Estrogen receptor β exerts tumor repressive functions in human malignant pleural mesothelioma via EGFR inactivation and affects response to gefitinib

Background

The role of estrogen and estrogen receptors in oncogenesis has been investigated in various malignancies. Recently our group identified estrogen receptor beta (ERβ) expression as an independent prognostic factor in the progression of human Malignant Pleural Mesothelioma (MMe), but the underlying mechanism by which ERβ expression in tumors determines clinical outcome remains largely unknown. This study is aimed at investigating the molecular mechanisms of ERβ action in MMe cells and disclosing the potential translational implications of these results.

Methods

We modulated ERβ expression in REN and MSTO-211H MMe cell lines and evaluated cell proliferation and EGF receptor (EGFR) activation.

Results

Our data indicate that ERβ knockdown in ER positive cells confers a more invasive phenotype, increases anchorage independent proliferation and elevates the constitutive activation of EGFR-coupled signal transduction pathways. Conversely, re-expression of ERβ in ER negative cells confers a more epithelioid phenotype, decreases their capacity for anchorage independent growth and down-modulates proliferative signal transduction pathways. We identify a physical interaction between ERβ, EGFR and caveolin 1 that results in an altered internalization and in a selective reduced activation of EGFR-coupled signaling, when ERβ is over-expressed. We also demonstrate that differential expression of ERβ influences MMe tumor cell responsiveness to the therapeutic agent: Gefitinib.

Conclusions

This study describes a role for ERβ in the modulation of cell proliferation and EGFR activation and provides a rationale to facilitate the targeting of a subgroup of MMe patients who would benefit most from therapy with Gefitinib alone or in combination with Akt inhibitors.     

Aging negatively affects estrogens-mediated effects on nitric oxide bioavailability by shifting ERα/ERβ balance in female mice

Aims

Aging is among the major causes for the lack of cardiovascular protection by estrogen (E2) during postmenopause. Our study aims to determine the mechanisms whereby aging changes E2 effects on nitric oxide (NO) production in a mouse model of accelerated senescence (SAM).

Methods and Results

Although we found no differences on NO production in females SAM prone (SAMP, aged) compared to SAM resistant (SAMR, young), by either DAF-2 fluorescence or plasmatic nitrite/nitrate (NO2/NO3), in both cases, E2 treatment increased NO production in SAMR but had no effect in SAMP. Those results are in agreement with changes of eNOS protein and gene expression. E2 up-regulated eNOS expression in SAMR but not in SAMP. E2 is also known to increase NO by decreasing its catabolism by superoxide anion (O₂ ^-). Interestingly, E2 treatment decreased O₂ ⁻ production in young females, while increased O₂ ⁻ in aged ones. Furthermore, we observed that aging changed expression ratio of estrogen receptors (ERβ/ERα) and levels of DNA methylation. Increased ratio ERβ/ERα in aged females is associated to a lack of estrogen modulation of NO production and with a reversal in its antioxidant effect to a pro-oxidant profile.

Conclusions

Together, our data suggest that aging has detrimental effects on E2-mediated benefits on NO bioavailability, partially by affecting the ability of E2 to induce up regulation of eNOS and decrease of O₂ ⁻. These modifications may be associated to aging-mediated modifications on global DNA methylation status, but not to a specific methylation at 5′flanking region of ERα gene.     

Extra-nuclear signaling of progesterone receptor to breast cancer cell movement and invasion through the actin cytoskeleton

Progesterone plays a role in breast cancer development and progression but the effects on breast cancer cell movement or invasion have not been fully explored. In this study, we investigate the actions of natural progesterone and of the synthetic progestin medroxyprogesterone acetate (MPA) on actin cytoskeleton remodeling and on breast cancer cell movement and invasion. In particular, we characterize the nongenomic signaling cascades implicated in these actions. T47-D breast cancer cells display enhanced horizontal migration and invasion of three-dimensional matrices in the presence of both progestins. Exposure to the hormones triggers a rapid remodeling of the actin cytoskeleton and the formation of membrane ruffles required for cell movement, which are dependent on the rapid phosphorylation of the actin-regulatory protein moesin. The extra-cellular small GTPase RhoA/Rho-associated kinase (ROCK-2) cascade plays central role in progesterone- and MPA-induced moesin activation, cell migration and invasion. In the presence of progesterone, progesterone receptor A (PRA) interacts with the G protein G alpha(13), while MPA drives PR to interact with tyrosine kinase c-Src and to activate phosphatidylinositol-3 kinase, leading to the activation of RhoA/ROCK-2. In conclusion, our findings manifest that progesterone and MPA promote breast cancer cell movement via rapid actin cytoskeleton remodeling, which are mediated by moesin activation. These events are triggered by RhoA/ROCK-2 cascade through partially differing pathways by the two compounds. These results provide original mechanistic explanations for the effects of progestins on breast cancer progression and highlight potential targets to treat endocrine-sensitive breast cancers.     

Family history and breast cancer hormone receptor status in a Spanish cohort

Background

Breast cancer is a heterogenous disease that impacts racial/ethnic groups differently. Differences in genetic composition, lifestyles, reproductive factors, or environmental exposures may contribute to the differential presentation of breast cancer among Hispanic women.

Materials and Methods

A population-based study was conducted in the city of Santiago de Compostela, Spain. A total of 645 women diagnosed with operable invasive breast cancer between 1992 and 2005 participated in the study. Data on demographics, breast cancer risk factors, and clinico-pathological characteristics of the tumors were collected. Hormone receptor negative tumors were compared with hormone receptor postive tumors on their clinico-pathological characteristics as well as risk factor profiles.

Results

Among the 645 breast cancer patients, 78% were estrogen receptor-positive (ER+) or progesterone receptor-positive (PR+), and 22% were ER−&PR−. Women with a family history of breast cancer were more likely to have ER−&PR− tumors than women without a family history (Odds ratio, 1.43; 95% confidence interval, 0.91–2.26). This association was limited to cancers diagnosed before age 50 (Odds ratio, 2.79; 95% confidence interval, 1.34–5.81).

Conclusions

An increased proportion of ER−&PR− breast cancer was observed among younger Spanish women with a family history of the disease.     

RhoA/ROCK-dependent moesin phosphorylation regulates AGE-induced endothelial cellular response

Background

The role of advanced glycation end products (AGEs) in the development of diabetes, especially diabetic complications, has been emphasized in many reports. Accumulation of AGEs in the vasculature triggers a series of morphological and functional changes in endothelial cells (ECs) and induces an increase of endothelial permeability. This study was to investigate the involvement of RhoA/ROCK-dependent moesin phosphorylation in endothelial abnormalities induced by AGEs.

Methods

Using human dermal microvascular endothelial cells (HMVECs), the effects of human serum albumin modified-AGEs (AGE-HSA) on the endothelium were assessed by measuring monolayer permeability and staining of F-actin in HMVECs. Activations of RhoA and ROCK were determined by a luminescence-based assay and immunoblotting. Transfection of recombinant adenovirus that was dominant negative for RhoA (RhoA N19) was done to down-regulate RhoA expression, while adenovirus with constitutively activated RhoA (RhoA L63) was transfected to cause overexpression of RhoA in HMVECs. H-1152 was employed to specifically block activation of ROCK. Co-immunoprecipitation was used to further confirm the interaction of ROCK and its downstream target moesin. To identify AGE/ROCK-induced phosphorylation site in moesin, two mutants pcDNA3/HA-moesinT^558A and pcDNA3/HA-moesinT^558D were applied in endothelial cells.

Results

The results showed that AGE-HSA increased the permeability of HMVEC monolayer and triggered the formation of F-actin-positive stress fibers. AGE-HSA enhanced RhoA activity as well as phosphorylation of ROCK in a time- and dose-dependent manner. Down-regulation of RhoA expression with RhoA N19 transfection abolished these AGE-induced changes, while transfection of RhoA L63 reproduced the AGE-evoked changes. H-1152 attenuated the AGE-induced alteration in monolayer permeability and cytoskeleton. The results also confirmed the AGE-induced direct interaction of ROCK and moesin. Thr558 was further identified as the phosphorylating site of moesin in AGE-evoked endothelial responses.

Conclusion

These results confirm the involvement of RhoA/ROCK pathway and subsequent moesin Thr558 phosphorylation in AGE-mediated endothelial dysfunction.     

The cholesterol metabolite 25-hydroxycholesterol activates estrogen receptor α-mediated signaling in cancer cells and in cardiomyocytes

Background

The hydroxylated derivatives of cholesterol, such as the oxysterols, play important roles in lipid metabolism. In particular, 25-hydroxycholesterol (25HC) has been implicated in a variety of metabolic events including cholesterol homeostasis and atherosclerosis. 25HC is detectable in human plasma after ingestion of a meal rich in oxysterols and following a dietary cholesterol challenge. In addition, the levels of oxysterols, including 25HC, have been found to be elevated in hypercholesterolemic serum.

Methodology/Principal Findings

Here, we demonstrate that the estrogen receptor (ER) α mediates gene expression changes and growth responses induced by 25HC in breast and ovarian cancer cells. Moreover, 25HC exhibits the ERα-dependent ability like 17β-estradiol (E2) to inhibit the up-regulation of HIF-1α and connective tissue growth factor by hypoxic conditions in cardiomyocytes and rat heart preparations and to prevent the hypoxia-induced apoptosis.

Conclusions/Significance

The estrogen action exerted by 25HC may be considered as an additional factor involved in the progression of breast and ovarian tumors. Moreover, the estrogen-like activity of 25HC elicited in the cardiovascular system may play a role against hypoxic environments.     

Microtubules Regulate Migratory Polarity through Rho/ROCK Signaling in T Cells

Background

Migrating leukocytes normally have a polarized morphology with an actin-rich lamellipodium at the front and a uropod at the rear. Microtubules (MTs) are required for persistent migration and chemotaxis, but how they affect cell polarity is not known.

Methodology/Principal Findings

Here we report that T cells treated with nocodazole to disrupt MTs are unable to form a stable uropod or lamellipodium, and instead often move by membrane blebbing with reduced migratory persistence. However, uropod-localized receptors and ezrin/radixin/moesin proteins still cluster in nocodazole-treated cells, indicating that MTs are required specifically for uropod stability. Nocodazole stimulates RhoA activity, and inhibition of the RhoA target ROCK allows nocodazole-treated cells to re-establish lamellipodia and uropods and persistent migratory polarity. ROCK inhibition decreases nocodazole-induced membrane blebbing and stabilizes MTs. The myosin inhibitor blebbistatin also stabilizes MTs, indicating that RhoA/ROCK act through myosin II to destabilize MTs.

Conclusions/Significance

Our results indicate that RhoA/ROCK signaling normally contributes to migration by affecting both actomyosin contractility and MT stability. We propose that regulation of MT stability and RhoA/ROCK activity is a mechanism to alter T-cell migratory behavior from lamellipodium-based persistent migration to bleb-based migration with frequent turning.     

Estrogen receptor alpha expression in podocytes mediates protection against apoptosis in-vitro and in-vivo

Context/Objective

Epidemiological studies have demonstrated that women have a significantly better prognosis in chronic renal diseases compared to men. This suggests critical influences of gender hormones on glomerular structure and function. We examined potential direct protective effects of estradiol on podocytes.

Methods

Expression of estrogen receptor alpha (ERα) was examined in podocytes in vitro and in vivo. Receptor localization was shown using Western blot of separated nuclear and cytoplasmatic protein fractions. Podocytes were treated with Puromycin aminonucleoside (PAN, apoptosis induction), estradiol, or both in combination. Apoptotic cells were detected with Hoechst nuclear staining and Annexin-FITC flow cytometry. To visualize mitochondrial membrane potential depolarization as an indicator for apoptosis, cells were stained with tetramethyl rhodamine methylester (TMRM). Estradiol-induced phosphorylation of ERK1/2 and p38 MAPK was examined by Western blot. Glomeruli of ERα knock-out mice and wild-type controls were analysed by histomorphometry and immunohistochemistry.

Results

ERα was consistently expressed in human and murine podocytes. Estradiol stimulated ERα protein expression, reduced PAN-induced apoptosis in vitro by 26.5±24.6% or 56.6±5.9% (flow cytometry or Hoechst-staining, respectively; both p<0.05), and restored PAN-induced mitochondrial membrane potential depolarization. Estradiol enhanced ERK1/2 phosphorylation. In ERα knockout mice, podocyte number was reduced compared to controls (female/male: 80/86 vs. 132/135 podocytes per glomerulus, p<0.05). Podocyte volume was enhanced in ERα knockout mice (female/male: 429/371 µm³ vs. 264/223 µm³ in controls, p<0.05). Tgfβ1 and collagen type IV expression were increased in knockout mice, indicating glomerular damage.

Conclusions

Podocytes express ERα, whose activation leads to a significant protection against experimentally induced apoptosis. Possible underlying mechanisms include stabilization of mitochondrial membrane potential and activation of MAPK signalling. Characteristic morphological changes indicating glomerulopathy in ERα knock-out mice support the in vivo relevance of the ERα for podocyte viability and function. Thus, our findings provide a novel model for the protective influence of female gender on chronic glomerular diseases.     

MicroRNA-152 mediates DNMT1-regulated DNA methylation in the estrogen receptor α gene

Background

Estrogen receptor α (ERα) has been shown to protect against atherosclerosis. Methylation of the ERα gene can reduce ERα expression leading to a higher risk for cardiovascular disease. Recently, microRNAs have been found to regulate DNA methyltransferases (DNMTs) and thus control methylation status in several genes. We first searched for microRNAs involved in DNMT-associated DNA methylation in the ERα gene. We also tested whether statin and a traditional Chinese medicine (San-Huang-Xie-Xin-Tang, SHXXT) could exert a therapeutic effect on microRNA, DNMT and ERα methylation.

Methodology/Principal Findings

The ERα expression was decreased and ERα methylation was increased in LPS-treated human aortic smooth muscle cells (HASMCs) and the aorta from rats under a high-fat diet. microRNA-152 was found to be down regulated in the LPS-treated HASMCs. We validated that microRNA-152 can knock down DNMT1 in HASMCs leading to hypermethylation of the ERα gene. Statin had no effect on microRNA-152, DNMT1 or ERα expression. On the contrary, SHXXT could restore microRNA-152, decrease DNMT1 and increase ERα expression in both cellular and animal studies.

Conclusions/Significance

The present study showed that microRNA-152 decreases under the pro-atherosclerotic conditions. The reduced microRNA-152 can lose an inhibitory effect on DNA methyltransferase, which leads to hypermethylation of the ERα gene and a decrease of ERα level. Although statin can not reverse these cascade proatherosclerotic changes, the SHXXT shows a promising effect to inhibit this unwanted signaling pathway.     

Gαi2- and Gαi3-specific regulation of voltage-dependent L-type calcium channels in cardiomyocytes

Background

Two pertussis toxin sensitive G_i proteins, G_i2 and G_i3, are expressed in cardiomyocytes and upregulated in heart failure. It has been proposed that the highly homologous G_i isoforms are functionally distinct. To test for isoform-specific functions of G_i proteins, we examined their role in the regulation of cardiac L-type voltage-dependent calcium channels (L-VDCC).

Methods

Ventricular tissues and isolated myocytes were obtained from mice with targeted deletion of either Gα_i2 (Gα_i2 ^−/−) or Gα_i3 (Gα_i3 ^−/−). mRNA levels of Gα_i/o isoforms and L-VDCC subunits were quantified by real-time PCR. Gα_i and Ca_vα₁ protein levels as well as protein kinase B/Akt and extracellular signal-regulated kinases 1/2 (ERK1/2) phosphorylation levels were assessed by immunoblot analysis. L-VDCC function was assessed by whole-cell and single-channel current recordings.

Results

In cardiac tissue from Gα_i2 ^−/− mice, Gα_i3 mRNA and protein expression was upregulated to 187±21% and 567±59%, respectively. In Gα_i3 ^−/− mouse hearts, Gα_i2 mRNA (127±5%) and protein (131±10%) levels were slightly enhanced. Interestingly, L-VDCC current density in cardiomyocytes from Gα_i2 ^−/− mice was lowered (−7.9±0.6 pA/pF, n = 11, p<0.05) compared to wild-type cells (−10.7±0.5 pA/pF, n = 22), whereas it was increased in myocytes from Gα_i3 ^−/− mice (−14.3±0.8 pA/pF, n = 14, p<0.05). Steady-state inactivation was shifted to negative potentials, and recovery kinetics slowed in the absence of Gα_i2 (but not of Gα_i3) and following treatment with pertussis toxin in Gα_i3 ^−/−. The pore forming Ca_vα₁ protein level was unchanged in all mouse models analyzed, similar to mRNA levels of Ca_vα₁ and Ca_vβ₂ subunits. Interestingly, at the cellular signalling level, phosphorylation assays revealed abolished carbachol-triggered activation of ERK1/2 in mice lacking Gα_i2.

Conclusion

Our data provide novel evidence for an isoform-specific modulation of L-VDCC by Gα_i proteins. In particular, loss of Gα_i2 is reflected by alterations in channel kinetics and likely involves an impairment of the ERK1/2 signalling pathway.     

Functional Adaptation in Female Rats: The Role of Estrogen Signaling

Background

Sex steroids have direct effects on the skeleton. Estrogen acts on the skeleton via the classical genomic estrogen receptors alpha and beta (ERα and ERβ), a membrane ER, and the non-genomic G-protein coupled estrogen receptor (GPER). GPER is distributed throughout the nervous system, but little is known about its effects on bone. In male rats, adaptation to loading is neuronally regulated, but this has not been studied in females.

Methodology/Principal Findings

We used the rat ulna end-loading model to induce an adaptive modeling response in ovariectomized (OVX) female Sprague-Dawley rats. Rats were treated with a placebo, estrogen (17β-estradiol), or G-1, a GPER-specific agonist. Fourteen days after OVX, rats underwent unilateral cyclic loading of the right ulna; half of the rats in each group had brachial plexus anesthesia (BPA) of the loaded limb before loading. Ten days after loading, serum estrogen concentrations, dorsal root ganglion (DRG) gene expression of ERα, ERβ, GPER, CGRPα, TRPV1, TRPV4 and TRPA1, and load-induced skeletal responses were quantified. We hypothesized that estrogen and G-1 treatment would influence skeletal responses to cyclic loading through a neuronal mechanism. We found that estrogen suppresses periosteal bone formation in female rats. This physiological effect is not GPER-mediated. We also found that absolute mechanosensitivity in female rats was decreased, when compared with male rats. Blocking of adaptive bone formation by BPA in Placebo OVX females was reduced.

Conclusions

Estrogen acts to decrease periosteal bone formation in female rats in vivo. This effect is not GPER-mediated. Gender differences in absolute bone mechanosensitivity exist in young Sprague-Dawley rats with reduced mechanosensitivity in females, although underlying bone formation rate associated with growth likely influences this observation. In contrast to female and male rats, central neuronal signals had a diminished effect on adaptive bone formation in estrogen-deficient female rats.     

Subtype specific elevated expression of hyaluronidase-1 (HYAL-1) in epithelial ovarian cancer

Background

Epithelial ovarian cancer (EOC) is morphologically heterogeneous being classified as serous, endometrioid, clear cell, or mucinous. Molecular genetic analysis has suggested a role for tumor suppressor genes located at chromosome 3p in serous EOC pathogenesis. Our objective was to evaluate the expression of HYAL1, located at chromosome 3p21.3, in these EOC subtypes, and to investigate its correlation with the expression of steroid hormone receptors.

Methodology/Principal Findings

We determined the mRNA expression of HYAL1, estrogen receptor (ER)-α, ERβ and progesterone receptor (PR) in EOC tumor samples and cell lines using quantitative RT-PCR. We also examined the expression of these genes in a publicly available microarray dataset. HYAL-1 enzyme activity was measured in EOC cell lines and in plasma samples from patients. We found that HYAL1 mRNA expression was elevated in clear cell and mucinous EOC tissue samples, but not in serous and endometrioid samples, normal ovaries or benign tumors. Similar results were obtained by two different techniques and with tissue sample cohorts from two independent institutions. Concordantly, HYAL1 mRNA levels and enzymatic activity were elevated only in EOC cell lines derived from clear cell and mucinous subtypes. We also showed that HYAL1 mRNA was inversely correlated to that of ERα specifically in clear cell and mucinous EOCs. Additionally, ectopic expression of ERα in a clear cell EOC cell line (ER- and PR-negative) induced 50% reduction of HYAL1 mRNA expression, supporting a role of ERα in HYAL1 gene regulation. Significantly, HYAL-1 activity was also high in the plasma of patients with these EOC subtypes.

Conclusions/Significance

This is the first report showing high HYAL-1 levels in EOC and demonstrating HYAL1 gene repression by ERα. Our results identify Hyaluronidase-1 as a potential target/biomarker for clear cell and mucinous EOCs and especially in tumors with low ERα levels.     

Complex Regulation of PKCβ2 and PDK-1/AKT by ROCK2 in Diabetic Heart

Objectives

The RhoA/ROCK pathway contributes to diabetic cardiomyopathy in part by promoting the sustained activation of PKCβ2 but the details of their interaction are unclear. The purpose of this study was to investigate if over-activation of ROCK in the diabetic heart leads to direct phosphorylation and activation of PKCβ2, and to determine if their interaction affects PDK-1/Akt signaling.

Methods

Regulation by ROCK of PKCβ2 and related kinases was investigated by Western blotting and co-immunoprecipitation in whole hearts and isolated cardiomyocytes from 12 to 14-week diabetic rats. Direct ROCK2 phosphorylation of PKCβ2 was examined in vitro. siRNA silencing was used to confirm role of ROCK2 in PKCβ2 phosphorylation in vascular smooth muscle cells cultured in high glucose. Furthermore, the effect of ROCK inhibition on GLUT4 translocation was determined in isolated cardiomyocytes by confocal microscopy.

Results

Expression of ROCK2 and expression and phosphorylation of PKCβ2 were increased in diabetic hearts. A physical interaction between the two kinases was demonstrated by reciprocal immunoprecipitation, while ROCK2 directly phosphorylated PKCβ2 at T641 in vitro. ROCK2 siRNA in vascular smooth muscle cells or inhibition of ROCK in diabetic hearts reduced PKCβ2 T641 phosphorylation, and this was associated with attenuation of PKCβ2 activity. PKCβ2 also formed a complex with PDK-1 and its target AKT, and ROCK inhibition resulted in upregulation of the phosphorylation of PDK-1 and AKT, and increased translocation of glucose transporter 4 (GLUT4) to the plasma membrane in diabetic hearts.

Conclusion

This study demonstrates that over-activation of ROCK2 contributes to diabetic cardiomyopathy by multiple mechanisms, including direct phosphorylation and activation of PKCβ2 and interference with the PDK-1-mediated phosphorylation and activation of AKT and translocation of GLUT4. This suggests that ROCK2 is a critical node in the development of diabetic cardiomyopathy and may be an effective target to improve cardiac function in diabetes.     

Subcellular localization of total and activated Src kinase in African American and Caucasian breast cancer

Background

Src, a non-receptor tyrosine kinase is elevated in cancer with expression and activity correlated with cell proliferation, adhesion, survival, motility, metastasis and angiogenesis. There is limited data on Src expression and subcellular localization in breast cancer and no information about expression in racial/ethnic groups.

Methodology/Principal Findings

The present study evaluated Src expression, activity, and subcellular localization in triple negative breast cancer (TNBC) and ERα positive breast cancer (ER+BC), cancer tissue and adjacent normal epithelial ducts, and Caucasian and African American cases. 79 paraffin embedded breast carcinoma cases were obtained from Tulane University Hospital between 2007–2009. 39 cases represented TNBC (33-African Americans, 4-Caucasians, 2-unknowns) and 40 cases represented ER+BC (21-African Americans, 16-Caucasians, 3-unknowns). Immunohistochemistry was used to measure staining distribution and intensity of total Src and activated phospho-SrcY416 (p-Y416Src) in carcinoma tissue and adjacent normal mammary ducts. In TNBC and ER+BC, total Src was significantly higher in cancer compared to adjacent normal ducts (P<0.0001) in both cell membrane and cytoplasm. In membranes, p-Y416Src was elevated in cancer compared to normal ducts. Total Src in the tumor cytoplasm was significantly higher in TNBC compared to ER+BC (P = 0.0028); conversely, p-Y416Src in the tumor cell membranes was higher in TNBC compared to ER+BC (P = 0.0106). Comparison between African American (n = 21) and Caucasian ER+BC (n = 16) revealed no significant difference in expression and localization of total Src and p-Y416Src. TNBC cases positive for lymph node metastasis showed elevated membrane p-Y416Src compared to lymph node negative TNBC (P = 0.027).

Conclusion/Significance

Total Src and p-Y416Src were expressed higher in cancer compared to adjacent normal ducts. Cytoplasmic total Src and membrane p-Y416Src were significantly higher in TNBC compared to ER+BC. TNBC cases with lymph node metastasis showed elevated membrane p-Y416Src. Taken together, Src was elevated in the membrane and cytoplasm of more aggressive TNBC.     

Specific interaction of Gαi3 with the Oa1 G-protein coupled receptor controls the size and density of melanosomes in retinal pigment epithelium

Background

Ocular albinism type 1, an X-linked disease characterized by the presence of enlarged melanosomes in the retinal pigment epithelium (RPE) and abnormal crossing of axons at the optic chiasm, is caused by mutations in the OA1 gene. The protein product of this gene is a G-protein-coupled receptor (GPCR) localized in RPE melanosomes. The Oa1-/- mouse model of ocular albinism reproduces the human disease. Oa1 has been shown to immunoprecipitate with the Gαi subunit of heterotrimeric G proteins from human skin melanocytes. However, the Gαi subfamily has three highly homologous members, Gαi1, Gαi2 and Gαi3 and it is possible that one or more of them partners with Oa1. We had previously shown by in-vivo studies that Gαi3-/- and Oa1-/- mice have similar RPE phenotype and decussation patterns. In this paper we analyze the specificity of the Oa1-Gαi interaction.

Methodology

By using the genetic mouse models Gαi1-/-, Gαi2-/-, Gαi3-/- and the double knockout Gαi1-/-, Gαi3-/- that lack functional Gαi1, Gαi2, Gαi3, or both Gαi1 and Gαi3 proteins, respectively, we show that Gαi3 is critical for the maintenance of a normal melanosomal phenotype and that its absence is associated with changes in melanosomal size and density. GST-pull-down and immunoprecipitation assays conclusively demonstrate that Gαi3 is the only Gαi that binds to Oa1. Western blots show that Gαi3 expression is barely detectable in the Oa1-/- RPE, strongly supporting a previously unsuspected role for Gαi3 in melanosomal biogenesis.

Conclusion

Our results identify the Oa1 transducer Gαi3 as the first downstream component in the Oa1 signaling pathway.     

17β-Hydroxysteroid dehydrogenase type 14 is a predictive marker for tamoxifen response in oestrogen receptor positive breast cancer

Introduction

17β-hydroxysteroid dehydrogenases (17βHSDs) are important enzymes regulating the pool of bioactive steroids in the breast. The current study was undertaken in order to evaluate implications of 17βHSD14 in breast cancer, measuring 17βHSD14 protein expression in breast tumours.

Methods

An antibody targeting the 17βHSD14 antigen was generated and validated using HSD17B14-transfected cells and a peptide-neutralising assay. Tissue microarrays with tumours from 912 post-menopausal women diagnosed with lymph node-negative breast cancer, and randomised to adjuvant tamoxifen or no endocrine treatment, were analysed for 17βHSD14 protein expression with immunohistochemistry.

Results

Results were obtained from 847 tumours. Patients with oestrogen positive tumours with high 17βHSD14 expression had fewer local recurrences when treated with tamoxifen (HR 0.38; 95% C.I. 0.19–0.77, p = 0.007) compared to patients with lower tumoural 17βHSD14 expression, for whom tamoxifen did not reduce the number of local recurrences (HR 1.19; 95% C.I. 0.54–2.59; p = 0.66). No prognostic importance of 17βHSD14 was seen for systemically untreated patients.

Conclusions

Using a highly specific validated antibody for immunohistochemical analysis of a large number of breast tumours, we have shown that tumoural expression levels of 17βHSD14 can predict the outcome of adjuvant tamoxifen treatment in terms of local recurrence-free survival in patients with lymph node-negative ER+ breast cancer. The results need be verified to confirm any clinical relevance.