Indiana University-Purdue University Indianapolis (IUPUI) / Triple-negative breast cancers (TNBCs) are negative for the expression of estrogen (ER), progesterone (PR), and HER-2 receptors. TNBC accounts for 15% of all breast cancers and results in disproportionally higher mortality compared to ER & HER2-positive tumours. Moreover, there is a paucity of therapies for this subtype of breast cancer resulting primarily from an inadequate understanding of the transcriptional differences that differentiate TNBC from normal breast. To this end, we embarked on a comprehensive examination of the transcriptomes of TNBCs and normal breast tissues using next-generation whole transcriptome sequencing (RNA-Seq). By comparing RNA-seq data from these tissues, we report the presence of differentially expressed coding and non-coding genes, novel transcribed regions, and mutations not previously reported in breast cancer. From these data we have identified two major themes. First, BRCA1 mutations are well known to be associated with development of TNBC. From these data we have identified many genes that work in concert with BRCA1 that are dysregulated suggesting a role of BRCA1 associated genes with sporadic TNBC. In addition, we observe a mutational profile in genes also associated with BRCA1 and DNA repair that lend more evidence to its role. Second, we demonstrate that using microdissected normal epithelium maybe an optimal comparator when searching for novel therapeutic targets for TNBC. Previous studies have used other controls such as reduction mammoplasties, adjacent normal tissue, or other breast cancer subtypes, which may be sub-optimal and have lead to identifying ineffective therapeutic targets. Our data suggests that the comparison of microdissected ductal epithelium to TNBC can identify potential therapeutic targets that may lead to be better clinical efficacy. In summation, with these data, we provide a detailed transcriptional landscape of TNBC and normal breast that we believe will lead to a better understanding of this complex disease.

No description available.

No description available.

No description available.

Poly-(ADP-ribsoyl) Polymerases (PARPs) are a superfamily of enzymes comprised of 17 known isoforms. PARP inhibitors (PARPi) have shown success in clinical trials for the treatment of homologous recombination-deficient cancers. Though proven effective initially, tumors treated with PARPi eventually develop resistance. Combinatorial therapeutics targeting PARP and other pathways that may re-sensitize tumors to PARP inhibition, including PI3K/AKT/mTor pathway, and cell-cycle checkpoints (such as CDKs, CHK, and Wee) are being tested. In this context, the synthetic lethality of cyclin-dependent kinase 1 (CDK1) and PARP1 is known. Evaluation of PARP1 and CDK1 pharmacophores led to the development of the tetrahydro-arylazepinone (TAAP) scaffold as a potential dual PARP1/CDK1 inhibitor. We screened a handful of TAAP analogs against PARP1 in a cell-free assay that identified the low micromolar PARP1 inhibitor 1,2,3,4-tetrahydro-5H-benzo[e][1,4]-diazepin-5-one (TBAP), which served as the lead compound. The analogous 1,2,3,4-tetrahydro-5H-pyrido[2,3-e][1,4]-diazepin-5-one (TPAP) series showed a similar bioactivity profile. Satisfyingly, the N1-benzyl TPAP analogue showed activity in the low nanomolar range. The TAAP series (i.e., 6/7-membered scaffold) unfortunately lacked CDK1 inhibitory activity. Finally, many PARPi's show poor isoform-selectivity. The development of isoform-selective PARPi can clarify the specific function of each PARP isoform and may reduce the adverse side effects shown by PARPi. A handful of TAAP analogs were screened against 13 PARP isoforms, where some compounds demonstrated exquisite PARP1/2 selectivity. Concurrently, we discovered an inhibitor for PARP11, an isoform that lacks any known synthetic ligand. Future directions are suggested towards fine-tuning the structure-activity relationship of TAAP-isoform selective PARPi as well as developing a dual PARP1/CDK1 inhibitor. / Master of Science / The aim of this work is to explore the therapeutic potential of poly-(ADP-ribosyl) polymerase inhibitors (PARPi) for the treatment of ovarian and breast cancer, specifically triple negative breast cancer. Poly-(ADP-ribsoyl) Polymerases (PARPs) are a superfamily of enzymes comprised of 17 known isoforms. Currently, there are three FDA approved PARPi - olaparib, isoforms. Further, tumors have been shown to develop resistance to PARPi. Herein, we explored the 1,2,3,4-tetrahydro-5H-aryl[1,4]diazepin-5-one scaffold as a potential PARP1/2-selective rucaparib, and niraparib; however, these PARPi demonstrate non-selectivity amongst the PARP inhibitor and its possibility for targeted pharmacology with other kinases.

[ES] El cáncer de mama triple negativo representa el 10-20% de los casos de cáncer de mama. Este subtipo molecular se caracteriza por su gran agresividad y capacidad invasiva y se asocia a un mal pronóstico. La ausencia de receptores hormonales y de HER2 hace que la quimioterapia sea el principal tratamiento sistémico eficaz. Sin embargo, varias pacientes con cáncer de mama triple negativo recaen tras recibir quimioterapia. Por lo tanto, es crucial descifrar las características moleculares y buscar nuevas herramientas terapéuticas para este subtipo. En este sentido, los microRNAs (miRNAs) están desregulados en varios tipos de cáncer y emergen como potenciales moléculas pronósticas implicadas en procesos biológicos y en la respuesta a la quimioterapia. Esta tesis titulada "Papel de los microRNAs-449 en las características del cáncer de mama: de la regulación epigenética a la resistencia a la quimioterapia" se centra en el papel de la familia miRNA-449 (miRNA-449a, miRNA-449b-5p y miRNA-449c-5p) en la modulación de la agresividad del cáncer de mama y la respuesta a la quimioterapia. Este estudio aportó pruebas de la regulación a la baja de los miRNAs-449 en líneas celulares de cáncer de mama triple negativo y en pacientes, y sugirió la desacetilación de histonas de su región promotora como mecanismo inhibidor. En particular, se observó que las histonas desacetilasas HDAC1 y SIRT1 estaban reguladas al alza en las células de cáncer de mama triple negativo, y se sugirió su inhibición como mecanismo de inhibición. Los análisis in silico señalaron a ACSL4, una enzima activadora de ácidos grasos, como diana de miARNs-449. Esto concuerda con otros estudios que señalan una alteración del metabolismo de los lípidos como causa de la progresión del cáncer. Se observó una correlación inversa entre los miRNA-449 y la expresión de ACSL4 en líneas celulares de cáncer de mama triple negativo y en pacientes, y el ensayo de reportero de luciferasa confirmó por primera vez esta diana directa de ACSL4 por los miRNA-449a y miRNA-449b-5p. Además, la sobreexpresión del microRNA-449c-5p por sí sola también produjo una regulación a la baja de ACSL4, por lo que también se sugirió una relación indirecta a través de la modulación de moléculas desconocidas. La sobreexpresión de miRNAs-449 y la infraexpresión de ACSL4 inhibieron la proliferación celular, la migración al disminuir la capacidad de sufrir el proceso de transición epitelial-mesenquimal, y la formación de colonias al regular a la baja los marcadores de troncalidad. Estos resultados sugieren una inhibición miRNAs-449 de la agresividad del cáncer de mama a través de la regulación a la baja de ACSL4. Además, basándonos en la literatura previa y en los resultados publicados por nuestro laboratorio, se evaluó la implicación de los miRNAs-449 en la respuesta a la quimioterapia a través de esta nueva diana. Los miRNAs-449 fueron regulados a la baja en las células resistentes a la doxorrubicina. A su vez, se observó una sobreexpresión de ACSL4 en células resistentes a la doxorrubicina y en pacientes que recayeron tras un tratamiento con quimioterapia. El tratamiento posterior con doxorrubicina aumentó la expresión de miRNAs-449 pero disminuyó la de ACSL4 en las células sensibles a la doxorrubicina, pero no en las resistentes, sugiriendo así una implicación de miRNAs-449 y ACSL4 en la respuesta a la quimioterapia. En este estudio, observamos que la sobreexpresión de miRNAs-449 producía sensibilidad a la doxorrubicina a través de la regulación a la baja de ACSL4 mediante ensayos de viabilidad y apoptosis. Además, la inhibición de ACSL4 disminuyó la expresión de la bomba de extrusión de fármacos ABCG2, lo que condujo a un aumento de la acumulación de doxorrubicina en las células. La implicación del miRNAs-449 en la inhibición de la agresividad y la sensibilidad a la doxorrubicina sugiere su potencial uso como herramienta terapéutica en el cáncer de mama triple negativo. / [CA] El càncer de mama triple negatiu representa el 10-20% dels casos de càncer de mama. Aquest subtipus molecular es caracteritza per la seua gran agressivitat i capacitat invasiva i s'associa a un mal pronòstic. L'absència de receptors hormonals i d'HER2 fa que la quimioteràpia siga el principal tractament sistèmic eficaç. No obstant això, diverses pacients amb càncer de mama triple negatiu recauen després de rebre quimioteràpia. Per tant, és crucial desxifrar les característiques moleculars i buscar noves eines terapèutiques per a aquest subtipus. En aquest sentit, els microRNAs (miRNAs) estan desregulats en diversos tipus de càncer i emergeixen com a potencials molècules pronósticas implicades en processos biològics i en la resposta a la quimioteràpia. Aquesta tesi titulada "Paper dels microRNAs-449 en les característiques del càncer de mama: de la regulació epigenètica a la resistència a la quimioteràpia" se centra en el paper de la família miRNA-449 (miRNA-449a, miRNA-449b-5p i miRNA-449c-5p) en la modulació de l'agressivitat del càncer de mama i la resposta a la quimioteràpia. Aquest estudi va aportar proves de la regulació a la baixa dels miRNAs-449 en línies cel·lulars de càncer de mama triple negatiu i en pacients, i va suggerir la *desacetilación d'histones de la seua regió promotora com a mecanisme inhibidor. En particular, es va observar que les histones desacetilasas HDAC1 i SIRT1 estaven regulades a l'alça en les cèl·lules de càncer de mama triple negatiu, i es va suggerir la seua inhibició com a mecanisme d'inhibició. Les anàlisis in silico van assenyalar a ACSL4, un enzim activador d'àcids grassos, com a diana de miARNs-449. Això concorda amb altres estudis que assenyalen una alteració del metabolisme dels lípids com a causa de la progressió del càncer. Es va observar una correlació inversa entre els miRNA-449 i l'expressió d'ACSL4 en línies cel·lulars de càncer de mama triple negatiu i en pacients, i l'assaig de reporter de luciferasa va confirmar per primera vegada aquesta diana directa d'ACSL4 pels miRNA-449a i miRNA-449b-5p. A més, la sobreexpressió del miRNA-449c-5p per si sola també va produir una regulació a la baixa d'ACSL4, per la qual cosa també es va suggerir una relació indirecta a través de la modulació de molècules desconegudes. La sobreexpressió de miRNAs-449 i la infraexpressió d'ACSL4 van inhibir la proliferació cel·lular, la migració en disminuir la capacitat de patir el procés de transició epitelial-mesenquimal, i la formació de colònies en regular a la baixa els marcadors de troncalitat. Aquests resultats suggereixen una inhibició de l'agressivitat del càncer de mama a través de la regulació a la baixa d'ACSL4. A més, basant-nos en la literatura prèvia i en els resultats publicats pel nostre laboratori, es va avaluar la implicació dels miRNAs-449 en la resposta a la quimioteràpia a través d'aquesta nova diana. Els miRNAs-449 van ser regulats a la baixa en les cèl·lules resistents a la doxorubicina. Al seu torn, es va observar una sobreexpressió d'ACSL4 en cèl·lules resistents a la doxorubicina i en pacients que van recaure després d'un tractament amb quimioteràpia. El tractament posterior amb doxorubicina va augmentar l'expressió de micRNAs-449 però va disminuir la d'ACSL4 en les cèl·lules sensibles a la doxorubicina, però no en les resistents, suggerint així una implicació dels miRNAs-449 i ACSL4 en la resposta a la quimioteràpia. En aquest estudi, observem que la sobreexpressió dels miRNAs-449 produïa sensibilitat a la doxorubicina a través de la regulació a la baixa d'ACSL4 mitjançant assajos de viabilitat i apoptosi. A més, la inhibició d'ACSL4 va disminuir l'expressió de la bomba d'extrusió de fàrmacs ABCG2, la qual cosa va conduir a un augment de l'acumulació de doxorubicina en les cèl·lules. La implicació dels miRNAs-449 en la inhibició de l'agressivitat i la sensibilitat a la doxorubicina suggereix el seu potencial ús com a eina terapèutica en el càncer de mama triple negatiu. / [EN] Triple-negative breast cancer accounts for 10-20% of breast cancer cases. This molecular subtype is characterized by its highly aggressive and invasive capacity being associated with a bad prognosis. The lack of hormone and HER2 receptors makes chemotherapy the main systemic effective treatment. However, several triple-negative breast cancer patients relapse after receiving chemotherapy. Therefore, deciphering the molecular characteristics and searching for new therapeutic tools for this subtype is crucial. In this sense, microRNAs (miRNAs) are found dysregulated in several cancer types and emerge as potential prognostic molecules involved in biological processes and chemotherapy response. This thesis entitled "Role of microRNAs-449 in breast cancer features: From epigenetic regulation to chemotherapy resistance" is focused on the role of the miRNA-449 (miRNA-449a, miRNA-449b-5p, and miRNA-449c-5p) family in the modulation of breast cancer aggressiveness and chemotherapy response. This study provided evidence of microRNAs-449 downregulation in triple-negative breast cancer cell lines and patients and suggested histone deacetylation of its promoter region as an inhibitor mechanism. Particularly, the histone deacetylases HDAC1 and SIRT1 were found upregulated in triple-negative breast cancer cells, and their genetic and chemical inhibition increased microRNAs-449 expression. In addition, a negative feedback loop modulation between miRNAs-449 and HDAC1/SIRT1 was observed, thus contributing to the homeostasis or tumoral phenotype of cells. In silico analyses pointed out ACSL4, a fatty acid-activating enzyme, as target of miRNAs-449. This is in concordance with other studies that pointed out an altered lipid metabolism to sustain cancer progression. An inverse correlation between microRNAs-449 and ACSL4 expression was observed in triple-negative breast cancer cell lines and patients, and luciferase reporter assay confirmed this ACSL4 direct targeting by miRNA-449a and miRNA-449b-5p for the first time. Moreover, the microRNA-449c-5p overexpression alone also produced an ACSL4 downregulation, so an indirect relationship was also suggested through the modulation of unknown molecules. MiRNAs-449 overexpression and ACSL4 knockdown inhibited cell proliferation, migration by diminishing the ability to undergo the epithelial-mesenchymal transition process, and colony formation by downregulating markers of stemness. These results suggested a microRNAs-449 inhibition of breast cancer aggressiveness through ACSL4 downregulation. Furthermore, based on previous literature and published results by our laboratory, a miRNAs-449 implication in chemotherapy response was evaluated through this novel target. MiRNAs-449 were downregulated in doxorubicin-resistant cells. In turn, ACSL4 overexpression was observed in doxorubicin-resistant cells and patients who relapsed after chemotherapy-containing treatment. Subsequent doxorubicin treatment increased miRNAs-449 but decreased ACSL4 expression in doxorubicin-sensitive, but not in resistant cells, thus suggesting a miRNAs-449 and ACSL4 implication in chemotherapy response. In this study, we observed that miRNAs-449 overexpression produced doxorubicin sensitivity through ACSL4 downregulation by viability and apoptosis assay. In addition, ACSL4 inhibition decreased the drug extrusion pump ABCG2's expression, leading to an increased doxorubicin accumulation in cells. The involvement of miRNAs-449 in the inhibition of aggressiveness and sensitivity to doxorubicin suggests its potential use as a therapeutic tool in triple-negative breast cancer. / Torres Ruiz, S. (2023). Papel de la familia de microRNAs-449 y el gen CDC20B en la respuesta a tratamientos en cáncer de mama: organoides y cultivos primarios como modelos tumorales [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/198846

Doctor of Philosophy / Department of Biochemistry and Molecular Biophysics / Anna Zolkiewska / The ADAM (A Disintegrin and Metalloprotease) family of multi-domain proteins modulates a number of cellular signaling pathways in both normal and cancerous cells. ADAM12 has been shown to be a candidate cancer gene for breast cancer and its expression is up-regulated in breast tumors. The human ADAM12 transcript is alternatively spliced. One of these splice variants encodes a transmembrane ADAM12 isoform, ADAM12-L, which has been demonstrated to release cell signaling molecules from the cell surface. Another variant encodes a secreted protease, ADAM12-S, which cleaves extracellular matrix proteins and other secreted proteins. Although these variants are expressed from the same promoter, their relative expression levels are highly discordant. Here, I demonstrate variant-specific regulation of ADAM12 transcripts by microRNAs. Members of the microRNA-29 and microRNA-200 families target the unique 3’UTR of the ADAM12-L transcript and cause transcript degradation. Additionally, I show the presence of a novel ADAM12 splicing event in which 9 additional nucleotides are inserted in the region encoding the autoinhibitory pro-domain. I demonstrate that this novel variant is expressed in breast epithelial cells and breast cancer cell lines. The resulting protein isoform does not undergo proteolytic processing to activate the protease. Additionally, trafficking of the novel isoform to the cell surface is impaired and this isoform is localized to the endoplasmic reticulum. Finally, I determined a role for ADAM12-L in the progression of triple negative breast cancers (TNBCs). These tumors are lacking expression of hormone receptors and the HER2 receptor. HER2 is a member of the epidermal growth factor receptor (EGFR) family and the loss of the HER2 receptor causes tumors to rely on EGFR for propagating pro-growth signals. I show here that, in TNBC tumors, ADAM12-L expression is strongly correlated with poor patient prognosis and increased activation of EGFR. These data suggest that in TNBCs, ADAM12-L enhances tumor growth via EGFR activation. Collectively, the data presented here demonstrate (a) transcript-specific regulation of ADAM12 in breast cancer, (b) the existence of a novel splice variant and protein isoform with impaired cellular trafficking, and (c) an important role of the ADAM12-L isoform in EGFR activation in TNBC.

Introdução: O cãncer de mama com fenótipo triplo negativo é definido como um tumor com receptor de estrógno e progesterona negativos e human epidermal growth factor receptor 2 (HER2) negativo. Desde sua primeira descrição, alguns artigos tentaram descrever sua aparência radiológica, sem nenhum consenso. Estas pacientes podem ter características clínicas e imaginológicos distintas. Seria interessante verificar se as características radiológicas desses tumores são as mesmas relatadas no câncer de mama familiar e com mutação do gene BRCA1, muitas vezes associados a esse subtipo tumoral. Estas características que muitas vezes simulariam doenças benignas poderiam atrasar o diagnóstico precoce, refletindo a necessidade de seu conhecimento radiológico na prática. Neste contexto, este estudo poderia auxiliar no reconhecimento precoce de lesões mamárias e alertar o médico para solicitar uma biópsia, consequentemente, resultando num diagnóstico precoce. Objetivo: O principal objetivo desse estudo foi avaliar as características radiológicas dos carcinomas ductais invasivos de mama nos seguintes métodos de diagnóstico por imagem: mamografia, ultrassonografia e ressonância magnética, utilizando a padronização do American College of Radiology Breast Imaging Reporting and Data System (ACR BI-RADS®), comparando os casos de carcinomas com fenótipo triplo negativo dividindo-os em não basal e basal com o uso da citoqueratina 5. Pacientes e Métodos: Revisamos os arquivos de imagens do Instituto de Radiologia (InRad) da Faculdade de Medicina da Universidade de São Paulo (FMUSP) e do Instituto do Câncer do Estado de São Paulo (ICESP), durante o período da coleta de dados, que envolveu 12 anos. Trata-se de estudo descritivo observacional, realizado após a aprovação do comitê de ética. Foram avaliadas 6.952 resultados de biópsias cirúrgicas ou percutâneas por agulha grossa de mama e após a verificação do material radiológico e histológico, chegamos ao resultado de 106 casos. Todas as reações imunohistoquímicas foram lidas por dois patologistas e os exames radiológicos foram avaliados por dois radiologistas, todos especialistas em doenças da mama em suas respectivas áreas. Resultados: A maioria das pacientes incluídas foram mulheres brancas, com idade entre 24-81 anos. Na mamografia, a principal lesão observada foi o nódulo (74,7%), com margem espiculada (36,9%) e forma oval (60,0%), seguido pela assimetria focal (12,6%), lesões ocultas (9,2%) e assimetria global (3,4%). Nenhuma lesão principal se manifestou como microcalcificações. Os tumores avaliados pela ressonância magnética se manifestaram principalmente como lesão nodular (92,0%), com forma irregular (60,0%), margem irregular (44%) e realce heterogêneo (56,0%). A curva tipo 3 (91,7%) foi comumente observada. O isosinal em T2 foi mais freqüentemente observado (52,2%). A ausência de áreas císticas foi observada na maioria das lesões (47,8%). Todos os tumores avaliados pelo ultrassom foram vistos, principalmente como nódulos (98,1%), hipoecoicos (90,3%), com forma irregular (61,2%), margem mal definida (34,7%) e o reforço acústico posterior (32,0%) e halo ecogênico (46,6%) foram comumente observados. Conclusão: Não foram encontradas associações estatisticamente significativas das características tumorais e demográficas com os subtipos de tumores (p > 0,05) na mamografia, ultrassom e ressonância magnética. Houve correlaçãos inversa entre a idade e o tamanho do tumor. Apenas no USG os linfonodos positivos apresentaram em média estatisticamente maior tamanho tumoral associado que os linfonodos negativos (p=0,045). Em nossa série, observamos que embora algumas características tenham sido mais frequentes, não houveram características que mostraram diferença estatisticamente significante entre os subtipos. Assim, não se pode atribuir qualquer característica específica que possa melhorar a acurácia diagnóstica / Introduction: Breast cancer with triple negative phenotype is defined as estrogen receptor negative tumor, progesterone receptor negative and human epidermal growth factor receptor 2 (HER2) negative. Since its first description, some articles have tried to describe its radiological appearance, with no consensus. These patients can have adverse clinical and imaging characteristics. It would be interesting to check if the radiological characteristics of these tumors are the same related in the familiar breast cancer and associated to the mutation of gene BRCA1, many times associated to this tumor subtype. These characteristics, that many times would simulate benign diseases, could delay the early diagnosis, reflecting the need of its radiological knowledge in practice. In this context, this study could help in the early recognition of breast lesions and alert the doctor to require a biopsy, consequently resulting in an early diagnosis. Objectives: The main objective of this study was to evaluate the radiological characteristics of breast invasive ductal breast carcinomas with triple negative invasive phenotype in the following diagnostic image methods: mammography, ultrasound and magnetic resonance, using the American College of Radiology Breast Imaging Reporting and Data System (ACR BIRADS ®) criteria, divided in basal and not basal subtypes with the use of cytokeratin 5. Patients and Methods: We\'ve reviewed the images from the Radiology Institute (InRad) from Faculty of Medicine of the University of São Paulo (FMUSP) and from the Cancer Institute of São Paulo (ICESP), during the period of data collecting, which took 12 years. This aims to be an observational descriptive study, realized after the Ethic Committee\'s approval. 6952 results of breast surgical or percutaneous needle biopsies were evaluated and after checking the radiological and histological material, we came to the result of 106 cases. All the immunohistochemical reactions were read by two pathologists and the radiological exams were evaluated by two radiologists, all specialists in breast illnesses in their respective areas. Results: Most patients included were white women, aging between 24-81 years old. In the mammography, the main lesion observed was the nodule (74,7%), spiculated margin (36,9%) and oval shape (60%), followed by focal asymmetry (12,6%), hidden lesions (9,2%) and global asymmetry (3,4%). No main lesion showed microcalcifications. The tumors evaluated by magnetic resonance imaging showed mainly as nodule (92,0%), irregular shaped (60,0%), irregular margin (44,0%) and heterogeneous enhancement (56,0%). The curve type 3 (91,7%) was generally observed. The isosignal in T2 was most frequently observed (52,2%). The absence of cystic areas was observed in most of the lesions (47,8%). The tumors evaluated by the ultrasound were seen principally as nodules (98,1%), hypoechoic (90,3%), irregular shaped (61,2%), ill-defined margin (34,7%) and with posterior acoustic enhancement (32,0%) and echogenic halo (46,6%). Conclusion: There were no statistically significant associations of tumor and demographic characteristics with tumor subtypes (p > 0.05) in mammography, ultrasound and magnetic resonance imaging. There were inverse correlations between the age and the size of the tumor. Only in USG positive lymph nodes had on average larger tumor size with a statistically significant result that the negative lymph nodes (p = 0,045). In our series, we\'ve observed that, although some characteristics have been more frequent, there were no characteristics which showed difference statistically significant among the subtypes. Therefore, we can not attribute any specific characteristic that can improve the diagnostic accuracy

Stimuler la réponse immunitaire anti-tumorale constitue une voie d’avenir indiscutable pour le traitement des cancers. Aujourd'hui, les thérapies ciblées à base d'anticorps ont une place majeure dans l’immunothérapie des cancers du sein de par leur impact positif sur le pronostic des patientes. Cependant, les cancers du sein triple négatifs (TNBC) résistent aux innovations thérapeutiques actuelles, et, par défaut de traitement ciblé efficace, restent de sombre pronostic. Notre équipe développe des stratégies d’immunothérapie à base d'anticorps bispécifiques (bsFabs) conçus à partir de fragments d'anticorps de camélidés qui présentent la particularité de cibler simultanément les cellules immunitaires et tumorales. Ainsi, mon projet visait à évaluer le potentiel anti-tumoral de deux bsFabs sur des modèles précliniques de TNBC à travers leur capacité à activer et à rediriger le système immunitaire contre les cellules tumorales. La finalité du projet est de proposer un nouvel axe de thérapie ciblée susceptible d'améliorer le pronostic des patientes atteintes de TNBC. / Mounting evidence of the key contribution of NK cells in immunity against cancer has boosted the investigations on NK cell-based therapies. Among these strategies, monoclonal antibody-based therapeutics (mAbs) are currently the fastest growing segment of the medicine market. Despite therapeutic innovations, triple negative breast cancers (TNBC) remain insensitive to the current targeted or hormono-therapies. Our objective is to manipulate NK cell functions and tumor targets using an original format of nanobody-based bispecific antibodies (bsFab) to revert the dampened immune response for treating TNBC. Thus, we generate two bsFabs able to crosslink NK and tumor cells. NK antitumor effects driven by mAbs and bsFabs, alone or in combination, were investigated in vitro and in vivo on preclinical TNBC models. Here, we demonstrate the potential of bsFabs to enlarge the number of patients eligible for breast cancer immunotherapy and prompt to consider combination strategies.

Breast cancer is the most prevalent cancer in women in the UK with over 50,000 new cases diagnosed each year. Almost all breast cancer deaths are due to metastatic disease. The RUNX1-CBFbeta transcription factor complex has been implicated in the development of human breast cancer and recent evidence from our laboratory indicated that it might have a role in metastasis. The aim of this project was therefore to determine the role of the RUNX1 transcription factor in breast cancer metastasis. Initial experiments to knockdown RUNX1 by shRNA also decreased the expression of RUNX2. Therefore, due to the off-target effect of shRUNX1, CRISPR-Cas9n was used to establish a RUNX1-negative cell line by targeting the first exon of the RUNX1 gene. Migration and invasion capacity of the cells decreased in the absence of RUNX1 and it was comparable to the absence of RUNX2 and CBFbeta respectively, which are known to play roles in migration and invasion of MDA-MB-231 cells. The cells also formed spherical clusters in 3D culture which was associated with the changes in cell morphology from stellate to round shape in the absence of RUNX1. The expression of the metastasis-related genes MMP13, MMP9, OPN and SLUG also decreased in parallel with the loss of the mesenchymal-like phenotype whilst the expression of the epithelial markers cytokeratin, desmoplakin and E-cadherin increased concomitantly. Importantly, re-expression of RUNX1 in the RUNX1-negative cell lines using an inducible expression system rescued migration and invasion. Therefore, RUNX1 is required to maintain the mesenchymal-like phenotype of MDA-MB-231 cells and hence is important for the epithelial to mesenchyme transition (EMT), a key characteristic of metastatic cells. The transcription factor SLUG is a known regulator of EMT. Data obtained shows that RUNX1 down-regulates the expression of SLUG. ChIP analysis demonstrated that RUNX1 was bound to the SLUG promoter and RUNX1 was subsequently shown to activate the promoter activity. Finally, experiments to inhibit the activity of the RUNX transcription factors pharmacologically showed changes in cell differentiation and also affected cell viability, possibly by off-target effects. Taken together, data presented in this work demonstrates that RUNX1 is required for EMT in the metastatic breast cancer cells and it is therefore a potential therapeutic target to prevent breast cancer metastasis.