Hypoxia and hyperglycaemia determine why some endometrial tumours fail to respond to metformin.

BACKGROUND: High expression of Ki67, a proliferation marker, is associated with reduced endometrial cancer-specific survival. Pre-surgical metformin reduces tumour Ki-67 expression in some women with endometrial cancer. Metformin's anti-cancer activity may relate to effects on cellular energy metabolism. Since tumour hypoxia and glucose availability are major cellular redox determinants, we evaluated their role in endometrial cancer response to metformin. METHODS: Endometrial cancer biopsies from women treated with pre-surgical metformin were tested for the hypoxia markers, HIF-1α and CA-9. Endometrial cancer cell lines were treated with metformin in variable glucose concentrations in normoxia or hypoxia and cell viability, mitochondrial biogenesis, function and energy metabolism were assessed. RESULTS: In women treated with metformin (n = 28), Ki-67 response was lower in hypoxic tumours. Metformin showed minimal cytostatic effects towards Ishikawa and HEC1A cells in conventional medium (25 mM glucose). In low glucose (5.5 mM), a dose-dependent cytostatic effect was observed in normoxia but attenuated in hypoxia. Tumours treated with metformin showed increased mitochondrial mass (n = 25), while in cultured cells metformin decreased mitochondrial function. Metformin targets mitochondrial respiration, however, in hypoxic, high glucose conditions, there was a switch to glycolytic metabolism and decreased metformin response. CONCLUSIONS: Understanding the metabolic adaptations of endometrial tumours may identify patients likely to derive clinical benefit from metformin.

Cancer stem cells (CSCs): metabolic strategies for their identification and eradication.

Phenotypic and functional heterogeneity is one of the most relevant features of cancer cells within different tumor types and is responsible for treatment failure. Cancer stem cells (CSCs) are a population of cells with stem cell-like properties that are considered to be the root cause of tumor heterogeneity, because of their ability to generate the full repertoire of cancer cell types. Moreover, CSCs have been invoked as the main drivers of metastatic dissemination and therapeutic resistance. As such, targeting CSCs may be a useful strategy to improve the effectiveness of classical anticancer therapies. Recently, metabolism has been considered as a relevant player in CSC biology, and indeed, oncogenic alterations trigger the metabolite-driven dissemination of CSCs. More interestingly, the action of metabolic pathways in CSC maintenance might not be merely a consequence of genomic alterations. Indeed, certain metabotypic phenotypes may play a causative role in maintaining the stem traits, acting as an orchestrator of stemness. Here, we review the current studies on the metabolic features of CSCs, focusing on the biochemical energy pathways involved in CSC maintenance and propagation. We provide a detailed overview of the plastic metabolic behavior of CSCs in response to microenvironment changes, genetic aberrations, and pharmacological stressors. In addition, we describe the potential of comprehensive metabolic approaches to identify and selectively eradicate CSCs, together with the possibility to 'force' CSCs within certain metabolic dependences, in order to effectively target such metabolic biochemical inflexibilities. Finally, we focus on targeting mitochondria to halt CSC dissemination and effectively eradicate cancer.

Bergamot natural products eradicate cancer stem cells (CSCs) by targeting mevalonate, Rho-GDI-signalling and mitochondrial metabolism.

Here, we show that a 2:1 mixture of Brutieridin and Melitidin, termed "BMF", has a statin-like properties, which blocks the action of the rate-limiting enzyme for mevalonate biosynthesis, namely HMGR (3-hydroxy-3-methylglutaryl-CoA-reductase). Moreover, our results indicate that BMF functionally inhibits several key characteristics of CSCs. More specifically, BMF effectively i) reduced ALDH activity, ii) blocked mammosphere formation and iii) inhibited the activation of CSC-associated signalling pathways (STAT1/3, Notch and Wnt/beta-catenin) targeting Rho-GDI-signalling. In addition, BMF metabolically inhibited mitochondrial respiration (OXPHOS) and fatty acid oxidation (FAO). Importantly, BMF did not show the same toxic side-effects in normal fibroblasts that were observed with statins. Lastly, we show that high expression of the mRNA species encoding HMGR is associated with poor clinical outcome in breast cancer patients, providing a potential companion diagnostic for BMF-directed personalized therapy.

GPER mediates the angiocrine actions induced by IGF1 through the HIF-1α/VEGF pathway in the breast tumor microenvironment.

BACKGROUND: The G protein estrogen receptor GPER/GPR30 mediates estrogen action in breast cancer cells as well as in breast cancer-associated fibroblasts (CAFs), which are key components of microenvironment driving tumor progression. GPER is a transcriptional target of hypoxia inducible factor 1 alpha (HIF-1α) and activates VEGF expression and angiogenesis in hypoxic breast tumor microenvironment. Furthermore, IGF1/IGF1R signaling, which has angiogenic effects, has been shown to activate GPER in breast cancer cells. METHODS: We analyzed gene expression data from published studies representing almost 5000 breast cancer patients to investigate whether GPER and IGF1 signaling establish an angiocrine gene signature in breast cancer patients. Next, we used GPER-positive but estrogen receptor (ER)-negative primary CAF cells derived from patient breast tumours and SKBR3 breast cancer cells to investigate the role of GPER in the regulation of VEGF expression and angiogenesis triggered by IGF1. We performed gene expression and promoter studies, western blotting and immunofluorescence analysis, gene silencing strategies and endothelial tube formation assays to evaluate the involvement of the HIF-1α/GPER/VEGF signaling in the biological responses to IGF1. RESULTS: We first determined that GPER is co-expressed with IGF1R and with the vessel marker CD34 in human breast tumors (n = 4972). Next, we determined that IGF1/IGF1R signaling engages the ERK1/2 and AKT transduction pathways to induce the expression of HIF-1α and its targets GPER and VEGF. We found that a functional cooperation between HIF-1α and GPER is essential for the transcriptional activation of VEGF induced by IGF1. Finally, using conditioned medium from CAFs and SKBR3 cells stimulated with IGF1, we established that HIF-1α and GPER are both required for VEGF-induced human vascular endothelial cell tube formation. CONCLUSIONS: These findings shed new light on the essential role played by GPER in IGF1/IGF1R signaling that induces breast tumor angiogenesis. Targeting the multifaceted interactions between cancer cells and tumor microenvironment involving both GPCRs and growth factor receptors has potential in future combination anticancer therapies.

G Protein-Coupled Receptors at the Crossroad between Physiologic and Pathologic Angiogenesis: Old Paradigms and Emerging Concepts.

G protein-coupled receptors (GPCRs) have been implicated in transmitting signals across the extra- and intra-cellular compartments, thus allowing environmental stimuli to elicit critical biological responses. As GPCRs can be activated by an extensive range of factors including hormones, neurotransmitters, phospholipids and other stimuli, their involvement in a plethora of physiological functions is not surprising. Aberrant GPCR signaling has been regarded as a major contributor to diverse pathologic conditions, such as inflammatory, cardiovascular and neoplastic diseases. In this regard, solid tumors have been demonstrated to activate an angiogenic program that relies on GPCR action to support cancer growth and metastatic dissemination. Therefore, the manipulation of aberrant GPCR signaling could represent a promising target in anticancer therapy. Here, we highlight the GPCR-mediated angiogenic function focusing on the molecular mechanisms and transduction effectors driving the patho-physiological vasculogenesis. Specifically, we describe evidence for the role of heptahelic receptors and associated G proteins in promoting angiogenic responses in pathologic conditions, especially tumor angiogenesis and progression. Likewise, we discuss opportunities to manipulate aberrant GPCR-mediated angiogenic signaling for therapeutic benefit using innovative GPCR-targeted and patient-tailored pharmacological strategies.

Cancer stem cell metabolism.

Cancer is now viewed as a stem cell disease. There is still no consensus on the metabolic characteristics of cancer stem cells, with several studies indicating that they are mainly glycolytic and others pointing instead to mitochondrial metabolism as their principal source of energy. Cancer stem cells also seem to adapt their metabolism to microenvironmental changes by conveniently shifting energy production from one pathway to another, or by acquiring intermediate metabolic phenotypes. Determining the role of cancer stem cell metabolism in carcinogenesis has become a major focus in cancer research, and substantial efforts are conducted towards discovering clinical targets.

Loss of Sirt1 promotes prostatic intraepithelial neoplasia, reduces mitophagy, and delays PARK2 translocation to mitochondria.

Prostatic intraepithelial neoplasia is a precursor to prostate cancer. Herein, deletion of the NAD(+)-dependent histone deacetylase Sirt1 induced histological features of prostatic intraepithelial neoplasia at 7 months of age; these features were associated with increased cell proliferation and enhanced mitophagy. In human prostate cancer, lower Sirt1 expression in the luminal epithelium was associated with poor prognosis. Genetic deletion of Sirt1 increased mitochondrial superoxide dismutase 2 (Sod2) acetylation of lysine residue 68, thereby enhancing reactive oxygen species (ROS) production and reducing SOD2 activity. The PARK2 gene, which has several features of a tumor suppressor, encodes an E3 ubiquitin ligase that participates in removal of damaged mitochondria via mitophagy. Increased ROS in Sirt1(-/-) cells enhanced the recruitment of Park2 to the mitochondria, inducing mitophagy. Sirt1 restoration inhibited PARK2 translocation and ROS production requiring the Sirt1 catalytic domain. Thus, the NAD(+)-dependent inhibition of SOD2 activity and ROS by SIRT1 provides a gatekeeper function to reduce PARK2-mediated mitophagy and aberrant cell survival.

Homozygous T172T and Heterozygous G135C Variants of Homologous Recombination Repairing Protein RAD51 are Related to Sporadic Breast Cancer Susceptibility.

Breast cancer (BC) is the most common cancer and the second leading cause of death among women worldwide. Only 10% of BC cases have been related to genetic predisposition. Rad51, a homologous recombination (HR) protein plays an important role in HR in meiosis and repairing DNA double-strand breaks. Expression of RAD51 may be a predictive biomarker in certain types of cancers. The exact mechanisms involved in the regulation of RAD51 expression are not fully understood, but certain transcription factors have been suggested to be the tuning mechanism of its expression. In this study, we propose that polymorphisms in the 5'-UTR promoter region of the RAD51 gene are prognostic factors for BC development. Direct sequencing of 106 samples from sporadic BC patients and 54 samples from a control group was performed. FFPE samples were the choice of sample collection, which might be a limitation of our study. Homologous variant T172T alone was found to be significantly associated with BC risk (OR 3.717, 95% CI 2.283-6.052, p < 0.0001). On the other hand, heterozygous G135C did not show any significant relationship with risk of sporadic BC (OR 1.598, 95% CI 0.5638-4.528, p > 0.05). Moreover, both variants; homozygous T172T and heterozygous G135C together; showed a significant relationship with sporadic BC susceptibility.

Catabolic cancer-associated fibroblasts transfer energy and biomass to anabolic cancer cells, fueling tumor growth.

Fibroblasts are the most abundant "non-cancerous" cells in tumors. However, it remains largely unknown how these cancer-associated fibroblasts (CAFs) promote tumor growth and metastasis, driving chemotherapy resistance and poor clinical outcome. This review summarizes new findings on CAF signaling pathways and their emerging metabolic phenotypes that promote tumor growth. Although it is well established that altered cancer metabolism enhances tumor growth, little is known about the role of fibroblast metabolism in tumor growth. New studies reveal that metabolic coupling occurs between catabolic fibroblasts and anabolic cancer cells, in many types of human tumors, including breast, prostate, and head & neck cancers, as well as lymphomas. These catabolic phenotypes observed in CAFs are secondary to a ROS-induced metabolic stress response. Mechanistically, this occurs via HIF1-alpha and NFκB signaling, driving oxidative stress, autophagy, glycolysis and senescence in stromal fibroblasts. These catabolic CAFs then create a nutrient-rich microenvironment, to metabolically support tumor growth, via the local stromal generation of mitochondrial fuels (lactate, ketone bodies, fatty acids, glutamine, and other amino acids). New biomarkers of this catabolic CAF phenotype (such as caveolin-1 (Cav-1) and MCT4), which are reversible upon treatment with anti-oxidants, are strong predictors of poor clinical outcome in various types of human cancers. How cancer cells metabolically reprogram fibroblasts can also help us to understand the effects of cancer cells at an organismal level, explaining para-neoplastic phenomena, such as cancer cachexia. In conclusion, cancer should be viewed more as a systemic disease, that engages the host-organism in various forms of energy-transfer and metabolic co-operation, across a whole-body "ecosystem".

Deficiency of hyccin, a newly identified membrane protein, causes hypomyelination and congenital cataract.

We describe a new autosomal recessive white matter disorder ('hypomyelination and congenital cataract') characterized by hypomyelination of the central and peripheral nervous system, progressive neurological impairment and congenital cataract. We identified mutations in five affected families, resulting in a deficiency of hyccin, a newly identified 521-amino acid membrane protein. Our study highlights the essential role of hyccin in central and peripheral myelination.

Ablation of calcineurin Aß reveals hyperlipidemia and signaling cross-talks with phosphodiesterases.

Insulin resistance, hyperlipidemia, and cardiovascular complications are common dysregulations of metabolic syndrome. Transplant patients treated with immunosuppressant drugs such as cyclosporine A (CsA), an inhibitor of calcineurin phosphatase, frequently develop similar metabolic complications. Although calcineurin is known to mediate insulin sensitivity by regulating ß-cell growth and adipokine gene transcription, its role in lipid homeostasis is poorly understood. Here, we examined lipid homeostasis in mice lacking calcineurin Aß (CnAß(-/-)). We show that mice lacking calcineurin Aß are hyperlipidemic and develop age-dependent insulin resistance. Hyperlipidemia found in CnAß(-/-) mice is, in part, due to increased lipolysis in adipose tissues, a process mediated by ß-adrenergic G-protein-coupled receptor signaling pathways. CnAß(-/-) mice also exhibit additional pathophysiological phenotypes caused by the potentiated GPCR signaling pathways. A cell autonomous mechanism with sustained cAMP/PKA activation is found in CnAß(-/-) mice or upon CsA treatment to inhibit calcineurin. Increased PKA activation and cAMP accumulation in CnAß(-/-) mice, however, are sensitive to phosphodiesterase inhibitor. Indeed, we show that calcineurin regulates degradation of phosphodiesterase 3B, in addition to phosphodiesterase 4D. These results establish a role for calcineurin in lipid homeostasis. These data also indicate that potentiated cAMP signaling pathway may provide an alternative molecular pathogenesis for the metabolic complications elicited by CsA in transplant patients.

Transmission FT-IR chemical imaging on glass substrates: applications in infrared spectral histopathology.

Fourier transform-infrared (FT-IR) chemical imaging in transmission mode has traditionally been performed on expensive mid-IR transparent windows such as barium/calcium fluoride, which are more fragile than glass, making preparation in the histopathology laboratories more cumbersome. A solution is presented here by using cheap glass substrates for the FT-IR chemical imaging, which has a high-wavenumber transmission window allowing measurement of the C-H, N-H, and O-H stretches occurring at ca. 2500-3800 cm(-1). The "fingerprint" region of the IR spectrum occurring below 1800 cm(-1) is not obtainable; however, we demonstrate that a wealth of information is contained in the high wavenumber range using 71 patients on a breast tissue microarray (TMA) as a model for investigation. Importantly, we demonstrate that the tissue can be classified into four basic tissue cell types and that using just the epithelial cells, reasonable discrimination of normal and malignant tissue can be found.

Cav1 suppresses tumor growth and metastasis in a murine model of cutaneous SCC through modulation of MAPK/AP-1 activation.

Caveolin-1 (Cav1) is a scaffolding protein that serves to regulate the activity of several signaling molecules. Its loss has been implicated in the pathogenesis of several types of cancer, but its role in the development and progression of cutaneous squamous cell carcinoma (cSCC) remains largely unexplored. Herein, we use the keratinocyte cell line PAM212, a murine model of cSCC, to determine the function of Cav1 in skin tumor biology. We first show that Cav1 overexpression decreases cell and tumor growth, whereas Cav1 knockdown increases these attributes in PAM212 cells. In addition, Cav1 knockdown increases the invasive ability and incidence of spontaneous lymph node metastasis. Finally, we demonstrate that Cav1 knockdown increases extracellular signaling-related kinase 1/2 mitogen-activated protein kinase/activator protein-1 pathway activation. We attribute the growth and invasive advantage conferred by Cav1 knockdown to increased expression of activator protein-1 transcriptional targets, including cyclin D1 and keratin 18, which show inverse expression in PAM212 based on the expression level of Cav1. In summary, we demonstrate that loss of Cav1 affects several characteristics associated with aggressive human skin tumors and that this protein may be an important modulator of tumor growth and invasion in cSCC.

Caveolin-1 deficiency induces spontaneous endothelial-to-mesenchymal transition in murine pulmonary endothelial cells in vitro.

It was previously demonstrated that transforming growth factor ß (TGF-ß) induces endothelial-to-mesenchymal transition (EndoMT) in murine lung endothelial cells (ECs) in vitro. Owing to the important role of caveolin-1 (CAV1) in TGF-ß receptor internalization and TGF-ß signaling, the participation of CAV1 in the induction of EndoMT in murine lung ECs was investigated. Pulmonary ECs were isolated from wild-type and Cav1 knockout mice using immunomagnetic methods with sequential anti-CD31 and anti-CD102 antibody selection followed by in vitro culture and treatment with TGF-ß1. EndoMT was assessed by semiquantitative RT-PCR for Acta2, Col1a1, Snai1, and Snai2; by immunofluorescence for α-smooth muscle actin; and by Western blot analysis for α-smooth muscle actin, SNAIL1, SNAIL2, and the α2 chain of type I collagen. The same studies were performed in Cav1(-/-) pulmonary ECs after restoration of functional CAV1 domains using a cell-permeable CAV1 scaffolding domain peptide. Pulmonary ECs from Cav1 knockout mice displayed high levels of spontaneous Acta2, Col1A, Snai1, and Snai2 expression, which increased after TGF-ß treatment. Spontaneous and TGF-ß1-stimulated EndoMT were abrogated by the restoration of functional CAV1 domains using a cell-permeable peptide. The findings suggest that CAV1 regulation of EndoMT may play a role in the development of fibroproliferative vasculopathies.

Imaging of small-animal models of infectious diseases.

Infectious diseases are the second leading cause of death worldwide. Noninvasive small-animal imaging has become an important research tool for preclinical studies of infectious diseases. Imaging studies permit enhanced information through longitudinal studies of the same animal during the infection. Herein, we briefly review recent studies of animal models of infectious disease that have used imaging modalities.

Alterations in glucose homeostasis in a murine model of Chagas disease.

Chagas disease, caused by Trypanosoma cruzi, is an important cause of morbidity and mortality primarily resulting from cardiac dysfunction, although T. cruzi infection results in inflammation and cell destruction in many organs. We found that T. cruzi (Brazil strain) infection of mice results in pancreatic inflammation and parasitism within pancreatic ß-cells with apparent sparing of α cells and leads to the disruption of pancreatic islet architecture, ß-cell dysfunction, and surprisingly, hypoglycemia. Blood glucose and insulin levels were reduced in infected mice during acute infection and insulin levels remained low into the chronic phase. In response to the hypoglycemia, glucagon levels 30 days postinfection were elevated, indicating normal α-cell function. Administration of L-arginine and a ß-adrenergic receptor agonist (CL316, 243, respectively) resulted in a diminished insulin response during the acute and chronic phases. Insulin granules were docked, but the lack of insulin secretion suggested an inability of granules to fuse at the plasma membrane of pancreatic ß-cells. In the liver, there was a concomitant reduced expression of glucose-6-phosphatase mRNA and glucose production from pyruvate (pyruvate tolerance test), demonstrating defective hepatic gluconeogenesis as a cause for the T. cruzi-induced hypoglycemia, despite reduced insulin, but elevated glucagon levels. The data establishes a complex, multi-tissue relationship between T. cruzi infection, Chagas disease, and host glucose homeostasis.

Endothelial caveolin-1 plays a major role in the development of atherosclerosis.

Clinical studies have established the important impact of atherosclerotic disease in Western societies. This disease is characterized by the accumulation of lipids and the migration of various cell types in the sub-endothelial space of blood vessels. As demonstrated by many studies, endothelial cells play an essential role in the development of this disease. The endothelium acts as a gatekeeper of blood vessel integrity and cardiovascular health status. For instance, the transfer of lipids via the transport of lipoproteins in the arterial intima is believed to be mediated by endothelial cells through a process termed transcytosis. In addition, lipoproteins that accumulate in the sub-endothelial space may also be modified, in a process that can direct the activation of endothelial cells. These steps are essential for the initiation of an atherosclerotic plaque and may be mediated, at least in part, by caveolae and their associated protein caveolin-1. In the present study, we evaluate the role of caveolin-1/caveolae in the regulation of these two steps in endothelial cells. Our data clearly demonstrate that caveolin-1 is involved in the regulation of lipoprotein transcytosis across endothelial cells and in the regulation of vascular inflammation.

Caveolin-1 regulates the anti-atherogenic properties of macrophages.

Atherosclerosis is a complex disease initiated by the vascular accumulation of lipoproteins in the sub-endothelial space, followed by the infiltration of monocytes into the arterial intima. Caveolin-1 (Cav-1) plays an essential role in the regulation of cellular cholesterol metabolism and of various signaling pathways. In order to study specifically the role of macrophage Cav-1 in atherosclerosis, we used Cav-1 (-/-) Apoe (-/-) mice and transplanted them with bone marrow (BM) cells obtained from Cav-1 (+/+) Apoe (-/-) or Cav-1 (-/-) Apoe (-/-) mice and vice versa. We found that Cav-1 (+/+) mice harboring Cav-1 (-/-) BM-derived macrophages developed significantly larger lesions than Cav-1 (+/+) mice harboring Cav-1 (+/+) BM-derived macrophages. Cav-1 (-/-) macrophages were more susceptible to apoptosis and more prone to induce inflammation. The present study provides clear evidence that the absence of Cav-1 in macrophage is pro-atherogenic, whereas its absence in endothelial cells protects against atherosclerotic lesion formation. These findings demonstrate the cell-specific role of Cav-1 during the development of this disease.

Pressure-overload-induced subcellular relocalization/oxidation of soluble guanylyl cyclase in the heart modulates enzyme stimulation.

RATIONALE: Soluble guanylyl cyclase (sGC) generates cyclic guanosine monophophate (cGMP) upon activation by nitric oxide (NO). Cardiac NO-sGC-cGMP signaling blunts cardiac stress responses, including pressure-overload-induced hypertrophy. The latter itself depresses signaling through this pathway by reducing NO generation and enhancing cGMP hydrolysis. OBJECTIVE: We tested the hypothesis that the sGC response to NO also declines with pressure-overload stress and assessed the role of heme-oxidation and altered intracellular compartmentation of sGC as potential mechanisms. METHODS AND RESULTS: C57BL/6 mice subjected to transverse aortic constriction (TAC) developed cardiac hypertrophy and dysfunction. NO-stimulated sGC activity was markedly depressed, whereas NO- and heme-independent sGC activation by BAY 60-2770 was preserved. Total sGCα(1) and ß(1) expression were unchanged by TAC; however, sGCß(1) subunits shifted out of caveolin-enriched microdomains. NO-stimulated sGC activity was 2- to 3-fold greater in Cav3-containing lipid raft versus nonlipid raft domains in control and 6-fold greater after TAC. In contrast, BAY 60-2770 responses were >10 fold higher in non-Cav3 domains with and without TAC, declining about 60% after TAC within each compartment. Mice genetically lacking Cav3 had reduced NO- and BAY-stimulated sGC activity in microdomains containing Cav3 for controls but no change within non-Cav3-enriched domains. CONCLUSIONS: Pressure overload depresses NO/heme-dependent sGC activation in the heart, consistent with enhanced oxidation. The data reveal a novel additional mechanism for reduced NO-coupled sGC activity related to dynamic shifts in membrane microdomain localization, with Cav3-microdomains protecting sGC from heme-oxidation and facilitating NO responsiveness. Translocation of sGC out of this domain favors sGC oxidation and contributes to depressed NO-stimulated sGC activity.

Loss of caveolin-1 in prostate cancer stroma correlates with reduced relapse-free survival and is functionally relevant to tumour progression.

Levels of caveolin-1 (Cav-1) in tumour epithelial cells increase during prostate cancer progression. Conversely, Cav-1 expression in the stroma can decline in advanced and metastatic prostate cancer. In a large cohort of 724 prostate cancers, we observed significantly decreased levels of stromal Cav-1 in concordance with increased Gleason score (p = 0.012). Importantly, reduced expression of Cav-1 in the stroma correlated with reduced relapse-free survival (p = 0.009), suggesting a role for stromal Cav-1 in inhibiting advanced disease. Silencing of Cav-1 by shRNA in WPMY-1 prostate fibroblasts resulted in up-regulation of Akt phosphorylation, and significantly altered expression of genes involved in angiogenesis, invasion, and metastasis, including a > 2.5-fold increase in TGF-ß1 and γ-synuclein (SNCG) gene expression. Moreover, silencing of Cav-1 induced migration of prostate cancer cells when stromal cells were used as attractants. Pharmacological inhibition of Akt caused down-regulation of TGF-ß1 and SNCG, suggesting that loss of Cav-1 in the stroma can influence Akt-mediated signalling in the tumour microenvironment. Cav-1-depleted stromal cells exhibited increased levels of intracellular cholesterol, a precursor for androgen biosynthesis, steroidogenic enzymes, and testosterone. These findings suggest that loss of Cav-1 in the tumour microenvironment contributes to the metastatic behaviour of tumour cells by a mechanism that involves up-regulation of TGF-ß1 and SNCG through Akt activation. They also suggest that intracrine production of androgens, a process relevant to castration resistance, may occur in the stroma.