Tubers from patients with tuberous sclerosis complex are characterized by changes in microtubule biology through ROCK2 signalling.

Most patients with tuberous sclerosis complex (TSC) develop cortical tubers that cause severe neurological disabilities. It has been suggested that defects in neuronal differentiation and/or migration underlie the appearance of tubers. However, the precise molecular alterations remain largely unknown. Here, by combining cytological and immunohistochemical analyses of tubers from nine TSC patients (four of them diagnosed with TSC2 germline mutations), we show that alteration of microtubule biology through ROCK2 signalling contributes to TSC neuropathology. All tubers showed a larger number of binucleated neurons than expected relative to control cortex. An excess of normal and altered cytokinetic figures was also commonly observed. Analysis of centrosomal markers suggested increased microtubule nucleation capacity, which was supported by the analysis of an expression dataset from cortical tubers and control cortex, and subsequently linked to under-expression of Rho-associated coiled-coil containing kinase 2 (ROCK2). Thus, augmented microtubule nucleation capacity was observed in mouse embryonic fibroblasts and human fibroblasts deficient in the Tsc2/TSC2 gene product, tuberin. Consistent with ROCK2 under-expression, microtubule acetylation was found to be increased with tuberin deficiency; this alteration was abrogated by rapamycin treatment and mimicked by HDAC6 inhibition. Together, the results of this study support the hypothesis that loss of TSC2 expression can alter microtubule organization and dynamics, which, in turn, deregulate cell division and potentially impair neuronal differentiation.

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.

Interplay between BRCA1 and RHAMM regulates epithelial apicobasal polarization and may influence risk of breast cancer.

Differentiated mammary epithelium shows apicobasal polarity, and loss of tissue organization is an early hallmark of breast carcinogenesis. In BRCA1 mutation carriers, accumulation of stem and progenitor cells in normal breast tissue and increased risk of developing tumors of basal-like type suggest that BRCA1 regulates stem/progenitor cell proliferation and differentiation. However, the function of BRCA1 in this process and its link to carcinogenesis remain unknown. Here we depict a molecular mechanism involving BRCA1 and RHAMM that regulates apicobasal polarity and, when perturbed, may increase risk of breast cancer. Starting from complementary genetic analyses across families and populations, we identified common genetic variation at the low-penetrance susceptibility HMMR locus (encoding for RHAMM) that modifies breast cancer risk among BRCA1, but probably not BRCA2, mutation carriers: n = 7,584, weighted hazard ratio ((w)HR) = 1.09 (95% CI 1.02-1.16), p(trend) = 0.017; and n = 3,965, (w)HR = 1.04 (95% CI 0.94-1.16), p(trend) = 0.43; respectively. Subsequently, studies of MCF10A apicobasal polarization revealed a central role for BRCA1 and RHAMM, together with AURKA and TPX2, in essential reorganization of microtubules. Mechanistically, reorganization is facilitated by BRCA1 and impaired by AURKA, which is regulated by negative feedback involving RHAMM and TPX2. Taken together, our data provide fundamental insight into apicobasal polarization through BRCA1 function, which may explain the expanded cell subsets and characteristic tumor type accompanying BRCA1 mutation, while also linking this process to sporadic breast cancer through perturbation of HMMR/RHAMM.

A novel inhibitor of fatty acid synthase shows activity against HER2+ breast cancer xenografts and is active in anti-HER2 drug-resistant cell lines.

INTRODUCTION: Inhibiting the enzyme Fatty Acid Synthase (FASN) leads to apoptosis of breast carcinoma cells, and this is linked to human epidermal growth factor receptor 2 (HER2) signaling pathways in models of simultaneous expression of FASN and HER2. METHODS: In a xenograft model of breast carcinoma cells that are FASN+ and HER2+, we have characterised the anticancer activity and the toxicity profile of G28UCM, the lead compound of a novel family of synthetic FASN inhibitors. In vitro, we analysed the cellular and molecular interactions of combining G28UCM with anti-HER drugs. Finally, we tested the cytotoxic ability of G28UCM on breast cancer cells resistant to trastuzumab or lapatinib, that we developed in our laboratory. RESULTS: In vivo, G28UCM reduced the size of 5 out of 14 established xenografts. In the responding tumours, we observed inhibition of FASN activity, cleavage of poly-ADPribose polymerase (PARP) and a decrease of p-HER2, p- protein kinase B (AKT) and p-ERK1/2, which were not observed in the nonresponding tumours. In the G28UCM-treated animals, no significant toxicities occurred, and weight loss was not observed. In vitro, G28UCM showed marked synergistic interactions with trastuzumab, lapatinib, erlotinib or gefitinib (but not with cetuximab), which correlated with increases in apoptosis and with decreases in the activation of HER2, extracellular signal-regulated kinase (ERK)1/2 and AKT. In trastuzumab-resistant and in lapatinib-resistant breast cancer cells, in which trastuzumab and lapatinib were not effective, G28UCM retained the anticancer activity observed in the parental cells. CONCLUSIONS: G28UCM inhibits fatty acid synthase (FASN) activity and the growth of breast carcinoma xenografts in vivo, and is active in cells with acquired resistance to anti-HER2 drugs, which make it a candidate for further pre-clinical development.

Exploring the link between MORF4L1 and risk of breast cancer.

INTRODUCTION: Proteins encoded by Fanconi anemia (FA) and/or breast cancer (BrCa) susceptibility genes cooperate in a common DNA damage repair signaling pathway. To gain deeper insight into this pathway and its influence on cancer risk, we searched for novel components through protein physical interaction screens. METHODS: Protein physical interactions were screened using the yeast two-hybrid system. Co-affinity purifications and endogenous co-immunoprecipitation assays were performed to corroborate interactions. Biochemical and functional assays in human, mouse and Caenorhabditis elegans models were carried out to characterize pathway components. Thirteen FANCD2-monoubiquitinylation-positive FA cell lines excluded for genetic defects in the downstream pathway components and 300 familial BrCa patients negative for BRCA1/2 mutations were analyzed for genetic mutations. Common genetic variants were genotyped in 9,573 BRCA1/2 mutation carriers for associations with BrCa risk. RESULTS: A previously identified co-purifying protein with PALB2 was identified, MRG15 (MORF4L1 gene). Results in human, mouse and C. elegans models delineate molecular and functional relationships with BRCA2, PALB2, RAD51 and RPA1 that suggest a role for MRG15 in the repair of DNA double-strand breaks. Mrg15-deficient murine embryonic fibroblasts showed moderate sensitivity to γ-irradiation relative to controls and reduced formation of Rad51 nuclear foci. Examination of mutants of MRG15 and BRCA2 C. elegans orthologs revealed phenocopy by accumulation of RPA-1 (human RPA1) nuclear foci and aberrant chromosomal compactions in meiotic cells. However, no alterations or mutations were identified for MRG15/MORF4L1 in unclassified FA patients and BrCa familial cases. Finally, no significant associations between common MORF4L1 variants and BrCa risk for BRCA1 or BRCA2 mutation carriers were identified: rs7164529, Ptrend = 0.45 and 0.05, P2df = 0.51 and 0.14, respectively; and rs10519219, Ptrend = 0.92 and 0.72, P2df = 0.76 and 0.07, respectively. CONCLUSIONS: While the present study expands on the role of MRG15 in the control of genomic stability, weak associations cannot be ruled out for potential low-penetrance variants at MORF4L1 and BrCa risk among BRCA2 mutation carriers.

Economic Impact of Low Adherence to COPD Management Guidelines in Spain.

OBJECTIVE: The objective of this study was to assess the non-adherence level of Spanish clinical practice to guideline recommendations for the treatment of chronic obstructive pulmonary disease (COPD) and to estimate the potential impact on pharmaceutical expenditure resulting from transitioning current treatment patterns according to guidelines. METHODS: A model was developed to compare current prescribing patterns with two alternative scenarios: the first aligned with the Global Initiative for Chronic Obstructive Lung Disease (GOLD 2020) recommendations, and the second with the Spanish Guidelines for COPD (GesEPOC 2017). Current treatment practice was obtained from publications that describe treatment patterns by pulmonology departments in Spain. The economic impact between patterns was calculated from the perspective of the Spanish National Health System (NHS), considering the annual pharmacological costs of COPD inhaled maintenance therapy. Two additional analyses were performed: one that included current prescribing patterns of patients managed by pulmonology and primary care centers in Spain (published aggregated data); and another that only considered the appropriate use of inhaled corticosteroids (ICS) treatment according to guidelines. RESULTS: It was estimated that 54% and 38% of patients were not treated in line with GOLD and GesEPOC recommendations, respectively, mainly due to a broader use of ICS-based therapies. Adapting treatment to recommendations could provide a potential annual cost-saving of €17,792,022 (according to GOLD) and €5,881,785 (according to GesEPOC). In scenario analysis 1, a 26% of non-adherence to GesEPOC guideline was observed with a potential annual pharmacological cost-saving of €2,707,554. In scenario analysis 2, considering only inappropriate use of ICS treatment, an annual cost-saving of €17,863,750 (according to GOLD) and €9,904,409 (according to GesEPOC) was calculated. CONCLUSION: More than a third of treatments for COPD patients in Spain are not prescribed in accordance with guideline recommendations. The adaptation of clinical practice to guideline recommendations could provide important cost-savings for the Spanish NHS.

Correction: Lymphangioleiomyomatosis Biomarkers Linked to Lung Metastatic Potential and Cell Stemness.

[This corrects the article DOI: 10.1371/journal.pone.0132546.].

Lymphangioleiomyomatosis Biomarkers Linked to Lung Metastatic Potential and Cell Stemness.

Lymphangioleiomyomatosis (LAM) is a rare lung-metastasizing neoplasm caused by the proliferation of smooth muscle-like cells that commonly carry loss-of-function mutations in either the tuberous sclerosis complex 1 or 2 (TSC1 or TSC2) genes. While allosteric inhibition of the mechanistic target of rapamycin (mTOR) has shown substantial clinical benefit, complementary therapies are required to improve response and/or to treat specific patients. However, there is a lack of LAM biomarkers that could potentially be used to monitor the disease and to develop other targeted therapies. We hypothesized that the mediators of cancer metastasis to lung, particularly in breast cancer, also play a relevant role in LAM. Analyses across independent breast cancer datasets revealed associations between low TSC1/2 expression, altered mTOR complex 1 (mTORC1) pathway signaling, and metastasis to lung. Subsequently, immunohistochemical analyses of 23 LAM lesions revealed positivity in all cases for the lung metastasis mediators fascin 1 (FSCN1) and inhibitor of DNA binding 1 (ID1). Moreover, assessment of breast cancer stem or luminal progenitor cell biomarkers showed positivity in most LAM tissue for the aldehyde dehydrogenase 1 (ALDH1), integrin-ß3 (ITGB3/CD61), and/or the sex-determining region Y-box 9 (SOX9) proteins. The immunohistochemical analyses also provided evidence of heterogeneity between and within LAM cases. The analysis of Tsc2-deficient cells revealed relative over-expression of FSCN1 and ID1; however, Tsc2-deficient cells did not show higher sensitivity to ID1-based cancer inhibitors. Collectively, the results of this study reveal novel LAM biomarkers linked to breast cancer metastasis to lung and to cell stemness, which in turn might guide the assessment of additional or complementary therapeutic opportunities for LAM.

Cancer develops, progresses and responds to therapies through restricted perturbation of the protein-protein interaction network.

The products of genes mutated or differentially expressed in cancer tend to occupy central positions within the network of protein-protein interactions, or the interactome network. Integration of different types of gene and protein relationships has considerably increased the understanding of the mechanisms of carcinogenesis, while also enhancing the applicability of expression signatures. In this scenario, however, it remains unknown how cancer develops, progresses and responds to therapies in a potentially controlled manner at the systems level. Here, by applying the concepts of load transfer and cascading failures in power grids, we examine the impact and transmission of cancer-related gene expression changes in the interactome network. Relative to random perturbations, this study reveals topological robustness associated with all cancer conditions. In addition, experimental perturbation of a central cancer node, which consists of over-expression of the α-synuclein (SNCA) protein in MCF7 breast cancer cells, also reveals robustness. Conversely, a search for proteins with an opposite topological impact identifies the autophagy pathway. Mechanistically, the existence of smaller shortest paths among cancer-related proteins appears to be a topological feature that partially contributes to the restricted perturbation of the network. Together, the results of this study suggest that cancer develops, progresses and responds to therapies following controlled, restricted perturbation of the interactome network.

Novel Inhibitors of Fatty Acid Synthase with Anticancer Activity.

PURPOSE: Fatty acid synthase (FASN) is overexpressed in human breast carcinoma. The natural polyphenol (-)-epigallocatechin-3-gallate blocks in vitro FASN activity and leads to apoptosis in breast cancer cells without any effects on carnitine palmitoyltransferase-1 (CPT-1) activity, and in vivo, does not decrease body weight. We synthesized a panel of new polyphenolic compounds and tested their effects on breast cancer models. EXPERIMENTAL DESIGN: We evaluated the in vitro effects of the compounds on breast cancer cell growth (SK-Br3, MCF-7, and MDA-MB-231), apoptosis [as assessed by cleavage of poly(ADP-ribose) polymerase], cell signaling (HER2, ERK1/2, and AKT), and fatty acid metabolism enzymes (FASN and CPT-1). In vivo, we have evaluated their antitumor activity and their effect on body weight in a mice model of BT474 breast cancer cells. RESULTS: Two compounds potently inhibited FASN activity and showed high cytotoxicity. Moreover, the compounds induced apoptosis and caused a marked decrease in the active forms of HER2, AKT, and ERK1/2 proteins. Interestingly, the compounds did not stimulate CPT-1 activity in vitro. We show evidence that one of the FASN inhibitors blocked the growth of BT474 breast cancer xenografts and did not induce weight loss in vivo. CONCLUSIONS: The synthesized polyphenolic compounds represent a novel class of FASN inhibitors, with in vitro and in vivo anticancer activity, that do not exhibit cross-activation of beta-oxidation and do not induce weight loss in animals. One of the compounds blocked the growth of breast cancer xenografts. These FASN inhibitors may represent new agents for breast cancer treatment. (Clin Cancer Res 2009;15(24):7608-15).

Biological convergence of cancer signatures.

Gene expression profiling has identified cancer prognostic and predictive signatures with superior performance to conventional histopathological or clinical parameters. Consequently, signatures are being incorporated into clinical practice and will soon influence everyday decisions in oncology. However, the slight overlap in the gene identity between signatures for the same cancer type or condition raises questions about their biological and clinical implications. To clarify these issues, better understanding of the molecular properties and possible interactions underlying apparently dissimilar signatures is needed. Here, we evaluated whether the signatures of 24 independent studies are related at the genome, transcriptome or proteome levels. Significant associations were consistently observed across these molecular layers, which suggest the existence of a common cancer cell phenotype. Convergence on cell proliferation and death supports the pivotal involvement of these processes in prognosis, metastasis and treatment response. In addition, functional and molecular associations were identified with the immune response in different cancer types and conditions that complement the contribution of cell proliferation and death. Examination of additional, independent, cancer datasets corroborated our observations. This study proposes a comprehensive strategy for interpreting cancer signatures that reveals common design principles and systems-level properties.

Pre-clinical validation of early molecular markers of sensitivity to aromatase inhibitors in a mouse model of post-menopausal hormone-sensitive breast cancer.

INTRODUCTION: Changes in breast cancer cell biology following hormonal treatment have been claimed as promising predictor markers of clinical benefit even outperforming clinical response. From previous work we selected 10 genes showing both a well known regulation by oestrogen and a high level of early transcriptional regulation following therapy with aromatase inhibitors. Here we use an animal breast cancer model to explore the feasibility of the determination of their expression in minimally invasive samples and to further assess the magnitude of their regulation by letrozole. ANIMAL AND METHODS: Aromatase inhibitor sensitive breast cancer tumours were grown in athymic mice under supplement with androstenedione. Following initial tumour growth animals were assigned to a control group or to receive letrozole at two different dosages. Fine needle aspirates were obtained at the moment of treatment assignation and one week later. Expression of the following genes at both time points was determined: Ki-67, Cyclin D1, pS2, Trefoil Factor 3, PDZ domain containing 1, Ubiquitin-conjugating enzyme E2C, Stanniocalcin 2, Topoisomerase 2 alfa, MAN1A1 and FAS. RESULTS: Fine needles aspirates were found to be a feasible and reproducible technique for RNA extraction. Trefoil Factor 3, pS2, Cyclin D1 and Stanniocalcin 2 were significantly downregulated by letrozole. Among them pS2 appears to be most sensitive to aromatase inhibitor treatment even differentiating sub-optimal from optimal letrozole dosage. DISCUSSION: We present pre-clinical evidence to justify the exploration in clinical trials of pS2, Trefoil factor 3, Cyclin D1 and Stanniocalcin as dynamic markers of oestrogen-driven pathway activation.

Human KIR2DL5 is an inhibitory receptor expressed on the surface of NK and T lymphocyte subsets.

Human NK cells, by means of a repertoire of clonally distributed killer cell Ig-like receptors (KIR), survey the expression of individual self HLA class I molecules, which is often altered in infections and tumors. KIR2DL5 (CD158f) is the last identified KIR gene and, with KIR2DL4, constitutes a structurally divergent lineage conserved in different primate species. Research on KIR2DL5 has thus far been limited to its genetic aspects due to a lack of reagents to detect its product. We report here the identification and characterization of the receptor encoded by KIR2DL5 using a newly generated specific mAb that recognizes its most commonly expressed allele, KIR2DL5A*001. KIR2DL5 displays a variegated distribution on the surface of CD56(dim) NK cells. This contrasts with the expression pattern of its structural homolog KIR2DL4 (ubiquitous transcription, surface expression restricted to CD56(bright) NK cells) and resembles the profile of KIR recognizing classical HLA class I molecules. Like other MHC class I receptors, KIR2DL5 is also found in a variable proportion of T lymphocytes. KIR2DL5 is detected on the cell surface as a monomer of approximately 60 kDa that, upon tyrosine phosphorylation, recruits the Src homology region 2-containing protein tyrosine phosphatase-2 and, to a lesser extent, Src homology region 2-containing protein tyrosine phosphatase-1. Ab-mediated cross-linking of KIR2DL5 inhibits NK cell cytotoxicity against murine FcR+ P815 cells. KIR2DL5 is thus an inhibitory receptor gathering a combination of genetic, structural, and functional features unique among KIR, which suggests that KIR2DL5 plays a specialized role in innate immunity.

Molecular characterization of a t(9;12)(p21;q13) balanced chromosome translocation in combination with integrative genomics analysis identifies C9orf14 as a candidate tumor-suppressor.

A large number of nevi (LNN) is a high risk phenotypic trait for developing cutaneous malignant melanoma (CMM). In this study, the breakpoints of a t(9;12)(p21;q13) balanced chromosome translocation were finely mapped in a family with LNN and CMM. Molecular characterization of the 9p21 breakpoint identified a novel gene C9orf14 expressed in melanocytes disrupted by the translocation. Integrative analysis of functional genomics data was applied to determine the role of C9orf14 in CMM development. An analysis of genome-wide DNA copy number alterations in melanoma tumors revealed the loss of the C9orf14 locus, located proximal to CDKN2A, in approximately one-fourth of tumors. Analysis of gene expression data in cancer cell lines and melanoma tumors suggests a loss of C9orf14 expression in melanoma tumorigenesis. Taken together, our results indicate that C9orf14 is a candidate tumor-suppressor for nevus development and late stage melanoma at 9p21, a region frequently deleted in different types of human cancers.

IREM-1 is a novel inhibitory receptor expressed by myeloid cells.

Using a three-hybrid strategy, we have identified a novel cell surface molecule which interacts with the Src homology 2 (SH2) domains of SH2 domain-containing protein tyrosine phosphatase 1 (SHP-1), termed "immune receptor expressed on myeloid cells 1" (IREM-1). The full-length cDNA coding for a polypeptide of 290 amino acids presents an extracellular single V-type Ig domain, a transmembrane region and a cytoplasmic tail with five tyrosine residues, two of which are in the context of an immunoreceptor tyrosine-based inhibitory motif. Moreover, cDNA encoding for three other splicing forms of IREM-1, named IREM-1 splice variant (Sv)1, Sv2 and Sv3 were cloned by reverse transcription (RT)-PCR. The gene encoding for IREM-1 contains nine exons, is located on human chromosome 17 (17q25.1) and is homologous to previously identified molecules termed CMRF-35 and IRp60. RT-PCR, northern blot and FACS analysis with specific monoclonal antibodies indicated that IREM-1 is expressed on monocytes, granulocytes, and myeloid leukemia cell lines. Western blot analysis confirmed the recruitment of SHP-1 to IREM-1 and demonstrated that phosphotyrosine residue 205 is the main docking site for this interaction. Finally, cross-linking of IREM-1 results in the inhibition of FcRepsilon-induced activation. Our results indicate that IREM-1 is a novel inhibitory receptor of the Ig superfamily in myeloid cells.

Molecular characterization of a novel immune receptor restricted to the monocytic lineage.

Homology basic local alignment search tool search was conducted using a sequence encoding for a novel inhibitory receptor (IREM-1) cloned in our laboratory and a previously described homologous sequence termed CMRF-35. On the basis of this information, we cloned a full length cDNA corresponding to a novel member of this family, termed immune receptor expressed by myeloid cells 2 (IREM-2). The gene, located in chromosome 17q25.1, encodes for a protein of 205 aa that contains an extracellular region comprising an Ig-like domain and a transmembrane region with a positively charged amino acid residue (lysine), that predicted its putative association with an adapter molecule. Indeed, the interaction between IREM-2 and DAP-12 was confirmed in transfected COS-7 cells. By generating specific Abs and using bone marrow and PBMCs, we observed that IREM-2 expression appeared to be restricted to mature hemopoietic cells of the monocytic and myeloid dendritic cell lineages. In vitro differentiation to macrophages or immature dendritic cells down-regulated IREM-2 expression. Upon engagement with the specific mAbs, IREM-2 expressed in rat basophilic leukemia cells together with DAP-12, induced NFAT transcriptional activity; moreover, IREM-2 engagement on monocytes induced TNF-alpha production. Taken together, our results indicate that IREM-2 is a novel activating receptor of the Ig-superfamily in the monocytic lineage.