Stéphanie Gofflot

Identification of HTF (HER2 transcription factor) as an AP-2 (activator protein-2) transcription factor and contribution of the HTF binding site to ERBB2 gene overexpression

The ERBB2 gene is overexpressed in 30% of human breast cancers and this is correlated with poor prognosis. Overexpression of the ERBB2 gene is due to increased transcription and gene amplification. Our previous studies have identified a new cis element in the ERBB2 promoter which is involved in the genes overexpression. This cis element, located 501 bp upstream from the main ERBB2 transcription initiation site, binds a transcription factor called HTF (HER2 transcription factor). We report here the identification of HTF as an AP-2 (activator protein-2) transcription factor. The new cis element is bound by AP-2 with high affinity, compared with a previously described AP-2 binding site located 284 bp downstream. Co-transfection of an AP-2alpha expression vector with a reporter vector containing the newly identified AP-2 binding site in front of a minimal ERBB2 promoter induced a dose-dependent increase in transcriptional activity. We examined the contribution of the new AP-2 binding site to ERBB2 overexpression. For this purpose we abolished the new and/or the previously described AP-2 binding sequence by site-directed mutagenesis. The results show that the two functional AP-2 sites in the first 700 bp of the ERBB2 promoter co-operate to achieve maximal transcriptional activity.

Immuno‐Quantitative Polymerase Chain Reaction for Detection and Quantitation of Prion Protein

Abstract Immuno‐polymerase chain reaction (PCR) is an extremely sensitive detection method, combining the specificity of antibody detection and the sensitivity of PCR. We have developed an immuno‐quantitative PCR (iqPCR), exploiting real‐time PCR technology, in order to improve this immuno‐detection method and make it quantitative. To illustrate the advantages of iqPCR, we have compared it with a conventional enzyme linked immuno sorbent assay (ELISA) technique in experiments aimed at detecting the cellular and the resistant form of prion protein in bovine brain extract. The iqPCR technique proved to be more sensitive than ELISA, so it could be a technique of choice for the diagnosis of infected animals both at an ante mortem and post‐mortem stage.

Asporin Is a Fibroblast-Derived TGF-β1 Inhibitor and a Tumor Suppressor Associated with Good Prognosis in Breast Cancer.

Background Breast cancer is a leading malignancy affecting the female population worldwide. Most morbidity is caused by metastases that remain incurable to date. TGF-β1 has been identified as a key driving force behind metastatic breast cancer, with promising therapeutic implications. Methods and Findings Employing immunohistochemistry (IHC) analysis, we report, to our knowledge for the first time, that asporin is overexpressed in the stroma of most human breast cancers and is not expressed in normal breast tissue. In vitro, asporin is secreted by breast fibroblasts upon exposure to conditioned medium from some but not all human breast cancer cells. While hormone receptor (HR) positive cells cause strong asporin expression, triple-negative breast cancer (TNBC) cells suppress it. Further, our findings show that soluble IL-1β, secreted by TNBC cells, is responsible for inhibiting asporin in normal and cancer-associated fibroblasts. Using recombinant protein, as well as a synthetic peptide fragment, we demonstrate the ability of asporin to inhibit TGF-β1-mediated SMAD2 phosphorylation, epithelial to mesenchymal transition, and stemness in breast cancer cells. In two in vivo murine models of TNBC, we observed that tumors expressing asporin exhibit significantly reduced growth (2-fold; p = 0.01) and metastatic properties (3-fold; p = 0.045). A retrospective IHC study performed on human breast carcinoma (n = 180) demonstrates that asporin expression is lowest in TNBC and HER2+ tumors, while HR+ tumors have significantly higher asporin expression (4-fold; p = 0.001). Assessment of asporin expression and patient outcome (n = 60; 10-y follow-up) shows that low protein levels in the primary breast lesion significantly delineate patients with bad outcome regardless of the tumor HR status (area under the curve = 0.87; 95% CI 0.78–0.96; p = 0.0001). Survival analysis, based on gene expression (n = 375; 25-y follow-up), confirmed that low asporin levels are associated with a reduced likelihood of survival (hazard ratio = 0.58; 95% CI 0.37–0.91; p = 0.017). Although these data highlight the potential of asporin to serve as a prognostic marker, confirmation of the clinical value would require a prospective study on a much larger patient cohort. Conclusions Our data show that asporin is a stroma-derived inhibitor of TGF-β1 and a tumor suppressor in breast cancer. High asporin expression is significantly associated with less aggressive tumors, stratifying patients according to the clinical outcome. Future pre-clinical studies should consider options for increasing asporin expression in TNBC as a promising strategy for targeted therapy.

c-Maf expression in angioimmunoblastic T-cell lymphoma reflects follicular helper T-cell derivation rather than oncogenesis

Vicky S Sabine Dana Faratian Tove Kirkegaard-Clausen John MS Bartlett Endocrine Cancer Group, Edinburgh Cancer Research Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, Edinburgh Breakthrough Research Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, Edinburgh, Scotland, UK, and Department of Breast Cancer Research, Institute of Cancer Biology, Danish Cancer Society, Copenhagen, Denmark 1. Kerr JF, Wyllie AH, Currie AR. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer 1972; 26; 239–257. 2. Ellis PA, Smith IE, Detre S et al. Reduced apoptosis and proliferation and increased Bcl-2 in residual breast cancer following preoperative chemotherapy. Breast Cancer Res. Treat. 1998; 48; 107–116. 3. Porter AG, Janicke RU. Emerging roles of caspase-3 in apoptosis. Cell Death Differ. 1999; 6; 99–104. 4. Kirkegaard T, Naresh A, Sabine VS et al. Expression of tumor necrosis factor alpha converting enzyme in endocrine cancers. Am. J. Clin. Pathol. 2008; 129; 735–743. 5. Faratian D, Kay C, Robson T et al. Automated image analysis for high-throughput quantitative detection of ER and PR expression levels in large-scale clinical studies: the TEAM trial experience. Histopathology 2009; 55; 587–593.

Myoferlin regulates cellular lipid metabolism and promotes metastases in triple-negative breast cancer.

Myoferlin is a multiple C2-domain-containing protein that regulates membrane repair, tyrosine kinase receptor function and endocytosis in myoblasts and endothelial cells. Recently it has been reported as overexpressed in several cancers and shown to contribute to proliferation, migration and invasion of cancer cells. We have previously demonstrated that myoferlin regulates epidermal growth factor receptor activity in breast cancer. In the current study, we report a consistent overexpression of myoferlin in triple-negative breast cancer cells (TNBC) over cells originating from other breast cancer subtypes. Using a combination of proteomics, metabolomics and electron microscopy, we demonstrate that myoferlin depletion results in marked alteration of endosomal system and metabolism. Mechanistically, myoferlin depletion caused impaired vesicle traffic that led to a misbalance of saturated/unsaturated fatty acids. This provoked mitochondrial dysfunction in TNBC cells. As a consequence of the major metabolic stress, TNBC cells rapidly triggered AMP activated protein kinase-mediated metabolic reprogramming to glycolysis. This reduced their ability to balance between oxidative phosphorylation and glycolysis, rendering TNBC cells metabolically inflexible, and more sensitive to metabolic drug targeting in vitro. In line with this, our in vivo findings demonstrated a significantly reduced capacity of myoferlin-deficient TNBC cells to metastasise to lungs. The significance of this observation was further supported by clinical data, showing that TNBC patients whose tumors overexpress myoferlin have worst distant metastasis-free and overall survivals. This novel insight into myoferlin function establishes an important link between vesicle traffic, cancer metabolism and progression, offering new diagnostic and therapeutic concepts to develop treatments for TNBC patients.

Surveillance of effects of HPV vaccination in Belgium

BACKGROUND Early effects of HPV (human papillomavirus) vaccination are reflected by changes observable in young women attending cervical cancer screening. SUBJECT AND METHODS The SEHIB study included HPV geno-typing of ∼6000 continuous and 650 pathological cervical cell specimen as well as biopsies, collected from women in Belgium in 2010-2014. Data were linked to vaccination status. RESULTS HPV vaccination offered protection among women aged <30years against infection with HPV16 (vaccine effectiveness [VE]=67%, 95% CI: 48-79%), HPV18 (VE=93%, 95% CI: 52-99%), and high-risk HPV (VE=16%, 95% CI: 2-29%). Vaccination protected also against cytological lesions. Vaccination protected against histologically confirmed lesions: significantly lower absolute risks of CIN1+ (risk difference [RD]=-1.6%, 95% CI: -2.6% to -0.7%) and CIN3+ associated with HPV16/18 (RD=-0.3%, 95% CI -0.6% to -0.1%). Vaccine effectiveness decreased with age. Protection against HPV16 and 18 infection was significant in all age groups, however no protection was observed against cytological lesions associated with these types in age-group 25-29. CONCLUSION The SEHIB study demonstrates the effectiveness of HPV vaccination in Belgian young women in particular in age group 18-19. Declining effectiveness with increasing age may be explained by higher tendency of women already exposed to infection to get the vaccine.

Characterization of an antibody panel for immunohistochemical analysis of canine muscle cells

Immunohistochemistry is an indispensable tool in the assessment and characterization of lineage-specific differentiation of grafted cells in cell-based-therapy. This strategy is under investigation for the treatment of many muscle disorders and different animals such as dogs are used as models to study the tissue regeneration. The aim of the present study was to characterize an antibody panel for the analysis of canine muscle cells, useful in routinely processed formalin-fixed paraffin-embedded tissues. Overall, 12 antibodies (8 mouse monoclonal and 4 goat polyclonal), validated for use on human tissues tested for cross-reactivity on canine smooth muscle (bladder, intestine, and uterus), skeletal muscle and heart. Specific staining was achieved with eight antibodies, of which six were cytoplasmic markers (desmin, HDAC8, MHC, SMA, Troponin I and Troponin T) and two were cardiac nuclear markers (GATA-4 and Nkx-2.5). This antibody panel may be useful not only for the evaluation of cell-based therapies in muscle disorders, but also for the evaluation of canine soft tissue neoplasms in veterinary pathology.

A specific immune and lymphatic profile characterizes the pre-metastatic state of the sentinel lymph node in patients with early cervical cancer

ABSTRACT The lymph node (LN) pre-metastatic niche is faintly characterized in lymphophilic human neoplasia, although LN metastasis is considered as the strongest prognostic marker of patient survival. Due to its specific dissemination through a complex bilateral pelvic lymphatic system, early cervical cancer is a relevant candidate for investigating the early nodal metastatic process. In the present study, we analyzed in-depth both the lymphatic vasculature and the immune climate of pre-metastatic sentinel LN (SLN), in 48 cases of FIGO stage IB1 cervical neoplasms. An original digital image analysis methodology was used to objectively determine whole slide densities and spatial distributions of immunostained structures. We observed a marked increase in lymphatic vessel density (LVD) and a specific capsular and subcapsular distribution in pre-metastatic SLN when compared with non-sentinel counterparts. Such features persisted in the presence of nodal metastatic colonization. The inflammatory profile attested by CD8+, Foxp3, CD20 and PD-1expression was also significantly increased in pre-metastatic SLN. Remarkably, the densities of CD20+ B cells and PD-1 expressing germinal centers were positively correlated with LVD. All together, these data strongly support the existence of a pre-metastatic dialog between the primary tumor and the first nodal relay. Both lymphatic and immune responses contribute to the elaboration of a specific pre-metastatic microenvironment in human SLN. Moreover, this work provides evidence that, in the context of early cervical cancer, a pre-metastatic lymphangiogenesis occurs within the SLN (pre-metastatic niche) and is associated with a specific humoral immune response.

MT4-MMP and EGFR expression levels are key biomarkers for breast cancer patient response to chemotherapy and erlotinib.

Background:Triple-negative breast cancers (TNBC) are heterogeneous cancers with poor prognosis. We aimed to determine the clinical relevance of membrane type-4 matrix metalloproteinase (MT4-MMP), a membrane type matrix metalloproteinase that interacts with epidermal growth factor receptor (EGFR) overexpressed in >50% of TNBC.Methods:We conducted a retrospective immunohistochemical analysis on human TNBC samples (n=81) and validated our findings in in vitro and in vivo assays.Results:Membrane type-4 matrix metalloproteinase and EGFR are produced in 72.5% of TNBC samples, whereas those proteins are faintly produced by healthy tissues. Unexpectedly, tumour relapse after chemotherapy was reduced in samples highly positive for MT4-MMP. Mechanistically, this is ascribed to a higher sensitivity of MT4-MMP-producing cells to alkylating or intercalating chemotherapeutic agents, as assessed in vitro. In sharp contrast, MT4-MMP expression did not affect tumour cell sensitivity to paclitaxel that interferes with protease trafficking. Importantly, MT4-MMP expression sensitised cancer cells to erlotinib, a tyrosine kinase EGFR inhibitor. In a pre-clinical model, the growth of MT4-MMP overexpressing xenografts, but not of control ones, was reduced by epirubicin or erlotinib. The combination of suboptimal drug doses blocked drastically the growth of MT4-MMP-producing tumours.Conclusions:We demonstrate that MT4-MMP defines a sub-population of TNBC sensitive to a combination of DNA-targeting chemotherapeutic agents and anti-EGFR drugs.

Myoferlin controls mitochondrial structure and activity in pancreatic ductal adenocarcinoma, and affects tumor aggressiveness

Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related death. Therapeutic options remain very limited and are based on classical chemotherapies. Energy metabolism reprogramming appears as an emerging hallmark of cancer and is considered a therapeutic target with considerable potential. Myoferlin, a ferlin family member protein overexpressed in PDAC, is involved in plasma membrane biology and has a tumor-promoting function. In the continuity of our previous studies, we investigated the role of myoferlin in the context of energy metabolism in PDAC. We used selected PDAC tumor samples and PDAC cell lines together with small interfering RNA technology to study the role of myoferlin in energetic metabolism. In PDAC patients, we showed that myoferlin expression is negatively correlated with overall survival and with glycolytic activity evaluated by 18F-deoxyglucose positron emission tomography. We found out that myoferlin is more abundant in lipogenic pancreatic cancer cell lines and is required to maintain a branched mitochondrial structure and a high oxidative phosphorylation activity. The observed mitochondrial fission induced by myoferlin depletion led to a decrease of cell proliferation, ATP production, and autophagy induction, thus indicating an essential role of myoferlin for PDAC cell fitness. The metabolic phenotype switch generated by myoferlin silencing could open up a new perspective in the development of therapeutic strategies, especially in the context of energy metabolism.