Androgens repress the expression of the angiogenesis inhibitor thrombospondin-1 in normal and neoplastic prostate.

In order to understand why the angiogenesis inhibitor thrombospondin-1 (TSP1) is often, although not always, associated with prostatic tumors, we have investigated its relationship with the testosterone and the vasculature on which both normal and tumorigenic prostatic epithelia depend. In vivo, androgen withdrawal led to increased TSP1 production and decreased vascularization in the normal rat prostate which was reversed by androgen replacement. Androgen repression of TSP1 production occurred at the transcriptional level and was dependent on the presence of the first intron of the TSP1 gene. In an experimental model of prostate tumorigenesis, TSP1, when delivered by admixed stromal fibroblasts, markedly delayed LNCaP tumor growth and limited tumor vascularization. However, prolonged exposure to TSP1 resulted in the growth of tumors secreting high levels of vascular endothelial growth factor in the bloodstream of tumor-bearing animals and tumor growth was no longer sensitive to TSP1 inhibitory effects. Clinical evidence also suggested that prostate carcinomas are able to adapt to escape the antiangiogenic effects of TSP1. In human androgen-dependent localized prostate carcinomas, TSP1 expression was inversely correlated with blood vessel density. Androgen deprivation in patients with hormone-responsive tumors led to increased TSP1 expression and vascular regression. In contrast, despite a sustained expression in the tumor bed, TSP1 was no longer associated with decreased vascularization in hormone-refractory prostate tumors. Overall, these results suggest that the high in situ TSP1 exposure triggered by androgen deprivation in patients with prostate cancer could lead to early tumor resistance. Such patients could benefit from a combination of androgen deprivation and antiangiogenic therapy in order to minimize the induction of such tumor escape.

SiRNA-mediated inhibition of vascular endothelial growth factor severely limits tumor resistance to antiangiogenic thrombospondin-1 and slows tumor vascularization and growth.

In the past few years, several laboratories have developed antiangiogenic molecules that starve tumors by targeting their vasculature and we have shown that, when produced in tumors, the antiangiogenic molecule thrombospondin-1 (TSP1) reduces the vascularization and delays tumor onset. Yet over time, tumor cells producing active TSP1 do eventually form exponentially growing tumors. These tumors are composed of cells secreting unusually high amounts of the angiogenic stimulator vascular endothelial growth factor (VEGF) that are sufficient to overcome the inhibitory TSP1. Here, we use short double-stranded RNA (siRNA) to trigger RNA interference and thereby impair the synthesis of VEGF and ask if this inability to produce VEGF prevents the development of TSP1 resistance. Systemic in vivo administration of crude anti-VEGF siRNA reduced the growth of unaltered fibrosarcoma tumor cells, and when the anti-VEGF siRNA was expressed from tumor cells themselves, such inhibition was synergistic with the inhibitory effects derived from TSP1 secretion by the tumor cells. Anti-VEGF siRNA delayed the emergence of TSP1-resistant tumors and strikingly reduced their subsequent growth rate.

Pak3 promotes cell cycle exit and differentiation of ß-cells in the embryonic pancreas and is necessary to maintain glucose homeostasis in adult mice.

The transcription factor neurogenin3 (Ngn3) triggers islet cell differentiation in the developing pancreas. However, little is known about the molecular mechanisms coupling cell cycle exit and differentiation in Ngn3(+) islet progenitors. We identified a novel effector of Ngn3 endocrinogenic function, the p21 protein-activated kinase Pak3, known to control neuronal differentiation and implicated in X-linked intellectual disability in humans. We show that Pak3 expression is initiated in Ngn3(+) endocrine progenitor cells and next maintained in maturing hormone-expressing cells during pancreas development as well as in adult islet cells. In Pak3-deficient embryos, the proliferation of Ngn3(+) progenitors and ß-cells is transiently increased concomitantly with an upregulation of Ccnd1. ß-Cell differentiation is impaired at E15.5 but resumes at later stages. Pak3-deficient mice do not develop overt diabetes but are glucose intolerant under high-fat diet (HFD). In the intestine, Pak3 is expressed in enteroendocrine cells but is not necessary for their differentiation. Our results indicate that Pak3 is a novel regulator of ß-cell differentiation and function. Pak3 acts downstream of Ngn3 to promote cell cycle exit and differentiation in the embryo by a mechanism that might involve repression of Ccnd1. In the adult, Pak3 is required for the proper control of glucose homeostasis under challenging HFD.

Molecular and cellular effects of vitamin B12 in brain, myocardium and liver through its role as co-factor of methionine synthase.

Vitamin B12 (cobalamin, cbl) is a cofactor of methionine synthase (MTR) in the synthesis of methionine, the precursor of the universal methyl donor S-Adenosylmethionine (SAM), which is involved in epigenomic regulatory mechanisms. We have established a neuronal cell model with stable expression of a transcobalamin-oleosin chimer and subsequent decreased cellular availability of vitamin B12, which produces reduced proliferation, increased apoptosis and accelerated differentiation through PP2A, NGF and TACE pathways. Anti-transcobalamin antibody or impaired transcobalamin receptor expression produce also impaired proliferation in other cells. Consistently, the transcription, protein expression and activity of MTR are increased in proliferating cells of skin and intestinal epitheliums, in rat intestine crypts and in proliferating CaCo2 cells, while MTR activity correlates with DNA methylation in rat intestine villi. Exposure to nitrous oxide in animal models identified impairment of MTR reaction as the most important metabolic cause of neurological manifestations of B12 deficiency. Early vitamin B12 and folate deprivation during gestation and lactation of a 'dam-progeny' rat model developed in our laboratory is associated with long-lasting disabilities of behavior and memory capacities, with persisting hallmarks related to increased apoptosis, impaired neurogenesis and altered plasticity. We found also an epigenomic deregulation of energy metabolism and fatty acids beta-oxidation in myocardium and liver, through imbalanced methylation/acetylation of PGC-1alpha and decreased expression of SIRT1. These nutrigenomic effects display similarities with the molecular mechanisms of fetal programming. Beside deficiency, B12 loading increases the expression of MTR through internal ribosome entry sites (IRES) and down-regulates MDR-1 gene expression. In conclusion, vitamin B12 influences cell proliferation, differentiation and apoptosis in brain. Vitamin B12 and folate combined deficiency impairs fatty acid oxidation and energy metabolism in liver and heart through epigenomic mechanisms related to imbalanced acetylation/methylation. Some but not all of these effects reflect the upstream role of vitamin B12 in SAM synthesis.

SIRNA-directed in vivo silencing of androgen receptor inhibits the growth of castration-resistant prostate carcinomas.

BACKGROUND: Prostate carcinomas are initially dependent on androgens, and castration or androgen antagonists inhibit their growth. After some time though, tumors become resistant and recur with a poor prognosis. The majority of resistant tumors still expresses a functional androgen receptor (AR), frequently amplified or mutated. METHODOLOGY/PRINCIPAL FINDINGS: To test the hypothesis that AR is not only expressed, but is still a key therapeutic target in advanced carcinomas, we injected siRNA targeting AR into mice bearing exponentially growing castration-resistant tumors. Quantification of siRNA into tumors and mouse tissues demonstrated their efficient uptake. This uptake silenced AR in the prostate, testes and tumors. AR silencing in tumors strongly inhibited their growth, and importantly, also markedly repressed the VEGF production and angiogenesis. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that carcinomas resistant to hormonal manipulations still depend on the expression of the androgen receptor for their development in vivo. The siRNA-directed silencing of AR, which allows targeting overexpressed as well as mutated isoforms, triggers a strong antitumoral and antiangiogenic effect. siRNA-directed silencing of this key gene in advanced and resistant prostate tumors opens promising new therapeutic perspectives and tools.