ER Markers Research Articles

9q31.2-rs865686 as a Susceptibility Locus for Estrogen Receptor-Positive Breast Cancer: Evidence from the Breast Cancer Association Consortium

Our recent genome-wide association study identified a novel breast cancer susceptibility locus at 9q31.2 (rs865686). To further investigate the rs865686-breast cancer association, we conducted a replication study within the Breast Cancer Association Consortium, which comprises 37 case-control studies (48,394 cases, 50,836 controls). This replication study provides additional strong evidence of an inverse association between rs865686 and breast cancer risk [study-adjusted per G-allele OR, 0.90; 95% confidence interval (CI), 0.88; 0.91, P = 2.01 × 10(-29)] among women of European ancestry. There were ethnic differences in the estimated minor (G)-allele frequency among controls [0.09, 0.30, and 0.38 among, respectively, Asians, Eastern Europeans, and other Europeans; P for heterogeneity (P(het)) = 1.3 × 10(-143)], but no evidence of ethnic differences in per allele OR (P(het) = 0.43). rs865686 was associated with estrogen receptor-positive (ER(+)) disease (per G-allele OR, 0.89; 95% CI, 0.86-0.91; P = 3.13 × 10(-22)) but less strongly, if at all, with ER-negative (ER(-)) disease (OR, 0.98; 95% CI, 0.94-1.02; P = 0.26; P(het) = 1.16 × 10(-6)), with no evidence of independent heterogeneity by progesterone receptor or HER2 status. The strength of the breast cancer association decreased with increasing age at diagnosis, with case-only analysis showing a trend in the number of copies of the G allele with increasing age at diagnosis (P for linear trend = 0.0095), but only among women with ER(+) tumors. This study is the first to show that rs865686 is a susceptibility marker for ER(+) breast cancer. The findings further support the view that genetic susceptibility varies according to tumor subtype.

Impairment of GABAB receptor dimer by endogenous 14-3-3ζ in chronic pain conditions

In the central nervous system, the inhibitory GABAB receptor is the archetype of heterodimeric G protein-coupled receptors (GPCRs). However, the regulation of GABAB dimerization, and more generally of GPCR oligomerization, remains largely unknown. We propose a novel mechanism for inhibition of GPCR activity through de-dimerization in pathological conditions. We show here that 14-3-3ζ, a GABAB1-binding protein, dissociates the GABAB heterodimer, resulting in the impairment of GABAB signalling in spinal neurons. In the dorsal spinal cord of neuropathic rats, 14-3-3ζ is overexpressed and weakens GABAB inhibition. Using anti-14-3-3ζ siRNA or competing peptides disrupts 14-3-3ζ/GABAB1 interaction and restores functional GABAB heterodimers in the dorsal horn. Importantly, both strategies greatly enhance the anti-nociceptive effect of intrathecal Baclofen in neuropathic rats. Taken together, our data provide the first example of endogenous regulation of a GPCR oligomeric state and demonstrate its functional impact on the pathophysiological process of neuropathic pain sensitization.

Intracellular Trafficking of FXYD1 (Phospholemman) and FXYD7 Proteins in Xenopus Oocytes and Mammalian Cells

FXYD proteins are a group of short single-span transmembrane proteins that interact with the Na(+)/K(+) ATPase and modulate its kinetic properties. This study characterizes intracellular trafficking of two FXYD family members, FXYD1 (phospholemman (PLM)) and FXYD7. Surface expression of PLM in Xenopus oocytes requires coexpression with the Na(+)/K(+) ATPase. On the other hand, the Na(+)/Ca(2+) exchanger, another PLM-interacting protein could not drive it to the cell surface. The Na(+)/K(+) ATPase-dependent surface expression of PLM could be facilitated by either a phosphorylation-mimicking mutation at Thr-69 or a truncation of three terminal arginine residues. Unlike PLM, FXYD7 could translocate to the cell surface of Xenopus oocytes independently of the coexpression of α1β1 Na(+)/K(+) ATPase. The Na(+)/K(+) ATPase-independent membrane translocation of FXYD7 requires O-glycosylation of at least two of three conserved threonines in its ectodomain. Subsequent experiments in mammalian cells confirmed the role of conserved extracellular threonine residues and demonstrated that FXYD7 protein, in which these have been mutated to alanine, is trapped in the endoplasmic reticulum and Golgi apparatus.

DNA repair metagene signature as a prognostic and predictive factor in molecular breast cancer subtypes.

1012 Background: We aimed to assess the prognostic and predictive role of DNA repair genes in breast cancer (bc) molecular subtypes. Methods: We evaluated Affymetrix gene expression profiling from untreated N- patients (N = 684), neoadjuvant treated tumors with taxanes- (N = 320), anthracyclines- (N = 211), and cisplatin- (N = 22) containing regimens. We assessed within 3 BC molecular subgroups (ER+/HER2-, HER2+, and ER-/HER2-) bimodality distribution, prognosis by association with distant relapse (N = 454, N = 105, and N = 125) and predictive value for likelihood of pathological complete response (pCR) (N = 208, N = 105, and N = 240). Moreover, we explored the function of relevant genes in BC cell lines. Results: Three genes (ERCC2, XRCC3, and RECQL4) showed bimodality in each bc subtype. Eight genes were associated with poor prognosis (including RECQL4) and 1 gene with good prognosis (ATM) [P < .0001] only in ER+/HER2- tumors. Our results suggest a subtype and treatment specific association with pCR although they did not satisfy stringent criteria for false discovery correction. In ER-/HER2- mismatch repair (MR) (MSH2 and MSH6) and MTMR15 genes were associated with response and resistance to taxane-containing regimens, respectively. TOP2A was the only gene associated with response to anthracycline but not taxanes in HER2+ tumors. RECQL4 had a positive trend with higher pCR in both ER+ and ER-/HER2- tumors. In in vitro studies we found that RECQL4 interacts with PARP1 and that the expression of these genes was correlated with sensitivity to chemotherapy and PARP inhibitors. Conclusions: We identified MR genes as potential predictive markers of response to taxanes-based regimens in ER-/HER2-. A novel gene RECQL4 showed bimodal distribution, prognostic value, and a trend for predictive association with response to taxanes-based chemotherapy, which was also confirmed by in vitro analysis. ATM deserves further evaluation as prognostic marker in ER+/HER2-.

Trafficking of renal betaine/GABA transporter (BGT1) is disrupted by mutation at T40

Cells in renal medulla accumulate several osmolytes to survive hypertonicity and this is reproduced in renal MDCK cells. In response to hypertonic stress (Hyp, 500 mOsm) for 24 h there is upregulation of BGT1 mRNA and protein synthesis followed by trafficking to the plasma membrane (PM) and a marked increase in betaine transport. PM trafficking motifs reside near the C‐terminus of BGT1 and truncation at K560 resulted in a protein that remained intracellular during Hyp stress, as expected. The K560Δ mutant expressed in MDCK cells was colocalized with an ER marker but not markers for endosomes or Golgi. Surprisingly, substitution of A at T40 of native BGT1, to block phosphorylation, also prevented trafficking to the PM during Hyp stress. The T40A mutant was not retained in the ER and colocalized with markers for Golgi and endosomes suggesting, indirectly, that it was not misfolded. Mutation of T40 to E or D, to mimic phosphorylation, restored normal trafficking to the PM. HEK cells transfected with K560Δ or T40A mutants had 10% of GABA transport activity of native BGT1, but normal transport activity was restored in cells expressing T40E. Data suggest that normal BGT1 trafficking (or PM retention) requires both C‐terminal motifs and phosphorylation at T40.

Antiprogestins in breast cancer treatment: are we ready?

Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death in females worldwide. It is accepted that breast cancer is not a single disease, but instead constitutes a spectrum of tumor subtypes with distinct cellular origins, somatic changes, and etiologies. Molecular gene expression studies have divided breast cancer into several categories, i.e. basal-like, ErbB2 enriched, normal breast-like (adipose tissue gene signature), luminal subtype A, luminal subtype B, and claudin-low. Chances are that as our knowledge increases, each of these types will also be subclassified. More than 66% of breast carcinomas express estrogen receptor alpha (ERα) and respond to antiestrogen therapies. Most of these ER+ tumors also express progesterone receptors (PRs), the expression of which has been considered as a reliable marker of a functional ER. In this paper we will review the evidence suggesting that PRs are valid targets for breast cancer therapy. Experimental data suggest that both PR isoforms (A and B) have different roles in breast cancer cell growth, and antiprogestins have already been clinically used in patients who have failed to other therapies. We hypothesize that antiprogestin therapy may be suitable for patients with high levels of PR-A. This paper will go over the experimental evidence of our laboratory and others supporting the use of antiprogestins in selected breast cancer patients.

P2-12-04: RACGAP1 mRNA Assay Outperforms Ki-67 as a Proliferation Marker in the FinHer Study Cohort.

Abstract Background: Molecular subtyping of breast cancer has become an integral part of standard evaluation of breast cancer patients. Their assessment requires combining data from analyses on ER, PR, HER2 and cell proliferation markers. However, their immunohistochemical (IHC) testing carries an up to 20% risk of erroneous results. Similarly, assessment of cell proliferation by Ki-67 staining is hampered by lack of standardization of laboratory methods and agreement on cut-offs. Here we tested the prognostic value of objective quantitation of ESR1, PgR, HER2 and the proliferation markers RACGAP1 using RT-qPCR and compared the results with local and central IHC assessments. Methods: RNA was extracted from FFPE tumor tissue of 917 patients who participated in the FinHer trial. ESR1, PgR, HER2 and RACGAP1 mRNA expression were measured using RT-qPCR. The molecular subtypes (luminal, HER2−enriched and triple-negative) were determined. Prognostic significance of proliferation markers was assessed using univariate and multivariate analyses. The RT-qPCR results were compared with local and central IHC results. Results: HER2 mRNA showed a bimodal distribution with 197 (21.4%) out of the 917 tumors being above the predefined cut-off. HER2 mRNA expression increased in parallel with HER protein expression. Overall concordance of HER2 mRNA testing with central IHC and CISH was good, while local IHC testing suffered higher false positive rates. RACGAP1 mRNA expression was the greater the higher the histological grade. ESR1 and PgR mRNA correlated negatively with the histological grade (r=-0.38 and r=-0.33; p<0.0001), whereas HER2 and RACGAP1 mRNA were correlated positively (r=0.10 and r=0.49; p=0.002 and p<0.0001, respectively). RACGAP1 mRNA was negatively associated with ESR1 and PgR mRNA (r=-0.17 and r=-0.26, respectively; p<0.0001 for each). Molecular subtypes determined by RT-qPCR using predefined cut-off values were highly prognostic for overall survival (OS) (p<0.001). The 5-year OS rate for patients with luminal cancer was 94% and 86% for HER2−enriched cancer and 84% for triple-negative cancer. In the subset of luminal tumors, high expression of RACGAP1 identified a population of patients who were at a high risk of death (5-year OS 82% versus 95%; p<0.0001). In a multivariate analysis RACGAP1 mRNA expression, nodal status and chemotherapy type were independent prognostic factors, whereas IHC of ER, PgR, Ki-67 and histological grade were not significant. Conclusions: Molecular subtyping of breast cancer by RT-qPCR using RNA isolated from FFPE tissue proved successfully in this large patient cohort. RACGAP1 mRNA expression distinguished high and low risk luminal breast cancers. In a multivariate analysis mRNA-based molecular markers outperformed the immunohistochemical markers ER, PgR and Ki-67. Of note, quantitative assessment of the proliferation marker RACGAP1 was superior to semiquantitative assessment of Ki-67 from routine FFPE tissues using IHC. We conclude that quantitative assessment of ESR1, PgR, HER2 and RACGAP1 mRNA by RT-qPCR is a robust and reproducible method to assess these key tumor biological factors from archival FFPE tumor tissue. RACGAP1 is novel cell proliferation marker in breast cancer that warrants further validation. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-12-04.

Abstract P045: Two Forms of Autophagy in the Heart: Constitutive Autophagy via an LC3-Dependent Pathway and Starvation-Induced Autophagy via a Rab9-Dependent Pathway

Autophagy is an intracellular bulk degradation process in which cytosolic proteins/organelles are sequestered into double-membrane vesicles termed autophagosomes to be fused with lysosomes for degradation. Recent evidence suggests that an alternative form of autophagy, which utilizes distinct sources of membrane for autophagosome formation, may exist. Whether alternative autophagy exists in the heart is unknown. Cardiac-specific Atg7 -KO ( Atg7 -CKO) and systemic Ulk1 -KO mice were subjected to starvation for 48 hours. LC3-II expression and p62 degradation were increased in wild-type (WT) mice after starvation (ST), indicating increased autophagy. In Atg7 -CKO mice, LC3-II expression was reduced and p62 was markedly increased at baseline and in response to ST, indicating inhibition of LC3-dependent autophagy. In contrast, in Ulk1 -KO mice, neither LC3-II nor p62 was affected at baseline or in response to ST. However, autolysosome formation evaluated by Lamp2/Rab9 staining, indicators of unconventional autophagy, was abolished. Furthermore, the level of polyubiquitinated protein was increased after ST. Echocardiographic analyses showed that fractional shortening (FS) was maintained after ST in WT mice (38%). However, in Atg7 -CKO mice, FS deteriorated at baseline (22%) but was not further affected by ST (20%). In contrast, in Ulk1 -KO mice, FS was relatively preserved at baseline (32%) but remarkably decreased during ST (19%). After glucose deprivation (GD) GFP-LC3 was co-localized with calnexin (ER marker) in Ad-sh-Control- and Ad-sh-Ulk1-transduced, but not in Ad-sh-Atg7-transduced, cultured cardiomyocytes (CMs) in vitro. In contrast, Lamp2 was co-localized with TGN46 (Golgi marker) in Ad-sh-Control and Ad-sh-Atg7-transduced, but not in Ad-sh-Ulk1-transduced, CMs after GD. These results suggest that the autophagosomes are derived from ER in LC3-dependent autophagy and Golgi in Rab9-dependent autophagy in CMs. Atg7 is required for maintaining cardiac function at baseline in an LC3-dependent manner, whereas Ulk1 is required for preserving cardiac function during ST, possibly through stimulation of Rab9-dependent autophagy. These results support the presence of alternative autophagy in the heart.

Processing of α-Defensin in Subcellular Fractionations of Promyelocytic Cell Line PLB-985

Abstract 2163 Introduction:α-defensins – in neutrophils also known as human neutrophil peptides (HNPs) – are the dominating proteins of neutrophil azurophil granules. They are synthesized in promyelocytes and myelocytes as proHNP, but only processed to mature HNP in promyelocytes. Yet, the mechanisms underlying the posttranslational processing of neutrophil defensins remain unsettled. Thus neither the processing enzyme of proHNP nor the localization of processing has been identified. We studied processing of proHNP in subcellular fractions of the human promyelocytic cell line PLB-985 and identified fractions in which processing occurs. Furthermore, different proteinase inhibitors were employed to test their ability to inhibit PLB-985 proteases from processing proHNP. Methods:Subcellular fractionation of PLB-985 cells was performed by nitrogen cavitation and sedimentation of the postnuclear supernatant on a two-layer Percoll density gradient. Fractions of 1 ml each were collected from the bottom of the gradient using a fraction collector. Myeloperoxidase (MPO) and calnexin were measured by western blotting in all fractions and used as markers for azurophil granules and endoplasmic reticulum respectively. 35S-labelled proHNP was obtained by affinity chromatography on medium from a prodefensin expressing cells line incubated overnight in medium containing 35S-cysteine/methionine. 35S-labelled proHNP was incubated with subcellular fractions from PLB-985 cells, subjected to SDS-Tricine-PAGE, and visualized by fluorography. Inhibition of proHNP processing was probed with a battery of protease inhibitors: PMSF, aprotinin, leupeptin, EDTA, pepstatin A, chymostatin, and E-64. For biosynthesis studies, PLB-985 cells were incubated with in medium containing 35S-cysteine/methionine before subcellular fractionation, immunoprecipitation, SDS-Tricine-PAGE, and fluorography. Results:35S-labelled proHNP was incubated with subcellular fractions from PLB-985 cells and tested by SDS-Tricine-PAGE and fluorography. Most extensive processing occurred in fractions positive for both calnexin (marker for ER) and MPO, which is actively synthesized in the promyelocyte. The heavier granules fractions were only positive for MPO and showed a lesser degree of processing. To test where proHNP is processed in promyelocytes, PLB-985 cells were pulsed overnight in medium containing 35S-cysteine/methionine. After 20h cells, subcellular fractionation was performed and fractions immunoprecipitated with an anti-defensin antibody coupled to Sepharose beads. Immunoprecipitate was subjected to SDS-Tricine-PAGE and fluorography, which showed proHNP in fractions with both calnexin and MPO, but not in heavier fractions representing granules. Processed HNP was present in all proHNP-containing fractions as well as in the protein dense granule fractions. 35S-labelled proHNP was incubated with PLB-985 lysate and protease inhibitors to test, which protease classes in the promyelocyte are capable of proHNP processing. Of the inhibitors tested, only the serine protease inhibitor PMSF and to a minor degree the aspartyl peptidase inhibitor pepstatin A showed significant inhibition of proHNP processing. Conclusion:We studied posttranslational processing of proHNP using a human promyelocytic cell line model. proHNP processing proteases were identified in pre-granular structures of the biosynthetic pathway as well as in granule fractions. Radiolabeling showed processed HNP early in the biosynthetic pathway as well as in granules, but proHNP was only detected in pre-granule fractions consistent with proHNP being processed before localization to granules. This is in accordance with recent findings indicating that processed HNP binds to the proteoglycan serglycin, which is primarily localized in the Golgi apparatus. Our findings showed the serine protease inhibitor PMSF to be a strong inhibitor of proHNP processing in the promyelocyte. This is in accordance with previous findings of the three major proteases of the promyelocyte – the serine proteases neutrophil elastase, cathepsin G, and proteinase 3 – being able to process proHNP in vitro. Disclosures:No relevant conflicts of interest to declare.

Changes in the ER, PgR, HER2, p53 and Ki-67 biological markers between primary and recurrent breast cancer: discordance rates and prognosis

BackgroundIn breast cancer, ER/PgR, HER2, and Ki-67 are important biological markers for predicting prognosis and making effective treatment decisions. In addition, changes in markers due to relapse are also clinically experienced; however, the frequency and clinical significance are still not fully understood. Thus, changes in markers and their correlations with prognosis were investigated.Patients and MethodsOut of the patients with relapse from 1997 to March 2011, there were 97 consecutive patients from whom the lesion was resected and evaluated by immunostaining. The biopsy sites were chest wall, lymph node, ipsilateral breast tumor recurrence, lungs, bones, ovaries and brain. The markers sought were ER, PgR, HER2, p53 and Ki-67.ResultsThe hormone receptor positive rate from the primary tumor to recurrence decreased from 63.9% to 57.7% and from 56.7% to 43.3% for ER and PgR, respectively. Changes in the positive/negative evaluation were seen at the rate of 10.3% and 25.8% for ER and PgR, respectively. The Ki-67 index increased significantly from a mean of 29.1% at primary tumor to 36.3% at relapse. When divided into 2 groups (< 50% and ≥50%), changes were seen in 24.7%. On the other hand, the rates of changes in HER2 and p53 positivity were 14.4% and 12.4%. The changes in subtypes were seen in 25%, however, the lowest rate of change was seen in the triple negative cases. Although there was no notable difference in the rate of change between disease-free interval (DFI) and PgR, Ki-67, p53 and HER2, there was a significant difference in the change rates in the ER. A multivariate analysis revealed that the status of distant metastasis and PgR level at relapse, and Ki-67 levels at primary tumor were all significant factors.ConclusionEstrogen receptor and PgR decreased while Ki-67 increased due to relapse; however, the rate of change was high for PgR and Ki-67. Change in the subtypes was seen in 25%. In addition, PgR at relapse and Ki-67 at primary tumor were significant factors for post-relapse prognosis while PgR becoming negative was a poor prognostic factor. These findings are important for making effective treatment decisions.

A Severe Form of Abetalipoproteinemia Caused by New Splicing Mutations of Microsomal Triglyceride Transfer Protein

The original article to which this Erratum refers was published in Human Mutation 32: 751–759 (2011). In this published article from Volume 32, Issue 7, Figures 1-5 do not appear clearly. Included in this erratum are high-resolution images for these figures. Diagnosis of abetalipoproteinemia. A: Upper gastrointestinal endoscopy performed in patient AM revealed a white aspect of duodenal villi, characteristic of lipid storage. B: Biopsy was performed in duodenum and processed for hematoxylin/eosin-staining. The arrow points at the presence of lipid droplets within the enterocytes. C, D: Serum from control or patient (AM) was analyzed using FPLC system. Lipoproteins were separated and total cholesterol (C) and triglycerides (D) were measured. Profiles are shown for control (Ctrl) and patient (AM). Proteins were only showed for patient in C. Note the difference of y-axis (lipid content) between control and patient. Sequence analysis of mutant MTTP transcription products. A, C: After mRNA extraction and reverse transcription from control and patient (AM) EBV-immortalized lymphocytes, PCR was performed to amplify exon 6 (A) and exon 10 (C) with 5′ and 3′ flanking regions. PCR products were analyzed by migration on agarose gel. Arrowhead points at the lower band found in patient compared to the control. B, D: Upper bands in the control and lower bands in patients were purified and then cloned into pCR2.1 vector before sequencing. In B, electrophoregrams showed the exon 6 and 5′ and 3′ flanking regions with exon 5 and 7 in the control. In the patient, the exon 6 is deleted, leading to a frameshift and a premature stop codon. In D, similarly, electrophoregrams showed the exon 10 and 5′ and 3′ flanking regions with exon 9 and 11 in the control. In the patient, the exon 10 is deleted. E: The amino acid sequences corresponding to exons 6 and 10 are shown underlined and in blue in the normal MTTP sequence. // determined the deletion of exons 6 or 10 in each mutant. For Δ6-MTTP, the frame-shift mutation results in an abnormal sequence described in red and in italic. MTTP expression and activity in duodenal biopsy. A: Duodenal biopsy from control (Ctrl) or patient (AM) and Caco-2 cell lysate were analyzed by SDS gel electrophoresis and Western blotting with antibodies against MTTP. B: The triglyceride transfer activity of MTTP was measured on duodenal biopsy from control or patient (AM). Values are expressed as the mean of three independent experiments; standard errors are indicated. Interaction of WT and mutant MTTP with PDI. A, B: HeLa cells were transfected or not with WT-MTTP, Δ6-MTTP or Δ10-MTTP tagged with EGFP (A) or 2xMyc (B). Cell lysates (100 µg) were analyzed by SDS gel electrophoresis and Western blotting with antibodies against GFP (A), Myc (B) and Rab5 as loading control. Arrowheads point at WT-MTTP, Δ6-MTTP or Δ10-MTTP and arrows point at nonspecific band (ns). Blots in B were scanned and the quantification is shown in C. Each experiment was repeated at least three times, and (C) shows a representative example. D, E: HeLa cells were transfected with 2xMyc-tagged WT-MTTP, Δ6-MTTP or Δ10-MTTP. Cell lysates (Lys) were subjected to immunoprecipitation with (+) or without (−) the anti-myc antibody (D) or the anti-PDI antibody (E). Analysis was performed by SDS gel electrophoresis and Western blotting with antibodies against Myc or PDI. Arrowheads point at immunoprecipitated WT-MTTP, Δ6-MTTP or Δ10-MTTP (D) or PDI (E) and arrows point at nonspecific band (ns). IgG HC represents heavy chain of anti-PDI antibody used for immunoprecipitation. Localization of WT and mutant MTTP and ER marker. A: HeLa cells were cotransfected with GFP-tagged WT-MTTP, Δ6-MTTP or Δ10-MTTP and a vector encoding DsRed-ER marker. Cells were then processed for fluorescence analysis. B: HeLa cells were transfected with GFP-tagged WT-MTTP, Δ6-MTTP or Δ10-MTTP and were then processed for immunofluorescence using anti-PDI antibody. Bar: 10 µm. The publisher regrets these errors.

A novel conserved targeting motif found in ABCA transporters mediates trafficking to early post-Golgi compartments

The ATP binding cassette, class A (ABCA) proteins are homologous polytopic transmembrane transporters that function as lipid pumps at distinct subcellular sites in a variety of cells. Located within the N terminus of these transporters, there exists a highly conserved xLxxKN motif of unknown function. To define its role, human ABCA3 was employed as a primary model representing ABCA transporters, while mouse ABCA1 was utilized to support major findings. Transfection studies showed colocalization of both transporters with surfactant protein C (SP-C), a marker peptide for successful protein targeting to lysosomal-like organelles. In contrast, alanine mutation of xLxxKN resulted in endoplasmic reticulum retention. As proof of principle, swapping xLxxKN for the known lysosomal targeting motif of SP-C resulted in post-Golgi targeting of the SP-C chimera. However, these products failed to reach their terminal processing compartments, suggesting that the xLxxKN motif only serves as a Golgi exit signal. We propose a model whereby an N-terminal signal sequence, xLxxKN, directs ABCA transporters to a post-Golgi vesicular sorting station where additional signals may be required for selective delivery of individual transporters to final subcellular destinations.

Is the 6 kDa tobacco etch viral protein a bona fide ERES marker?

The claim that the 6 kDa viral protein (VP) of Tobacco Etch Virus is a marker for ER exit sites (ERES) has been investigated. When transiently expressed as a CFP tagged fusion construct in tobacco mesophyll protoplasts, this integral membrane protein co-localizes with both the COPII coat protein YFP-SEC24 and the Golgi marker Man1-RFP. However, when over-expressed the VP locates to larger spherical structures which co-localize with neither ER nor Golgi markers. Nevertheless, deletion of the COPII interactive N-terminal D(X)E motif causes it to be broadly distributed throughout the ER, supporting the notion that this protein could be an ERES marker. Curiously, whereas brefeldin A (BFA) caused a typical Golgi-stack response (redistribution into the ER) of the VP in leaf epidermal cells, in protoplasts it resulted in the formation of structures identical to those formed by over-expression. However, anomalous results were obtained with protoplasts: when co-expressed with the non-cycling cis-Golgi marker Man1-RFP, a BFA-induced redistribution of the VP-CFP signal into the ER was observed, but, in the presence of the cycling Golgi marker ERD2-YFP, this did not occur. High resolution images of side-on views of Golgi stacks in epidermal cells showed that the 6 kDa VP-CFP signal overlapped considerably more with YFP-SEC24 than with Man1-RFP, indicating that the VP is proportionately more associated with ERES. However, based on a consideration of the structure of its cytoplasmic tail, the scenario that the VP collects at ERES and is transported to the cis-Golgi before being recycled back to the ER, is supported.

The Molecular Subtype Classification Is a Determinant of Sentinel Node Positivity in Early Breast Carcinoma

IntroductionSeveral authors have underscored a strong relation between the molecular subtypes and the axillary status of breast cancer patients. The aim of our work was to decipher the interaction between this classification and the probability of a positive sentinel node biopsy.Materials and MethodsOur dataset consisted of a total number of 2654 early-stage breast cancer patients. Patients treated at first by conservative breast surgery plus sentinel node biopsies were selected. A multivariate logistic regression model was trained and validated. Interaction covariate between ER and HER2 markers was a forced input of this model. The performance of the multivariate model in the training and the two validation sets was analyzed in terms of discrimination and calibration. Probability of axillary metastasis was detailed for each molecular subtype.ResultsThe interaction covariate between ER and HER2 status was a stronger predictor (p = 0.0031) of positive sentinel node biopsy than the ER status by itself (p = 0.016). A multivariate model to determine the probability of sentinel node positivity was defined with the following variables; tumour size, lympho-vascular invasion, molecular subtypes and age at diagnosis. This model showed similar results in terms of discrimination (AUC = 0.72/0.73/0.72) and calibration (HL p = 0.28/0.05/0.11) in the training and validation sets. The interaction between molecular subtypes, tumour size and sentinel nodes status was approximated.DiscussionWe showed that biologically-driven analyses are able to build new models with higher performance in terms of breast cancer axillary status prediction. The molecular subtype classification strongly interacts with the axillary and distant metastasis process.

A Methyl-Deviator Epigenotype of Estrogen Receptor–Positive Breast Carcinoma Is Associated with Malignant Biology

We broadly profiled DNA methylation in breast cancers (n = 351) and benign parenchyma (n = 47) for correspondence with disease phenotype, using FFPE diagnostic surgical pathology specimens. Exploratory analysis revealed a distinctive primary invasive carcinoma subclass featuring extreme global methylation deviation. Subsequently, we tested the correlation between methylation remodeling pervasiveness and malignant biological features. A methyl deviation index (MDI) was calculated for each lesion relative to terminal ductal-lobular unit baseline, and group comparisons revealed that high-grade and short-survival estrogen receptor-positive (ER(+)) cancers manifest a significantly higher MDI than low-grade and long-survival ER(+) cancers. In contrast, ER(-) cancers display a significantly lower MDI, revealing a striking epigenomic distinction between cancer hormone receptor subtypes. Kaplan-Meier survival curves of MDI-based risk classes showed significant divergence between low- and high-risk groups. MDI showed superior prognostic performance to crude methylation levels, and MDI retained prognostic significance (P < 0.01) in Cox multivariate analysis, including clinical stage and pathological grade. Most MDI targets individually are significant markers of ER(+) cancer survival. Lymphoid and mesenchymal indexes were not substantially different between ER(+) and ER(-) groups and do not explain MDI dichotomy. However, the mesenchymal index was associated with ER(+) cancer survival, and a high lymphoid index was associated with medullary carcinoma. Finally, a comparison between metastases and primary tumors suggests methylation patterns are established early and maintained through disease progression for both ER(+) and ER(-) tumors.

Requirement of antigen cross-presentation for lupus tissue injuries: essential role of endosomal trafficking and proteosomal degradation (44.24)

Abstract Objective: Our ‘self-organized criticality theory’ explaining the cause of SLE shows that full-matured CTL generated via antigen cross-presentation induces lupus tissue injuries. Here we examine the molecular details of antigen cross-presentation to clarify how effector CTL is generated. Methods: Bone marrow-derived DC (BMDC) from BALB/c mice was cultured with fluorescent-labeled OVA. EEA1, calnexin and Sec61 were detected by using immunofluorescent staining. MG132 or primaquine (PQ) was repeatedly co-immunized with OVA to inhibit proteasomal degradation or endosomal trafficking to cell surface, respectively. Proteinuria and IFNγ-producing CD8+ T cell in spleen were examined. Results: In BMDC, OVA co-localized with EEA1, an endosomal marker. However, OVA was gradually separated from EEA1. In contrast, OVA never co-localized with calnexin, a ER marker. Sec61, a translocon, was co-localized with OVA, which indicated that OVA was transported from endosome to cytoplasm via Sec61. MG132, which inhibits proteasomal degradation, and PQ, which inhibits endosomal trafficking to cell surface, abrogated generation of CTL and development of nephritis. Conclusion: Endosomal trafficking, bypassing ER, is important not only in antigen cross-presentation but also development of lupus nephritis. Export of antigen from endosome to cytoplasm via Sec61 seems to be the first step in antigen cross-presentation, and subsequent the generation of lupus nephritis.

157a HLA-C Associates with Env and Increases HIV-1 Infectivity

Introduction: Host cell proteins are specifically incorporated into HIV-1 envelope during budding. Virionic HLA-C reduces HIV-1 susceptibility to neutralizing antibodies (Cosma 1999). A polymorphism in the 5' region of the HLA-C gene has been associated to individual variations in set point viral loads (Fellay 2007), suggesting a role of HLA-C expression levels in modulating HIV-1 infectivity. We have reported (Matucci 2008; Baroni 2010) that HLA-C in the HIV-1 envelope associates to Env increasing viral infectivity of both R5 and X4 tropic viruses. The purpose of this study is to elucidate this interaction and exploit it for generating new immunogens capable of conferring protective immunity. Methods: Using recombinant HLA-C and Env molecules fused to fluorescent tags, we are studying their association using the bimolecular fluorescence complementation (BiFC) technique. BiFC allows the analysis of the interaction between associated proteins in living cells and to study their co-localization into cellular compartments. Env-deletion mutants and Env swapped domain recombinants are being tested to identify protein domains involved in their association. HLA-C coded by different alleles will be analyzed for their association with Env to study the influence of HLA-C polymorphisms in increasing HIV-1 infectivity. Results: Preliminary results on Env-HLA-C association and sub-cellular compartmentalization, using a specific marker for ER, reveal a direct proximity between these proteins, suggesting an early association at the ER level. Similarly, analysis of co-localization between Golgi apparatus and Env-HLA-C reveal the presence of the complementation signal between the two proteins in Golgi vesicles. Conclusions: BiFC assays allows an efficient visualization of HLA-C and HIV-1 Env association in living cells. Preliminary results obtained suggest an association between the two proteins at the ER and Golgi level. Understanding the interaction between HLA-C and HIV-1 Env might give valuable information for the design of new immunogens and/or compounds that, by reducing viral infectivity, may help controlling HIV-1 infection.

Molecular Characterization of the NPC1L1 Variants Identified from Cholesterol Low Absorbers

Niemann-Pick C1-like 1 (NPC1L1) is an essential protein for dietary cholesterol absorption. Nonsynonymous (NS) variants of NPC1L1 in humans have been suggested to associate with cholesterol absorption variations. However, information concerning the characteristics and mechanism of these variants in cholesterol uptake is limited. In this study, we analyzed the cholesterol uptake ability of the 19 reported NS variants of NPC1L1 identified from cholesterol low absorbers. Among these variants, L110F, R306C, A395V, G402S, T413M, R693C, R1214H, and R1268H could partially mediate cellular cholesterol uptake and were categorized as partially dysfunctional variants. The other 11 variants including T61M, N132S, D398G, R417W, G434R, T499M, S620C, I647N, G672R, S881L, and R1108W could barely facilitate cholesterol uptake, and were classified into the severely dysfunctional group. The partially dysfunctional variants showed mild defects in one or multiple aspects of cholesterol-regulated recycling, subcellular localization, glycosylation, and protein stability. The severely dysfunctional ones displayed remarkable defects in all these aspects and were rapidly degraded through the ER-associated degradation (ERAD) pathway. In vivo analyses using adenovirus-mediated expression in mouse liver confirmed that the S881L variant failed to localize to liver canalicular membrane, and the mice showed defects in biliary cholesterol re-absorption, while the G402S variant appeared to be similar to wild-type NPC1L1 in mouse liver. This study suggests that the dysfunction of the 19 variants on cholesterol absorption is due to the impairment of recycling, subcellular localization, glycosylation, or stability of NPC1L1.

Kre6 Protein Essential for Yeast Cell Wall β-1,6-Glucan Synthesis Accumulates at Sites of Polarized Growth

Saccharomyces cerevisiae Kre6 is a type II membrane protein with amino acid sequence homology with glycoside hydrolase and is essential for β-1,6-glucan synthesis as revealed by the mutant phenotype, but its biochemical function is still unknown. The localization of Kre6, determined by epitope tagging, is a matter of debate. We raised anti-Kre6 rabbit antiserum and examined the localization of Kre6 and its tagged protein by immunofluorescence microscopy, subcellular fractionation in sucrose density gradients, and immunoelectron microscopy. Integration of the results indicates that the majority of Kre6 is in the endoplasmic reticulum; however, a small but significant portion is also present in the secretory vesicle-like compartments and plasma membrane. Kre6 in the latter compartments is observed as strong signals that accumulate at the sites of polarized growth by immunofluorescence. The truncated Kre6 without the N-terminal 230-amino acid cytoplasmic region did not show this polarized accumulation and had a severe defect in β-1,6-glucan synthesis. This is the first evidence of a β-1,6-glucan-related protein showing the polarized membrane localization that correlates with its biological function.

Mitochondrial ROS fuel the inflammasome

Mitochondrial ROS fuel the inflammasome