Summary There is substantial heterogeneity among primary prostate cancers, evident in the spectrum of molecular abnormalities and its variable clinical course. As part of The Cancer Genome Atlas (TCGA), we present a comprehensive molecular analysis of 333 primary prostate carcinomas. Our results revealed a molecular taxonomy in which 74% of these tumors fell into one of seven subtypes defined by specific gene fusions (ERG, ETV1/4, FLI1) or mutations (SPOP, FOXA1, IDH1). Epigenetic profiles showed substantial heterogeneity, including an IDH1-mutant subset with a methylator phenotype. Androgen receptor (AR) activity varied widely and in a subtype-specific manner with SPOP and FOXA1 mutant tumors having the highest levels of AR-induced transcripts. 25% of the prostate cancers had a presumed actionable lesion in the PI3K or MAPK signaling pathways, and DNA repair genes were inactivated in 19%. Our analysis reveals molecular heterogeneity among primary prostate cancers, as well as potentially actionable molecular defects.

Androgen receptor (AR) signaling reprograms cellular metabolism to support prostate cancer (PCa) growth and survival. Another key regulator of cellular metabolism is mTOR, a kinase found in diverse protein complexes and cellular localizations, including the nucleus. However, whether nuclear mTOR plays a role in PCa progression and participates in direct transcriptional cross-talk with the AR is unknown. Here, via the intersection of gene expression, genomic, and metabolic studies, we reveal the existence of a nuclear mTOR-AR transcriptional axis integral to the metabolic rewiring of PCa cells. Androgens reprogram mTOR-chromatin associations in an AR-dependent manner in which activation of mTOR-dependent metabolic gene networks is essential for androgeninduced aerobic glycolysis and mitochondrial respiration. In models of castration-resistant PCa cells, mTOR was capable of transcriptionally regulating metabolic gene programs in the absence of androgens, highlighting a potential novel castration resistance mechanism to sustain cell metabolism even without a functional AR. Remarkably, we demonstrate that increased mTOR nuclear localization is indicative of poor prognosis in patients, with the highest levels detected in castration-resistant PCa tumors and metastases. Identification of a functional mTOR targeted multigene signature robustly discriminates between normal prostate tissues, primary tumors, and hormone refractory metastatic samples but is also predictive of cancer recurrence. This study thus underscores a paradigm shift from AR to nuclear mTOR as being the master transcriptional regulator of metabolism in PCa.

The murine Hyp model reproduces the characteristics of human X-linked hypophosphatemia (XLH), an inherited disease causing renal loss of phosphate (Pi), severe rickets and osteomalacia. A current hypothesis considers that a humoral factor may be responsible for the renal Pi loss, although in vitro experiments with renal cell models have failed to demonstrate the presence of such a factor in XLH or in the Hyp mouse model. To test this hypothesis directly, we prepared primary mouse proximal tubule cell cultures (MPTC), expressing normal features of proximal tubule cells. These cells possess high alkaline phosphatase activity, and respond to human parathyroid hormone fragment 1-34 (PTH) with a four- to sixfold increase in cAMP production but do not respond to either arginine vasopressin (AVP) or to salmon calcitonin (sCT). They also show sodium-dependent phosphate, glucose and amino acid uptake. The presence of 10% Hyp mouse serum in HAMF12/DMEM media (1 mM Pi) for the last 48 hours of culture of MPTC reduced Pi uptake (0.1 mM 32P-Pi in the presence of 140 mM NaCl) by 45.7 +/- 3.9% (P < 0.01) as compared to normal mouse serum. This effect of Hyp mouse serum was dose-dependent between 5 to 20% (final concentration) in culture media for the last 48 hours of culture (P < 0.01 by analysis of variance). This effect of Hyp mouse serum was also time-dependent, with a lag time of at least 12 hours. Indeed, no significant inhibition of Pi uptake could be detected with incubations less than 12 hours in the presence of 10% Hyp mouse serum, whereas a maximal effect was obtained after 24 hours of incubation and remained unchanged after 36 and 48 hours. The inhibition of phosphate uptake by Hyp mouse serum was specific, since neither sodium-dependent glucose nor alpha-aminobutyric acid uptake was modified under these conditions. MPTC cells showed a very nice adaptation to Pi concentration in the media; low Pi (0.4 mM final concentration in the presence of 10% serum) stimulated Pi uptake, whereas high Pi concentration (3 mM) reduced Pi uptake by these cells as compared to regular HAMF12/DMEM media containing 1 mM Pi. Normal and Hyp mouse serum both inhibited Pi uptake by MPTC following adaptation in low or normal Pi media, however, Hyp mouse serum always showed a stronger inhibition than normal serum. In contrast, adaptation of MPTC in high Pi media resulted in no inhibition of phosphate uptake either in the presence of normal or Hyp mouse serum. We next questioned whether conditioned media from confluent Hyp mouse primary osteoblast-like cell cultures could affect Pi uptake by MPTC. These osteoblast-like cells expressed high alkaline phosphatase and produced the bone specific protein, osteocalcin. When MPTC were treated for 48 hours with Hyp mouse bone cell media conditioned for the last 48 hours of cultures, Pi uptake was specifically inhibited by 30.5 +/- 4.1% (P < 0.025) as compared to normal mouse bone cell-conditioned media. This effect of primary Hyp mouse bone cell-conditioned media is specific for these cells since it was...

Background:Prostate cancer shows considerable heterogeneity in disease progression and we propose that markers expressed in tumour stroma may be reliable predictors of aggressive tumour subtypes.Methods:We have used Kaplan–Meier, univariate and multivariate analysis to correlate the expression of Asporin (ASPN) mRNA and protein with prostate cancer progression in independent cohorts. We used immunohistochemistry and H scoring to document stromal localisation of ASPN in a tissue microarray and mouse prostate cancer model, and correlated expression with reactive stroma, defined using Masson Trichrome staining. We used cell cultures of primary prostate cancer fibroblasts treated with serum-free conditioned media from prostate cancer cell lines to examine regulation of ASPN mRNA in tumour stromal cells.Results:We observed increased expression of ASPN mRNA in a data set derived from benign vs tumour microdissected tissue, and a correlation with biochemical recurrence using Kaplan–Meier and Cox proportional hazard analysis. ASPN protein localised to tumour stroma and elevated expression of ASPN was correlated with decreased time to biochemical recurrence, in a cohort of 326 patients with a median follow up of 9.6 years. Univariate and multivariate analysis demonstrated that ASPN was correlated with progression, as were Gleason score, and clinical stage. Additionally, ASPN expression correlated with the presence of reactive stroma, suggesting that it may be a stromal marker expressed in response to the presence of tumour cells and particularly with aggressive tumour subtypes. We observed expression of ASPN in the stroma of tumours induced by p53 inhibition in a mouse model of prostate cancer, and correlation with neuroendocrine marker expression. Finally, we demonstrated that ASPN transcript expression in normal and cancer fibroblasts was regulated by conditioned media derived from the PC3, but not LNCaP, prostate cancer cell lines.Conclusions:Our results suggest that ASPN is a stromally expressed biomarker that correlates with disease progression, and is observed in reactive stroma. ASPN expression in stroma may be part of a stromal response to aggressive tumour subtypes.

Androgen withdrawal is the most effective form of systemic therapy for men with advanced prostate cancer. Unfortunately, androgen-independent progression is inevitable, and the development of hormone-refractory disease and death occurs within 2 to 3 years in most men. The understanding of molecular mechanisms promoting the growth of androgenindependent prostate cancer cells is essential for the rational design of agents to treat advanced disease. We previously reported that Fer tyrosine kinase level correlates with the development of prostate cancer and aggressiveness of prostate cancer cell lines. Moreover, knocking down Fer expression interferes with prostate cancer cell growth in vitro. However, the mechanism by which Fer mediates prostate cancer progression remains elusive. We present here that Fer and phospho-Y705 signal transducer and activator of transcription 3 (STAT3) are barely detectable in human benign prostate tissues but constitutively expressed in the cytoplasm and nucleus of the same subsets of tumor cells in human prostate cancer. The interaction between STAT3 and Fer was observed in all prostate cancer cell lines tested, and this interaction is mediated via the Fer Src homology 2 domain and modulated by interleukin-6 (IL-6). Moreover, IL-6 triggered a rapid formation of Fer/gp130 and Fer/STAT3 complexes in a time-dependent manner and consistent with changes in Fer and STAT3 phosphorylation and cytoplasmic/nuclear distribution. The modulation of Fer expression/activation resulted in inhibitory or stimulatory effects on STAT3 phosphorylation, nuclear translocation, and transcriptional activation. These effects translated in IL-6 -mediated PC-3 cell growth. Taken together, these results support an important function of Fer in prostate