF-2 clearance. Our results suggest that molecules such as SOS have the potential to remove growth factors from tumor microenvironments and the approach offers an attractive area for further study.

PPARalpha agonist fenofibrate suppresses tumor growth through direct and indirect angiogenesis inhibition.

Angiogenesis and inflammation are central processes through which the tumor microenvironment influences tumor growth. We have demonstrated recently that peroxisome proliferator-activated receptor (PPAR)alpha deficiency in the host leads to overt inflammation that suppresses angiogenesis via excess production of thrombospondin (TSP)-1 and prevents tumor growth. Hence, we speculated that pharmacologic activation of PPARalpha would promote tumor growth. Surprisingly, the PPARalpha agonist fenofibrate potently suppressed primary tumor growth in mice. This effect was not mediated by cancer-cell-autonomous antiproliferative mechanisms but by the inhibition of angiogenesis and inflammation in the host tissue. Although PPARalpha-deficient tumors were still susceptible to fenofibrate, absence of PPARalpha in the host animal abrogated the potent antitumor effect of fenofibrate. In addition, fenofibrate suppressed endothelial cell proliferation and VEGF production, increased TSP-1 and endostatin, and inhibited corneal neovascularization. Thus, both genetic abrogation of PPARalpha as well as its activation by ligands cause tumor suppression via overlapping antiangiogenic pathways. These findings reveal the potential utility of the well tolerated PPARalpha agonists beyond their use as lipid-lowering drugs in anticancer therapy. Our results provide a mechanistic rationale for evaluating the clinical benefits of PPARalpha agonists in cancer treatment, alone and in combination with other therapies.