VAV3 mediates resistance to breast cancer endocrine therapy.

INTRODUCTION: Endocrine therapies targeting cell proliferation and survival mediated by estrogen receptor α (ERα) are among the most effective systemic treatments for ERα-positive breast cancer. However, most tumors initially responsive to these therapies acquire resistance through mechanisms that involve ERα transcriptional regulatory plasticity. Herein we identify VAV3 as a critical component in this process. METHODS: A cell-based chemical compound screen was carried out to identify therapeutic strategies against resistance to endocrine therapy. Binding to ERα was evaluated by molecular docking analyses, an agonist fluoligand assay and short hairpin (sh)RNA-mediated protein depletion. Microarray analyses were performed to identify altered gene expression. Western blot analysis of signaling and proliferation markers, and shRNA-mediated protein depletion in viability and clonogenic assays, were performed to delineate the role of VAV3. Genetic variation in VAV3 was assessed for association with the response to tamoxifen. Immunohistochemical analyses of VAV3 were carried out to determine its association with therapeutic response and different tumor markers. An analysis of gene expression association with drug sensitivity was carried out to identify a potential therapeutic approach based on differential VAV3 expression. RESULTS: The compound YC-1 was found to comparatively reduce the viability of cell models of acquired resistance. This effect was probably not due to activation of its canonical target (soluble guanylyl cyclase), but instead was likely a result of binding to ERα. VAV3 was selectively reduced upon exposure to YC-1 or ERα depletion, and, accordingly, VAV3 depletion comparatively reduced the viability of cell models of acquired resistance. In the clinical scenario, germline variation in VAV3 was associated with the response to tamoxifen in Japanese breast cancer patients (rs10494071 combined P value = 8.4 × 10-4). The allele association combined with gene expression analyses indicated that low VAV3 expression predicts better clinical outcome. Conversely, high nuclear VAV3 expression in tumor cells was associated with poorer endocrine therapy response. Based on VAV3 expression levels and the response to erlotinib in cancer cell lines, targeting EGFR signaling may be a promising therapeutic strategy. CONCLUSIONS: This study proposes VAV3 as a biomarker and a rationale for its use as a signaling target to prevent and/or overcome resistance to endocrine therapy in breast cancer.

Etiopathology of chronic tubular, glomerular and renovascular nephropathies: clinical implications.

Chronic kidney disease (CKD) comprises a group of pathologies in which the renal excretory function is chronically compromised. Most, but not all, forms of CKD are progressive and irreversible, pathological syndromes that start silently (i.e. no functional alterations are evident), continue through renal dysfunction and ends up in renal failure. At this point, kidney transplant or dialysis (renal replacement therapy, RRT) becomes necessary to prevent death derived from the inability of the kidneys to cleanse the blood and achieve hydroelectrolytic balance. Worldwide, nearly 1.5 million people need RRT, and the incidence of CKD has increased significantly over the last decades. Diabetes and hypertension are among the leading causes of end stage renal disease, although autoimmunity, renal atherosclerosis, certain infections, drugs and toxins, obstruction of the urinary tract, genetic alterations, and other insults may initiate the disease by damaging the glomerular, tubular, vascular or interstitial compartments of the kidneys. In all cases, CKD eventually compromises all these structures and gives rise to a similar phenotype regardless of etiology. This review describes with an integrative approach the pathophysiological process of tubulointerstitial, glomerular and renovascular diseases, and makes emphasis on the key cellular and molecular events involved. It further analyses the key mechanisms leading to a merging phenotype and pathophysiological scenario as etiologically distinct diseases progress. Finally clinical implications and future experimental and therapeutic perspectives are discussed.

Bypassing ubiquitination enables LAT recycling to the cell surface and enhanced signaling in T cells.

LAT molecules defective in ubiquitination have an increased half-life and induce enhanced signaling when expressed in T cells. In this study, we have examined the role of ubiquitination in regulating LAT endocytosis, recycling, and degradation in resting and stimulated T cells. By tracking and comparing plasma membrane-labeled wild type and ubiquitination-resistant 2KR LAT, we find that ubiquitination promotes the degradation of surface LAT in T cells. Activation of T cells increases LAT ubiquitination and promotes trafficking of internalized LAT to lysosomes for degradation. Ubiquitination of LAT does not change internalization rates from the cell surface, but prevents efficient recycling of LAT to the surface of T cells. Our study demonstrates that surface LAT levels are tightly controlled by ubiquitination. LAT in unstimulated cells lacks ubiquitin allowing for increased LAT stability and efficient T cell activation upon TCR triggering; ubiquitination leads to efficient removal of LAT after activation.

Activation of Erk1/2 and Akt following unilateral ureteral obstruction.

Chronic unilateral ureteral obstruction is a well characterized model of renal injury leading to tubulointerstitial fibrosis and distinct patterns of cell proliferation and apoptosis in the obstructed kidney. In this study we assessed the contribution of the mitogen activated protein kinase (MAPK)-ERK1/2 and the phosphatidylinositol 3 kinase (PI3K)-Akt pathways to early renal changes following unilateral obstruction. Increased activation of small Ras GTPase and its downstream effectors ERK1/2 and Akt was detected in ligated kidneys. The use of specific pharmacological inhibitors to either ERK1/2 or Akt activation led to decreased levels of fibroblast-myofibroblast markers in the interstitium while inhibition of PI3K reduced the number of proliferating cells and the amount of interstitial extracellular matrix deposition. Treatment with an ERK1/2 inhibitor diminished the number of apoptotic tubule and interstitial cells. Our results suggest a role for the MAPK-ERK1/2 and PI3K-Akt systems in early changes induced by ureteral obstruction and that inhibition of these signaling pathways may provide a novel approach to prevent progression of renal fibrosis.

Long-term nebivolol administration reduces renal fibrosis and prevents endothelial dysfunction in rats with hypertension induced by renal mass reduction.

OBJECTIVES: D/L-Nebivolol is a lypophilic beta1-adrenergic antagonist which is devoid of intrinsic sympathomimetic activity and can increase nitric oxide (NO) bioavailability with its subsequent vasodilating properties. The purpose of the present work was to assess the effect of long-term nebivolol administration on both renal damage and endothelial dysfunction induced by renal mass reduction (RMR) in rats. Atenolol, which does not increase NO bioavailability, was included in the study as a comparative beta-adrenoceptor antagonist. METHODS: Rats were subjected to both right nephrectomy and surgical removal of two-thirds of the left kidney in order to retain approximately one-sixth of the total renal mass. One week after ablation, rats were distributed randomly according to the following experimental groups: control group containing RMR rats without treatment; RMR rats treated daily with nebivolol for 6 months (drinking water, 8 mg/kg per day); and RMR rats treated daily with atenolol for 6 months (drinking water, 80 mg/kg per day). A group of sham-operated animals was also included. RESULTS: Administration of either nebivolol or atenolol similarly reduced arterial pressure in comparison with RMR untreated animals; however, animals receiving nebivolol presented lower levels of collagen type I expression as well as lower glomerular and interstitial fibrosis than those receiving atenolol. Urinary excretion of oxidative stress markers were also lower in animals receiving nebivolol than in rats treated with atenolol. Furthermore, nebivolol prevented RMR-induced endothelial dysfunction more efficiently than atenolol. CONCLUSIONS: Nebivolol protects against renal fibrosis, oxidative stress and endothelial dysfunction better than equivalent doses, in terms of arterial pressure reduction, of atenolol in a hypertensive model of renal damage induced by RMR.

Activation of small GTPase Ras and renal fibrosis.

Although mechanisms responsible for the initiation and development of renal fibrosis have been extensively studied, the intracellular signals involved in this phenomenon are still poorly understood. Ras proteins are the prototype members of a large family of small GTPases bound to membrane that control signalling pathways implicated in cellular growth, differentiation, proliferation and apoptosis. The purpose of the present manuscript is to review Ras signalling studies focusing on the possible role of Ras activation in renal fibrosis. A cell-specific expression of the three Ras isoforms (K-Ras, H-Ras and N-Ras) has been found in both normal and injured kidneys. Ras activation has been described in cultured mesangial cells or renal fibroblasts when challenged with several cytokines, high glucose medium or advanced glycation end-products. A role for K-Ras has been demonstrated in renal fibroblast proliferation. Mesangial cell proliferation induced by high glucose can be reverted by 3-hydroxy-3-methylglutaryl CoA reductase inhibitor which blocks the synthesis of prenyl groups and consequently Ras activation. In addition, increased Ras activation measured by Ras-GTP/total Ras ratio has been found in an experimental model of tubulointerstitial fibrosis induced by unilateral ureteral ligation. In overall, these data give enough evidence of a role for Ras activation in renal fibrosis.

Effect of angiotensin II and small GTPase Ras signaling pathway inhibition on early renal changes in a murine model of obstructive nephropathy.

Tubulointerstitial fibrosis is a major feature of chronic kidney disease. Unilateral ureteral obstruction (UUO) in rodents leads to the development of renal tubulointerstitial fibrosis consistent with histopathological changes observed in advanced chronic kidney disease in humans. The purpose of this study was to assess the effect of inhibiting angiotensin II receptors or Ras activation on early renal fibrotic changes induced by UUO. Animals either received angiotensin II or underwent UUO. UUO animals received either losartan, atorvastatin, and farnesyl transferase inhibitor (FTI) L-744,832, or chaetomellic acid A (ChA). Levels of activated Ras, phospho-ERK1/2, phospho-Akt, fibronectin, and α-smooth muscle actin were subsequently quantified in renal tissue by ELISA, Western blot, and/or immunohistochemistry. Our results demonstrate that administration of angiotensin II induces activation of the small GTPase Ras/Erk/Akt signaling system, suggesting an involvement of angiotensin II in the early obstruction-induced activation of renal Ras. Furthermore, upstream inhibition of Ras signalling by blocking either angiotensin AT1 type receptor or by inhibiting Ras prenylation (atorvastatin, FTI o ChA) reduced the activation of the Ras/Erk/Akt signaling system and decreased the early fibrotic response in the obstructed kidney. This study points out that pharmacological inhibition of Ras activation may hold promise as a future strategy in the prevention of renal fibrosis.

Common pathophysiological mechanisms of chronic kidney disease: therapeutic perspectives.

It is estimated that over 10% of the adult population in developed countries have some degree of chronic kidney disease (CKD). CKD is a progressive and irreversible deterioration of the renal excretory function that results in implementation of renal replacement therapy in the form of dialysis or renal transplant, which may also lead to death. CKD poses a growing problem to society as the incidence of the disease increases at an annual rate of 8%, and consumes up to 2% of the global health expenditure. CKD is caused by a variety of factors including diabetes, hypertension, infection, reduced blood supply to the kidneys, obstruction of the urinary tract and genetic alterations. The nephropathies associated with some of these conditions have been modeled in animals, this being crucial to understanding their pathophysiological mechanism and assessing prospective treatments at the preclinical level. This article reviews and updates the pathophysiological knowledge acquired primarily from experimental models and human studies of CKD. It also highlights the common mechanism(s) underlying the most relevant chronic nephropathies which lead to the appearance of a progressive, common renal phenotype regardless of aetiology. Based on this knowledge, a therapeutic horizon for the treatment of CKD is described. Present therapy primarily based upon renin-angiotensin inhibition, future diagnostics and therapeutic perspectives based upon anti-inflammatory, anti-fibrotic and hemodynamic approaches, new drugs targeting specific signaling pathways, and advances in gene and cell therapies, are all elaborated.