Les cancers du sein triple-négatif (TNBC) (RE-, RP-, HER2) représentent 15% des cas de cancer du sein. Les patientes atteintes de TNBC sont traitées uniquement par chimiothérapie. A l’heure actuelle, il n’existe aucune thérapie ciblée efficace. Malgré une chimiosensibilité initiale, les rechutes sont fréquentes. Ainsi de nouveaux traitements sont nécessaires pour soigner ces patientes. Dans le cancer du sein, l’aspartyl protéase cathepsine D (cath-D), un marqueur de mauvais pronostic, est surexprimée par les cellules cancéreuses et est hyper-secrétée dans le microenvironnement tumoral. La cath-D stimule la prolifération des cellules cancéreuses, la croissance invasive des fibroblastes, la croissance tumorale, l’angiogenèse tumorale et la formation des métastases. Différentes études ont mis en exergue le rôle oncogénique de la cath-D extracellulaire dans le cancer du sein, suggérant qu’elle serait une cible thérapeutique d’intérêt. Afin de neutraliser sélectivement la forme sécrétée de la cath-D, le laboratoire a généré des anticorps humains IgG1 dirigés contre la cath-D par un crible de phage display (International patent N° PCT/EP2016/061454).Les principaux objectifs de ma thèse ont été i) de valider la cath-D comme une cible extracellulaire d’intérêt pour les patientes atteintes de TNBC, ii) d’évaluer les effets thérapeutiques et iii) de caractériser les mécanismes d’action des anticorps humains anti-cath-D.Nous avons montré que des niveaux élevés d'ARNm de CTSD sont corrélés à une survie sans récidive plus courte. Par analyse protéomique et étude immunohistochimique anti-cath-D réalisée sur Tissue Micro-Array, nous avons observé que la cath-D extracellulaire est détectée dans le microenvironnement tumoral des TNBC contrairement au tissu mammaire normal. Nos résultats mettent ainsi en exergue que la cath-D serait un biomarqueur tumoral extracellulaire, suggérant que les patientes atteintes de TBNC pourraient bénéficier d’une thérapie par des anticorps anti-cath-D. Par des analyses de SPECT-CT (Single Photon Emission Computed Tomography) et de biodistribution, nous avons validé que les anticorps humains anti-cath-D, F1 et E2, s’accumulent dans les xénogreffes de la lignée TNBC MDA-MB-231 chez la souris athymique. Les anticorps F1 et E2 inhibent la croissance tumorale des xénogreffes MDA-MB-231 et améliorent la survie des souris athymiques. Notre meilleur anticorps anti-cath-D, F1, inhibe également la croissance tumorale de deux lignées de TNBC PDX (patient-tumor derived xenografts). Au niveau mécanistique, l’anticorps F1 module le microenvironnement immunitaire dans les tumeurs issues des xénogreffes MDA-MB-231. L’ensemble de nos résultats suggèrent qu’une immunothérapie avec des anticorps humains anti-cath-D pourrait être une nouvelle approche thérapeutique pour les patientes atteintes de TNBC. / Triple-negative breast cancer (TNBC) accounts for 15-20% of all breast cancer cases, and chemotherapy is the only available treatment. Thus identification of new therapeutic targets is required to improve TNBC outcome. In breast cancer, the aspartic protease cathepsin D (cath-D) is a marker of poor prognosis associated with metastatic risk. This protease is overexpressed by breast cancer cells and is abnormally hypersecreted into the tumor microenvironment. Cath-D affects both cancer and stromal cells in the breast tumor microenvironment by increasing the proliferation of breast cancer cells, fibroblast invasive outgrowth, tumor growth and angiogenesis, and metastasis formation. Many studies indicated that extracellular cath-D displays oncogenic activities, suggesting it could represent a novel therapeutic target in TNBC. In order to block its oncogenic actions, the laboratory generated human IgG1 antibodies against extracellular cath-D by phage display (International patent N° PCT/EP2016/061454). The aims of my PhD project was i) to validate the potential value of cath-D as a tumor-specific extracellular target in TNBC, ii) to evaluate the therapeutic activity, and iii) to characterize the mechanisms of action of these anti-cath-D human antibodies.We showed that elevated CTSD mRNA levels correlated with shorter recurrence-free survival. Using proteomics analysis and anti-cath-D immunohistochemistry performed on Tissue Micro-Array, we observed that extracellular cath-D was detected in the tumor microenvironment of TNBC, but not in matched normal breast stroma samples. Our results thus indicate that cath-D is a tumor cell-associated extracellular biomarker and strongly suggest that it could be a good candidate for antibody-based therapy in TNBC. We found that anti-cath-D human antibodies, F1 and E2, accumulated in TNBC MDA-MB-231 tumor xenografts in athymic mice by SPECT-CT (Single Photon Emission Computed Tomography) and biodistribution analysis. F1 and E2 antibodies inhibited tumor growth of MDA-MB-231 tumor xenografts and improved mice survival without apparent toxicity. F1, the best antibody candidate, inhibited tumor growth of two TNBC patient-derived xenografts (PDX). Mechanistically, F1 treatment modulates immune tumor microenvironment in the MDA-MB-231 tumor cell xenograft model. Together, our results indicate that antibody-based targeting of cath-D may have therapeutic efficacy for TNBC treatment.

Introduction Triple-negative breast cancer (TNBC) has limited treatment options, as chemotherapy is the only systemic therapy. This poses a challenge in the elderly population due to the limited research done, but also the increased risk for adverse events. Aim To describe the treatment pattern, identify the risk of hospitalization and potential risk factors for hospitalization within 1 year from diagnosis, and investigate the causes of death in elderly with TNBC and possible predictors for mortality.MethodsWe performed a registry-based cohort study using the BCBaSe database which links cases of breast cancer from 3 Swedish healthcare regions with socioeconomic factors, hospitalizations and causes of death. Women ≥ 70 years old with non-metastatic TNBC, between 1/1 2007 and 31/12 2012 were chosen (n = 413). Logistic regression and Cox proportional hazards regression analyses were used. Results Age, stage and comorbidities influenced administration of chemotherapy. The risk of hospitalization overall was increased in the group receiving chemotherapy (OR 2.35, 95% CI 1.30 – 4.26) mainly due to toxicities. Chemotherapy use was not associated with either breast cancer-specific (HR 0.95, 95% CI 0.51 – 1.79) or overall survival (HR 0.72, 95% CI 0.44 – 1.18). Stage at diagnosis and comorbidities were associated with both breast cancer-specific mortality and overall mortality, whereas age was only associated with overall mortality. Conclusions In elderly TNBC patients, chemotherapy use was associated with increased risk for hospitalization within 1 year from diagnosis without improving breast cancer-specific or overall survival. No benefit regarding mortality was seen in those who were administered chemotherapy.

acase@tulane.edu / Triple-negative breast cancer (TNBC) presents a clinical challenge due to the aggressive nature of the disease and a lack of targeted therapies. Constitutive activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway has been linked to chemoresistance and metastatic progression through distinct mechanisms, including activation of epithelial-to-mesenchymal transition (EMT) whereby cells adopt a motile and invasive phenotype through loss of epithelial markers, namely Cadherin 1/E-Cadherin (CDH1), and acquisition of mesenchymal markers, such as vimentin (VIM) and Cadherin 2/N-Cadherin (CDH2). While MAPK/ERK1/2 kinase inhibitors (MEKi) have shown promise as antitumor agents in the preclinical setting, application has had limited success clinically. Activation of compensatory signaling, potentially contributing to the emergence of drug resistance, has shifted the therapeutic strategy to combine MEK1/2 inhibitors with agents targeting oncoproteins (RAF) or parallel growth pathways (PI3K). Conventional MAPK family members have been well-characterized in modulation of cellular processes involved in tumor initiation and progression, yet the role of MEK5-ERK5 in cancer biology is not completely understood. Recent studies have highlighted the importance of the MEK5 pathway in metastatic progression of various cancer types, including those of the prostate, colon, bone and breast. Furthermore, elevated levels of ERK5 expression and activity observed in breast carcinomas are linked to worse prognosis in TNBC patients. The purpose of this work is to explore MEK5 regulation of the EMT axis and to evaluate a novel pan-MEK inhibitor on clinically aggressive TNBC cells. Our results show a distinction between the MEK1/2 and MEK5 cascades in maintenance of the mesenchymal phenotype, suggesting that the MEK5 pathway may be necessary and sufficient in EMT regulation while MEK1/2 signaling further sustains the mesenchymal state of TNBC cells. Furthermore, additive effects on MET induction are evident through the inhibition of both MEK1/2 and MEK5. Taken together, these data demonstrate the need for a better understanding of the individual roles of MEK1/2 and MEK5 signaling in breast cancer and provide rationale for combined targeting of these pathways to circumvent compensatory signaling and subsequent therapeutic resistance. / 1 / Van Hoang

Triple-negative breast cancer (TNBC) accounts for 15% of all diagnosed breast cancers nationally and affects African-American women 3 times more likely than any other ethnic group. Locally, African-American women in the New Orleans area see higher incidence of TNBC cases versus African-American women from the rest of the state of Louisiana, which represents an area of heightened public health interest for the metropolitan area. TNBC is a highly metastatic disease, and targeted therapies such as tamoxifen and herceptin are ineffective due to the lack of estrogen receptor (ER) and HER2/neu target expression in TNBC tumors. Chemotherapy remains the only effective drug therapy in TNBC cases. Evaluating new classes of drugs for clinical use against TNBC as well as furthering our understanding of underlying regulatory mechanisms in TNBC is a priority. Pan-deacetylase inhibitors (DACi), like panobinostat, have shown promise in clinical trials as therapies in other cancers. Pre-clinical data of panobinostat treatment in TNBC cell lines published by this lab has been positive so far, exhibiting a reduction in TNBC metastatic potential. DACi can alter multiple signaling pathways and are known to restore dysregulated microRNA (miRNA) expression patterns (miRnome) in cancers. MiRNA are a relatively new class of non-protein coding regulatory biomolecules that exhibit a variety of cancer-related properties, many still unknown in TNBC. Pan-DACi treatment combined with miRnome analysis in TNBC cell lines can be used to identify previously unknown miRNA cancer-related properties in TNBC. The specific aim of this project consists of using DACi treatment on TNBC cell lines in conjunction with miRnome analysis to identify previously undescribed anti-cancer miRNAs and elucidate their cancer-related properties in TNBC while uncovering affected cancer pathways, detecting miRNA targets, and revealing affected downstream components. Initial miRNA expression analysis of MDA-MB-231 TNBC cells treated with panobinostat or trichostatin A versus controls produced a list of potential anti-cancer miRNA candidates for further study. Among them, investigations into miR-203 and miR-335 produced unclear results as these were theorized to have anti-metastatic properties in TNBC yet enhanced cancer properties in our models and assays. Overexpression of mir-215 (a tumor suppressor in other cancers) unexpectedly enhanced tumor growth five fold in SCID mice xenografted with lentivirally-transduced MDA-MB-231 breast cancer cells stably overexpressing miR-215 (231/215+). Further qPCR analysis of 231/215+ cells uncovered upregulation of the breast cancer-associated lncRNA, HOTAIR; the breast cancer-associated miRNA, miR-196a; as well as the entire HOXC cluster in which they reside. This represents a previously unidentified regulatory mechanism of the HOXC cluster in humans. Additionally, miR-200b overexpression in MDA-MB-231 cells induced a change in cell morphology to an epithelial-like phenotype, reduced migration by 50%, and re-expressed the epithelial marker CDH1. This demonstrates a partial reversal of epithelial-mesenchymal transition (EMT), which indicates a reduction in metastatic potential by miR-200b overexpression in MDA-MB-231 cells. Additionally, these cells exhibited increased estrogen receptor alpha and related signaling pathways while also being susceptible to reduced proliferation with the anti-estrogen drug fulvestrant at high doses. Using pan-DACi treatment of TNBC cells to analyze changes in the miRnome for unknown cancer-related miRNA candidates suitable for further investigation in TNBC, we identified miR-215 overexpression in MDA-MB-231 cells as an oncogenic event that enhances tumor growth, cell proliferation, and HOXC cluster transcription while miR-200b is an anti-metastatic miRNA that partially reverses EMT and reduces fulvestrant resistance through re-expression of estrogen receptor signaling. / acase@tulane.edu

<p>Chemotherapy remains the only available treatment for triple-negative (TN) breast cancer. Although some TN breast cancers respond initially to neoadjuvant chemotherapy, the majority of patients die within three years of treatment due to recurrent tumor growth. Developing ex vivo models for TN breast cancer recurrence and defining responsible molecules will be crucial to developing effective combination therapies for TN breast cancer patients. We have developed an in vitro model of TN breast cancer dormancy/recurrence. Short-term exposure of tumor cells to chemotherapy at clinically relevant doses enriches for a dormant tumor cell population. Several days after removing chemotherapy, dormant tumor cells regain proliferative ability and establish colonies, resembling tumor recurrence. Tumor cells from "recurrent" colonies exhibit increased chemotherapy resistance, resembling therapy resistance of recurrent tumors in patients. Furthermore, we identify a novel signaling axis [nuclear bFGF/DNA-dependent protein kinase (DNA-PK)] supported by chemotherapy-enriched dormant TN breast cancer cells. This signaling axis drives accelerated DNA repair in chemo-residual TN breast cancer cells. Targeting this axis with either with a bFGF shRNA or DNA-PK small molecule inhibitor blocks recurrent colony formation. Using the Oncomine gene expression database, we found that bFGF expression in tumor samples from TN breast cancer patients predicts five year tumor recurrence following neoadjuvant chemotherapy treatment. Finally, we demonstrate that recurrent tumor cells exhibit increased invasiveness, reflecting the aggressive behavior of recurrent tumors in patients. Collectively, these studies identify a novel signaling axis in TN breast cancer that likely contributes to tumor recurrence and provide molecular targets for developing future therapeutics against TN breast cancer.</p> / Dissertation

abstract: In the U.S., breast cancer (BC) incidences among African American (AA) and CA (CA) women are similar, yet AA women have a significantly higher mortality rate. In addition, AA women often present with tumors at a younger age, with a higher tumor grade/stage and are more likely to be diagnosed with the highly aggressive triple-negative breast cancer (TNBC) subtype. Even within the TNBC subtype, AA women have a worse clinical outcome compared to CA. Although multiple socio-economic and lifestyle factors may contribute to these observed health disparities, it is essential that the underlying biological differences between CA and AA TNBC are identified. In this study, gene expression profiling was performed on archived FFPE samples, obtained from CA and AA women diagnosed with early stage TNBC. Initial analysis revealed a pattern of differential expression in the AA cohort compared to CA. Further molecular characterization results showed that the AA cohort segregated into 3-TNBC molecular subtypes; Basal-like (BL2), Immunomodulatory (IM) and Mesenchymal (M). Gene expression analyses resulted in 190 differentially expressed genes between the AA and CA cohorts. Pathway enrichment analysis demonstrated that differentially expressed genes were over-represented in cytoskeletal remodeling, cell adhesion, tight junctions, and immune response in the AA TNBC -cohort. Furthermore, genes in the Wnt/β-catenin pathway were over-expressed. These results were validated using RT-qPCR on an independent cohort of FFPE samples from AA and CA women with early stage TNBC, and identified Caveolin-1 (CAV1) as being significantly expressed in the AA-TNBC cohort. Furthermore, CAV1 was shown to be highly expressed in a cell line panel of TNBC, in particular, those of the mesenchymal and basal-like molecular subtype. Finally, silencing of CAV1 expression by siRNA resulted in a significant decrease in proliferation in each of the TNBC cell lines. These observations suggest that CAV1 expression may contribute to the more aggressive phenotype observed in AA women diagnosed with TNBC. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2015

abstract: In the United States, 12% of women are typically diagnosed with breast cancer, where 20-30% of these cases are identified as Triple Negative Breast Cancer (TNBC). In the state of Arizona, 810 deaths occur due to breast cancer and more than 4,600 cases are diagnosed every year (American Cancer Society). The lack of estrogen, progesterone, and HER2 receptors in TNBC makes discovery of targeted therapies further challenging. To tackle this issue, a novel multi-component drug vehicle is presented. Previously, we have shown that mitoxantrone, a DNA damaging drug, can sensitize TNBC cells to TRAIL, which is a protein that can selectively kill cancer cells. In this current study, we have formulated aminoglycoside-derived nanoparticles (liposomes) loaded with mitoxantrone, PARP inhibitors, for delivery to cancer cells. PARP inhibitors are helpful in preventing cancer cells from repairing their DNA following damage with other drugs (e.g. mitoxantrone). Various treatment liposome groups, consisting of lipid-containing polymers (lipopolymers) synthesized in our laboratory, were formulated and characterized for their size, surface charge, and stability. PARP inhibitors and treatment of cells for in-vitro and in-vivo experiments with these liposomes resulted in synergistic death of cancer cells. Finally, studies to evaluate the pre-clinical efficacy of these approaches using immuno-deficient mouse models of TNBC disease have been initiated. / Dissertation/Thesis / Masters Thesis Chemical Engineering 2018

Tumor heterogeneity observed between patients has made it challenging to develop universal or broadly effective cancer therapies. Therefore, an ever-growing movement within cancer research aims to tailor cancer therapies to individual patients or specific tumor subtypes. Tumor stratification is generally dictated by the genomic mutation status of the tumor cells themselves. Importantly, non-genetic influences – such as interactions between tumor cells and other components of the tumor microenvironment – have largely been ignored. Therefore, in an effort to increase treatment predictability and efficacy, we investigated how tumor-stroma interactions contribute to drug sensitivity and drug resistance. I designed a high throughput co-culture screening platform to measure how tumor-stroma interactions alter drug mediated cell death. I identified tumor-stroma interactions that strongly desensitize or sensitize cancer cells to various drug treatments. The directionality of these observed phenotypes was dependent on the stromal cell tissue of origin. Further study revealed that interactions between tumor cells and fibroblasts modulate apoptotic priming in tumor cells to mediate sensitivity to chemotherapeutics. The principles uncovered in this study have important implications on the use of drugs that are designed to enhance apoptosis. For example, based on our screening data, I hypothesized and experimentally validated that the effectiveness of BH3 mimetic compounds would be strongly dependent on the fibroblast growth environment. Taken together, our study highlights the importance of understanding how environmental interactions alter the drug responses of cancer cells and reveals a mechanism by which stromal cells drive broad spectrum changes in tumor cell sensitivities to common chemotherapeutics.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23469号 / 医博第4776号 / 新制||医||1053(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 溝脇 尚志, 教授 武藤 学, 教授 森田 智視 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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