Monitoring Glucocorticoid Receptor in Plasma-derived Extracellular Vesicles as a Marker of Resistance to Androgen Receptor Signaling Inhibition in Prostate Cancer.

Disease progression following androgen ablation was shown to be associated with upregulation of the glucocorticoid receptor (GR). Longitudinal monitoring of GR expression in circulating extracellular vesicles (EV) may reflect changes in the tumor cell and facilitates detection of acquired resistance. We utilized LNCaP, LREX cells and a patient-derived xenograft, MDA PDX 322-2-6a, for in vitro and in vivo experiments. Plasma-derived EVs were isolated from patients with localized high-risk prostate cancer undergoing androgen ablation. The mRNA levels of GR in EVs and their responsive genes were detected by transcriptome analysis, qRT-PCR and the protein levels by Western blot analysis. We detected changes in GR expression at mRNA and protein levels in EVs derived from LNCaP and LREX cells in in vitro studies. In in vivo experiments, LNCaP and the PDX MDA 322-2-6a-bearing mice were treated with enzalutamide. GR levels in plasma-derived EVs were increased only in those tumors that did not respond to enzalutamide. Treatment of mice bearing enzalutamide-resistant tumors with a GR inhibitor in combination with enzalutamide led to a transient pause in tumor growth in a subset of tumors and decreased GR levels intracellular and in plasma-derived EVs. In a subgroup of patients with high-risk localized prostate cancer treated with androgen signaling inhibition, GR was found upregulated in matching tissue and plasma EVs. These analyses showed that GR levels in plasma-derived EVs may be used for monitoring the transition of GR expression allowing for early detection of resistance to androgen ablation treatment. SIGNIFICANCE: Longitudinal monitoring of GR expression in plasma-derived EVs from patients with prostate cancer treated with androgen signaling inhibitors facilitates early detection of acquisition of resistance to androgen receptor signaling inhibition in individual patients.

Characterization of prostate cancer adrenal metastases: dependence upon androgen receptor signaling and steroid hormones.

BACKGROUND: Prostate cancer (PCa) typically spreads to the bone, and this distribution is attributed to the central role of the microenvironment in progression. However, metastasis to the adrenal glands, while not as common, does occur. The biology that accounts for adrenal metastases may be attributed to the unique local steroid metabolome and co-clinical characterization may elucidate the role steroid biosynthesis plays in PCa progression. METHODS: Three patients with metastatic PCa who had archived tumor tissue from an adrenalectomy were retrospectively identified, and one adrenal metastasis was developed into a xenograft (MDA-PCa-250). The adrenal metastases were characterized by performing somatic DNA whole exome sequencing (WES), RNA-Seq, immunohistochemistry (IHC), and steroid metabolite quantitation. The influence of steroid metabolites on adrenal metastasis cells and tumor growth was tested in vitro and in vivo. RESULTS: Clinically, adrenalectomy was performed during castration-resistant oligometastatic disease, and two men experienced resensitization to leuprolide. Somatic DNA WES revealed heterogeneous alterations in tumor suppressor and DNA damage repair pathway genes. Adrenal metastases had active androgen receptor (AR) signaling by IHC, and RNA-Seq supported a potential role for adrenal androgen precursor metabolism in activating the AR. Steroid quantitation suggested the adrenal androgen precursors were converted into testosterone in these metastases, and stable isotope tracing of an organoid from MDA-PCa-250 confirmed the capability of adrenal metastases to biosynthesize testosterone from adrenal precursors. In vitro testing of a cell line derived from MDA-PCa-250 showed that testosterone and cortisol stimulated tumor cell growth. In vivo experiments demonstrated that MDA-PCa-250 grew in intact mice with circulating testosterone, but not in castrated mice. CONCLUSIONS: PCa adrenal metastases depend upon AR signaling driven by androgen precursors, androstenedione and dehydroepiandrosterone, available in the microenvironment, despite the presence of heterogeneous somatic DNA alterations. Moreover, MDA-PCa-250 provides a preclinical model that can recapitulate the unique androgen-dependence of adrenal metastases. CLINICAL TRIAL REGISTRATION: This study does not report the clinical results of a clinical trial, but it does use samples from a completed clinical trial that is registered with clinicaltrials.gov (NCT01254864).

Targeting the mTOR Pathway for the Prevention of ER-Negative Breast Cancer.

PREVENTION RELEVANCE: Our results show that everolimus delays mammary tumor formation in multiple mouse models, suggesting that mTOR inhibitors will be useful for the prevention of ER-negative and triple-negative breast cancer in humans. See related Spotlight, p. 787.

Patterns of indolence in prostate cancer (Review).

Although prostate cancer is a major cause of cancer-related mortality worldwide, most patients will have a relatively indolent clinical course. Contrary to most other types of cancer, even the diagnosis of locally advanced or metastatic disease is not always lethal. The present review aimed to summarize what is known regarding the underlying mechanisms related to the indolent course of subsets of prostate cancer, at various stages. The data suggested that no specific gene alteration by itself was responsible for carcinogenesis or disease aggressiveness. However, pathway analysis identified genetic aberrations in multiple critical pathways that tend to accumulate over the course of the disease. The progression from indolence into aggressive disease is associated with a complex interplay in which genetic and epigenetic factors are involved. The effect of the immune tumor microenvironment is also very important. Emerging evidence has suggested that the upregulation of pathways related to cellular aging and senescence can identify patients with indolent disease. In addition, a number of tumors enter a long-lasting quiescent state. Further research will determine whether halting tumor evolution is a feasible option, and whether the life of patients can be markedly prolonged by inducing tumor senescence or long-term dormancy.

MTAP deficiency creates an exploitable target for antifolate therapy in 9p21-loss cancers.

Methylthioadenosine phosphorylase, an essential enzyme for the adenine salvage pathway, is often deficient (MTAPdef) in tumors with 9p21 loss and hypothetically renders tumors susceptible to synthetic lethality by antifolates targeting de novo purine synthesis. Here we report our single arm phase II trial (NCT02693717) that assesses pemetrexed in MTAPdef urothelial carcinoma (UC) with the primary endpoint of overall response rate (ORR). Three of 7 enrolled MTAPdef patients show response to pemetrexed (ORR 43%). Furthermore, a historic cohort shows 4 of 4 MTAPdef patients respond to pemetrexed as compared to 1 of 10 MTAP-proficient patients. In vitro and in vivo preclinical data using UC cell lines demonstrate increased sensitivity to pemetrexed by inducing DNA damage, and distorting nucleotide pools. In addition, MTAP-knockdown increases sensitivity to pemetrexed. Furthermore, in a lung adenocarcinoma retrospective cohort (N = 72) from the published BATTLE2 clinical trial (NCT01248247), MTAPdef associates with an improved response rate to pemetrexed. Our data demonstrate a synthetic lethal interaction between MTAPdef and de novo purine inhibition, which represents a promising therapeutic strategy for larger prospective trials.

Identifying the Metabolic Signatures of PPARD-Overexpressing Gastric Tumors.

Peroxisome proliferator-activated receptor delta (PPARD) is a nuclear receptor known to play an essential role in regulation of cell metabolism, cell proliferation, inflammation, and tumorigenesis in normal and cancer cells. Recently, we found that a newly generated villin-PPARD mouse model, in which PPARD is overexpressed in villin-positive gastric progenitor cells, demonstrated spontaneous development of large, invasive gastric tumors as the mice aged. However, the role of PPARD in regulation of downstream metabolism in normal gastric and tumor cells is elusive. The aim of the present study was to find PPARD-regulated downstream metabolic changes and to determine the potential significance of those changes to gastric tumorigenesis in mice. Hyperpolarized [1-13C] pyruvate magnetic resonance spectroscopy, nuclear magnetic resonance spectroscopy, and liquid chromatography-mass spectrometry were employed for metabolic profiling to determine the PPARD-regulated metabolite changes in PPARD mice at different ages during the development of gastric cancer, and the changes were compared to corresponding wild-type mice. Nuclear magnetic resonance spectroscopy-based metabolomic screening results showed higher levels of inosine monophosphate (p = 0.0054), uracil (p = 0.0205), phenylalanine (p = 0.017), glycine (p = 0.014), and isocitrate (p = 0.029) and lower levels of inosine (p = 0.0188) in 55-week-old PPARD mice than in 55-week-old wild-type mice. As the PPARD mice aged from 10 weeks to 35 weeks and 55 weeks, we observed significant changes in levels of the metabolites inosine monophosphate (p = 0.0054), adenosine monophosphate (p = 0.009), UDP-glucose (p = 0.0006), and oxypurinol (p = 0.039). Hyperpolarized [1-13C] pyruvate magnetic resonance spectroscopy performed to measure lactate flux in live 10-week-old PPARD mice with no gastric tumors and 35-week-old PPARD mice with gastric tumors did not reveal a significant difference in the ratio of lactate to total pyruvate plus lactate, indicating that this PPARD-induced spontaneous gastric tumor development does not require glycolysis as the main source of fuel for tumorigenesis. Liquid chromatography-mass spectrometry-based measurement of fatty acid levels showed lower linoleic acid, palmitic acid, oleic acid, and steric acid levels in 55-week-old PPARD mice than in 10-week-old PPARD mice, supporting fatty acid oxidation as a bioenergy source for PPARD-expressing gastric tumors.

Estrogen Promotes Resistance to Bevacizumab in Murine Models of NSCLC.

INTRODUCTION: Subgroup analyses from clinical studies have suggested that among patients with metastatic NSCLC receiving chemotherapy, females may derive less benefit from the addition of the vascular endothelial growth factor (VEGF) monoclonal antibody bevacizumab (BV) than males. This has raised the question of whether estrogen may affect the response to antiangiogenic therapy. METHODS: To address this, we investigated the effects of estrogen on tumor growth, angiogenesis, and the response to BV in human xenograft models of NSCLC. RESULTS: We observed that estrogen induced marked resistance to BV, which was accompanied by a 2.3-fold increase in tumor vascular pericyte coverage (p = 0.01) and an up-regulation of proangiogenic factors, VEGF and platelet-derived growth factor-BB. We also investigated the role of infiltrating myeloid cells, a population that has been associated with resistance to anti-VEGF therapies. We observed that estrogen induced a greater than twofold increase (p = 0.001) in the recruitment of tumor-infiltrating myeloid cells and concomitant increases in the myeloid recruitment factors, G-CSF and CXCL1. Blockade of the estrogen receptor pathway using fulvestrant resensitized tumors to VEGF targeting as evidenced by reduced tumor vasculature and an increase in overall survival in our NSCLC xenograft models. CONCLUSIONS: Collectively, these data provide evidence that estrogen may promote resistance to VEGF-targeted therapies, potentially by enhancing pericyte coverage and myeloid recruitment, and suggest that estrogen receptor blockade merits further investigation as an approach to enhance the effects of antiangiogenic therapy.

Prostate cancer cells survive anti-androgen and mitochondrial metabolic inhibitors by modulating glycolysis and mitochondrial metabolic activities.

BACKGROUND: Most cancer cells are more glycolytic even under aerobic conditions compared with their normal counterparts. Recent evidence of tumor cell metabolism, however, shows that some tumors also increase mitochondrial oxidative phosphorylation (ox-phos) at some disease states during progression and/or development of drug resistance. Our data show that anti-androgen enzalutamide (ENZA) resistant prostate cancer (PCa) cells use more mitochondrial metabolism leading to higher ox-phos as compared to the ENZA-sensitive cells and can become vulnerable to mitochondrial metabolism targeted therapies. METHODS: Seahorse assay, mass spectrometry and high resolution fluorescence confocal microscopy coupled with image analysis has been used to compare mitochondrial metabolism in ENZA-treated and -untreated anti-androgen-sensitive LNCaP and -resistant C4-2, CWR22ν1, and PCa2b cells. Ex vivo fluorescence microscopy and image analysis has been standardized to monitor mitochondrial electron transport (ETS) activity that likely increases ox-phos in circulating tumor cells (CTCs) isolated fom patients undergoing AR-targeted therapies. RESULTS: Our data show that PCa cells that are resistant to anti-androgen ENZA switch from glycolysis to ox-phos leading to an increased ETS activity. ENZA pretreated cells are more vulnerable to ETS component complex I inhibitor IACS-010759 (IACS) and mitochondrial glutaminase inhibitor CB-839 that reduces glutamate supply to tricarboxylic acid cycle. CTCs isolated from 6 of 20 patient blood samples showed relatively higher ETS activity than the rest of the patients. All six patients have developed ENZA resistance within less than 6 months of the sample collection. CONCLUSION: The enhanced growth inhibitory effects of mitochondrial metabolic inhibitors IACS and CB-839 in ENZA pretreated PCa cells provides a rationale for designing a drug combination trial. Patients can be selected for such trials by monitoring the mitochondrial ETS activities in their CTCs to maximize success.

A Phase II Study of Cabozantinib and Androgen Ablation in Patients with Hormone-Naïve Metastatic Prostate Cancer.

PURPOSE: Cabozantinib, an oral inhibitor of c-MET/VEGFR2 signaling, improved progression-free survival (mPFS) but not overall survival (OS) in metastatic castrate-resistant prostate cancer. We evaluated cabozantinib plus androgen deprivation therapy (ADT) in hormone-naïve metastatic prostate cancer (HNMPCa). PATIENTS AND METHODS: Patients received ADT plus cabozantinib starting at 60 mg daily. The primary endpoint was castrate-resistant PFS by radiographic criteria, clinical progression, or receipt of additional therapy. Secondary endpoints included OS, safety, radiographic responses, and biomarker modulation. RESULTS: Sixty-two patients received treatment. With a median follow-up of 31.2 months, the mPFS was 16.1 months (95% CI, 14.6-22.7 months), and mOS was not reached. Reductions in PSA ≥ 90%, bone-specific alkaline phosphatase ≥ 50%, and urine N-telopeptides ≥ 50% occurred in 83%, 87%, and 86% of evaluable patients, respectively. Responses in bone scan and measurable disease were observed in 81% of and 90% of evaluable patients, respectively. Most common grade 3 adverse events were hypertension (19%), diarrhea (6%), and thromboembolic events (6%), and dose reductions occurred in 85% of patients. Analysis of baseline cytokine and angiogenic factors (CAFs) revealed that higher plasma concentrations of Lumican, CXCL5, CD25, and CD30 were associated with shorter PFS as was high tumor expression of pFGFR1. CONCLUSIONS: Cabozantinib plus ADT has promising clinical activity in HNMPCa. CAF profiles and tissue markers suggest candidate prognostic and predictive markers of cabozantinib benefit and provide insights for rational therapy combinations.

Androgen Receptor Signaling in Castration-Resistant Prostate Cancer Alters Hyperpolarized Pyruvate to Lactate Conversion and Lactate Levels In Vivo.

PURPOSE: Androgen receptor (AR) signaling affects prostate cancer (PCa) growth, metabolism, and progression. Often, PCa progresses from androgen-sensitive to castration-resistant prostate cancer (CRPC) following androgen-deprivation therapy. Clinicopathologic and genomic characterizations of CRPC tumors lead to subdividing CRPC into two subtypes: (1) AR-dependent CRPC containing dysregulation of AR signaling alterations in AR such as amplification, point mutations, and/or generation of splice variants in the AR gene; and (2) an aggressive variant PCa (AVPC) subtype that is phenotypically similar to small cell prostate cancer and is defined by chemotherapy sensitivity, gain of neuroendocrine or pro-neural marker expression, loss of AR expression, and combined alterations of PTEN, TP53, and RB1 tumor suppressors. Previously, we reported patient-derived xenograft (PDX) animal models that contain characteristics of these CRPC subtypes. In this study, we have employed the PDX models to test metabolic alterations in the CRPC subtypes. PROCEDURES: Mass spectrometry and nuclear magnetic resonance analysis along with in vivo hyperpolarized 1-[13C]pyruvate spectroscopy experiments were performed on prostate PDX animal models. RESULTS: Using hyperpolarized 1-[13C]pyruvate conversion to 1-[13C]lactate in vivo as well as lactate measurements ex vivo, we have found increased lactate production in AR-dependent CRPC PDX models even under low-hormone levels (castrated mouse) compared to AR-negative AVPC PDX models. CONCLUSIONS: Our analysis underscores the potential of hyperpolarized metabolic imaging in determining the underlying biology and in vivo phenotyping of CRPC.