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

INTRODUÇÃO: O câncer de mama é uma importante causa de morbimortalidade, é conhecido por ser uma doença heterogênea. A caracterização clínica e molecular de seus subtipos é fundamental para nortear o prognóstico e o tratamento dessas pacientes. Novos estudos são necessários na melhor caracterização dos tumores triplo-negativos. O estudo da expressão das claudinas pode auxiliar na caracterização desses tumores. OBJETIVO: Investigar a associação da expressão das claudinas 1, 3, 4 e 7 e E-caderina com variáveis clínico-patológicas e fatores prognósticos, em uma série de tumores de mama triplo-negativos (RE-, RP- e HER2-). MÉTODOS: 80 tumores triplo-negativos foram analisados por imunoistoquímica automatizada para as claudinas 1, 3, 4, 7 e E-caderina. A expressão imunoistoquímica foi avaliada pelo escore H (intensidade multiplicada pela porcentagem de marcação). Foram avaliadas as associações entre características clínico-patólogicas e o escore H. Para a avaliação prognóstica das pacientes, curvas de Kaplan-Meier foram construídas a partir dos dados de seguimento das pacientes e do escore H. RESULTADOS: Foi encontrada associação significativa entre o alto escore H da CLDN-1 (HCLDN-1) e pacientes mais idosas e com a presença de necrose, alto escore H da E-caderina (HE-CAD) em pacientes mais jovens e baixo escore H da CLDN-7 (HCLDN-7) e Ki67 positivo. Além disso, pacientes com elevado HCLDN-1 tiveram menor sobrevida geral. Por outro lado, o elevado HCLDN-3 apresentou uma tendência à associação com maior sobrevida geral e sobrevida livre de doença. CONCLUSÕES: A expressão diferencial das claudinas e E-caderina podem auxiliar na caracterização clinico-patológica dos tumores triplo-negativos. Além disso, as claudinas 1 e 3 parecem ser fatores prognósticos para esses tumores. / INTRODUCTION: Breast cancer is a major cause of morbidity and mortality, is known to be a heterogeneous disease. The clinical and molecular characterization of its subtypes is critical to guide the prognosis and treatment of these patients. Further studies are needed for the best characterization of triple-negative tumors. The study of the expression of claudinas can aid in the characterization of these tumors. OBJECTIVE: To investigate the association of expression of claudinas 1, 3, 4 and 7 and E-cadherin with clinicopathological variables and prognosis in a series of triple-negative breast cancers (ER-, PR- and HER2-). METHODS: 80 triple negative tumors were analyzed by automated immunohistochemistry for the claudins 1, 3, 4, 7 and E-cadherin. The immunohistochemical expression was assessed by H-Score (intensity multiplied by the percentage of staining). We evaluated the associations between clinicopathological characteristics and H-Score. For the prognostic assessment of patients, Kaplan-Meier curves were constructed from the follow-up data of patients and H-Score. RESULTS: We found a significant association between high H-Score of CLDN-1 (HCLDN-1) and older patients and the presence of necrosis, high H-Score of E-cadherin (H-CAD) in younger patients and low H-Score CLDN-7-(7-HCLDN) and Ki67 positive. Furthermore, patients with high-HCLDN1 had a lower overall survival. On the other hand, the high HCLDN-3 showed a trend toward association with greater overall survival and disease-free survival. CONCLUSIONS: Differencial expression of claudins and E-cadherin can help in clinic-pathological characterization of triple-negative tumors. Futhermore, claudin 1 and 3 appear to be prognostic factor for these tumors.

INTRODUÇÃO: o câncer de mama é uma doença heterogênea. Há necessidade de critérios diagnósticos e prognósticos mais refinados. O emprego da imunohistoquímica, através do painel prognóstico, fez despontar a figura do carcinoma triplonegativo e, da mesma forma, a histogenética trouxe à evidência o carcinoma basal. Paralelamente, o conhecimento sobre a origem biológica das neoplasias e da sua heterogeneidade vem sendo acentuadamente debatido através do tema das células-tronco tumorais. OBJETIVOS: investigar a prevalência de carcinomas basais e pentanegativos, numa amostra de carcinomas triplonegativos, e estabelecer associações com a expressão de células-tronco tumorais nestes tumores. Verificar diferenças entre estes subtipos com as variáveis clinicopatológicas. MÉTODOS: 94 carcinomas mamários triplonegativos foram testados para CK5/6, HER1, CD44 e CD24. As expressões desses marcadores por imuno-histoquímica automatizada foram avaliadas através de escore simples de positividade (porcentagem de células) e correlacionadas com os dados clínico-patológicos e análise de sobrevivência. RESULTADOS: carcinomas basais apresentam maior grau tumoral que carcinomas pentanegativos (p=0,004). A negatividade para CD44 (p=0,007) e o perfil CD44- CD24+ (p=0,013) foram associados com maior invasão vascular entre carcinomas triplonegativos. A expressão de CD44 foi associada aos carcinomas basais (p=0,007). O perfil CD44-CD24-/low foi associado aos carcinomas pentanegativos (p=0,04). Nenhuma das variáveis em estudo foi associada com os desfechos clínicos. CONCLUSÃO: Carcinomas mamários basais e pentanegativos são subtipos tumorais bastante próximos. Nosso estudo é o primeiro desenhado especificamente para avaliar a presença células tronco-tumorais mamárias entre carcinomas basais e pentanegativos, onde o perfil CD44-CD24-/low foi associado ao subtipo pentanegativo, e o perfil CD44-CD24+ à invasão vascular, resultados que merecem confirmação por histogenética em estudos de maior porte. / INTRODUCTION: Breast cancer is a heterogeneous disease, and there is a need for more refined diagnostic and prognostic criteria. Immunohistochemistry, as a breast prognostic panel, has given rise to triple-negative carcinoma, as well as histogenetics highlighted basal carcinoma. Concomitantly, the understanding of the biological origins of neoplasia and its heterogeneity has been strongly debated through the theme of cancer stem cells. OBJECTIVES: To investigate the prevalence of basal and penta-negative carcinomas in a sample of triple-negative carcinomas and to establish associations with cancer stem cells (CD44/CD24 expression profiles) and the clinicopathological variables within these tumors. METHODS: Ninety-four triplenegative breast carcinomas were tested for CK5/6, HER1, CD44 and CD24. The expression of these markers was evaluated by automated immunohistochemistry using a simple positivity score (percentage of cells) and was correlated with the clinicopathological and survival analysis data. RESULTS: Basal carcinomas had higher tumor grades than penta-negative carcinomas (p=0.004). CD44 negativity (p=0.007) and the CD44-CD24+ phenotype (p=0.013) were associated with increased vascular invasion amongst the triple-negative carcinomas. CD44 expression was associated with basal carcinomas (p=0.007). The CD44-CD24-/low phenotype was associated with penta-negative carcinomas. None of the variables in the study were associated with clinical outcome. CONCLUSION: Basal and penta-negative breast carcinomas are closely related tumor subtypes. Our study is the first to be specifically designed to assess the presence of breast cancer stem cells in basal and pentanegative carcinomas. The CD44-CD24-/low phenotype was associated with the pentanegative subtype, and the CD44-CD24+ phenotype was associated with vascular invasion. These results require histogenetic confirmation in larger studies.

INTRODUÇÃO: Carcinomas de mama triplo-negativos (CMTN) constituem um grupo de tumores heterogêneos caracterizados por sobrevida pobre dos pacientes e falta de terapia-alvo. Receptor andrógeno (AR) tem sido descrito no CMTN, mas o impacto prognóstico da expressão nesse subgrupo não está bem claro. OBJETIVO: Investigar a associação do status de expressão AR analisado por imunohistoquímica em casos de CMTN com parâmetros clínicos (idade, sobrevida) e variáveis patológicas (tamanho do tumor, grau do tumor). MÉTODOS: Foram analisados 62 CMTN por imuno-histoquímica automatizada para receptor andrógeno. A imuno-histoquímica foi avaliada por dois diferentes patologistas e a expressão do biomarcador foi avaliada por H-Score (intensidade mais percentagem de coloração). Kaplan-Meyer foi o método usado para avaliar a sobrevida global, tendo as diferenças nas distribuições sido avaliadas com base na expressão do marcador. RESULTADOS: 26% dos CMTNs foram AR positivos (n = 16) e 74% AR negativos (n = 46). Todos os casos de AR positivos ocorreram em mulheres ≥ 40 anos enquanto 13% dos casos de AR negativos foram vistos em mulheres ≤ 40 anos. 93% (52/56) de todos os CMTNs foram carcinoma ductal invasivo do tipo não especial (ICNST) e 93% (14/15) de AR positivos dos casos de ICNST. O tamanho do tumor variou de 2 a 4,9 cm na maioria dos cânceres AR positivos (n = 8/13; 62%). 60% (n = 9/15) dos casos de AR positivos mostraram grau histológico 3 seguidos de 27% de tumores de grau 2. Não se observou diferença entre pacientes AR positivos e AR negativos quando comparados por idade, tamanho do tumor, grau do tumor e tipo do tumor (p > 0,17). A positividade imuno-histoquímica para ARs também não foi associada com melhor sobrevida (p = 0,737; n = 51) ou sobrevida livre da doença (p = 0,552; n = 45) em CMTNs. CONCLUSÃO: Sob o ponto de vista do prognóstico, a imunorreatividade do AR tem sido associada com melhor sobrevida das pacientes. Esse resultado não foi confirmado na presente série. Isso poderia estar relacionado com o pequeno tamanho amostral ou com uma baixa prevalência de casos de AR positivos especificamente nesse subtipo de câncer de mama. Estudos realizados com maiores amostras são necessários para investigar esse biomarcador em CMTNs. / INTRODUCTION: Triple negative breast carcinomas (TNBC) are a heterogeneous group of tumors characterized by poor patient survival and lack of targeted therapies. Androgen receptor (AR) has been described in TNBC but the prognostic impact of the expression in this subgroup of tumors is not clear. OBJECTIVE: To investigate the association of AR expression status by immunohistochemistry in TNBC cases with clinical (age, survival) and pathological variables (tumor size, tumor grade). METHODS: 62 TNBC were analyzed by automated immunohistochemistry for androgen receptor. Immunohistochemistry was scored by two investigators and biomarker expression was assessed by H-Score (intensity plus the percentage of staining). Kaplan-Meier was used to evaluate overall survival, where differences in distributions were evaluated based on marker expression. RESULTS: 26% of TNBC were AR-positive (n = 16) and 74% AR negative (n = 46). All AR-positive cases occurred in women ≥ 40 years, while 13% of AR-negative cases were seen in women ≤ 40 years. 93% (52/56) of all TNBC were infiltrating ductal carcinomas of no special type (ICNST) and 14/15 (93%) of AR-positive cases were ICNST. Tumor size varied from 2-4.9 cm in the majority of AR-positive cancers (n = 8/13; 62%). 60% (n = 9/15) of AR-positive cases showed histological grade 3 tumors, followed by 27% of grade 2 tumors. No differences were observed between AR-positive and AR-negative patients when compared for age, tumor size, tumor grade and tumor type (all p > 0.17). AR immunohistochemical positivity was also not associated with better overall survival (p = 0.737, n = 51) or disease-free survival (p = 0.552; n = 45) in TNBCs. CONCLUSION: From the prognostic point of view, AR immunoreactivity has been associated with better overall patient survival. Unfortunately, this result could not be confirmed in our series. This could be related to the relatively small series of analyzed samples and to the low prevalence of AR-positive cases in this specific breast cancer subtype. Further studies with bigger samples are needed to investigate this biomarker in TNBC.

Therapeutics targeting the PI3K (phosphatidylinositol 3-kinase) and the Ras/MAPK (mitogen-activated protein kinases) pathways have potential as non-toxic treatments for triple-negative breast cancer due to their frequent over-activation in several forms of cancer. Interestingly, the PI3K and Ras/MAPK pathways have been shown to incite cancer dormancy behavior individually and tumorigenic behavior in unison when induced in healthy breast epithelial cells (MCF-10A) in vivo. Tumorigenesis and metastasis are heavily reliant on the specific mechanical and adhesive properties of cells, including decreased stiffness, increased mechanosensitivity, and decreased adhesion. However, the describe cellular behaviors are poorly understood for dormant cancer phenotypes. Understanding the mechanical and adhesive behaviors of MCF-10A cells as a function of PI3K and/or Ras/MAPK pathway over-activation further explores the cross-talk enabling unique dormant and tumorigenic characteristics. Cellular viscoelasticity and adhesion were measured for MCF-10A cells with PTEN (phosphatase and tensin homolog) knockout and activated KRAS (Kristen rat sarcoma viral oncogene homolog) overexpression to activate the PI3K and Ras/MAPK pathways respectively with atomic force microscopy. PTEN knockout alone has no observable influence on cell adhesion but resulted in softer cells with less organized cytoskeleton. Activated KRAS overexpression increased cell stiffness and cell adhesion regardless of PTEN expression level. Moreover, the overexpression of activated KRAS enhanced the sensitivity of cells to the substrate stiffness. The findings suggest that the cancer-associated pathways PI3K and Ras/MAPK regulate cell adhesion and mechanics to promote tumor formation and metastasis. More importantly, the results that signify mutations of different molecular pathways associated with cancer dormancy regulate cell mechanics differently suggests that cell stiffness is a biomarker that detects and differentiates different types of dormant cancers.

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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Parmi les sous-types moléculaires de cancers du sein, le cancer du sein triple-négatif (TNBC) est caractérisé par un très mauvais pronostic et ne bénéficie actuellement d’aucune thérapie ciblée efficace. Dans ce projet, nous avons analysé le profil de certaines altérations oncogéniques dans les tumeurs TNBC et évalué le potentiel thérapeutique de leur ciblage à l’aide des modèles de xénogreffes (PDX).Nous avons d'abord démontré que le récepteur à activité tyrosine kinase RET est surexprimé dans une sous-population de tumeurs du sein TN et HER2+. Le ciblage de RET par son inhibiteur Vandetanib a été testé in vivo dans trois modèles de PDX TNBC et un modèle de PDX HER2+ caractérisés par des niveaux différents d’expression de RET et d’EGFR (les cibles principales du Vandetanib). Le Vandetanib a induit une régression tumorale dans les trois modèles de PDX surexprimant RET ou EGFR. L’effet du Vandetanib a été associé à une inhibition de la voie MAPK, une inhibition de l'angiogenèse et une induction de la nécrose.Nous avons également étudié les altérations de la voie PI3K/AKT/mTOR dans une large série de PDX de cancers du sein incluant des PDX TNBC. La voie PI3K/AKT/mTOR a été trouvée activée dans le cancer du sein triple-négatif. L’altération principalement retrouvée dans cette voie est la perte des deux suppresseurs de la voie, PTEN et/ou INPP4B. Sept des quinze modèles de PDX triple-négatifs testés ont montré une réponse à l’Everolimus. L'analyse des tumeurs traitées a montré que la phosphorylation post-traitement d’AKT est significativement plus fréquente dans les modèles répondeurs par rapport aux non-répondeurs. En conclusion, mon travail de thèse a permis de montrer que le Vandetanib et l'Everolimus pourraient être efficaces pour traiter le cancer du sein triple-négatif. Des études complémentaires sont nécessaires pour valider les biomarqueurs prédictifs de réponse à ces deux thérapies ciblées. / Among breast cancer subtypes, Triple-negative breast cancer (TNBC) has a very poor prognosis. There are currently no known targeted therapies for this subgroup of patients. In this project, we analyzed the profile of certain oncogenic alterations in the TN tumors and evaluated in vivo the therapeutic potential of targeting these alterations in TNBC.We first demonstrated that the tyrosine kinase receptor RET is overexpressed in a subset of TN and HER2+ tumors. Targeting RET by his inhibitor Vandetanib was tested in vivo in three PDX models of TNBC and one model of HER2+ BC with different expression levels of RET and EGFR. Vandetanib induced tumor regression in the three PDX models with high expression of RET or EGFR. Vandetanib effect was associated with inhibition of MAPK pathway, inhibition of angiogenesis and induction of necrosis. PI3K pathway alterations were investigated in an important number of BC PDX including TNBC PDX. PI3K pathway was shown to be activated in TNBC PDX possibly by the loss of the two pathway suppressors, PTEN and/or INPP4B. Treatment by Everolimus induced response in seven among the fifteen TNBC PDX tested. Analysis of treated tumors showed that post-treatment phosphorylation of AKT was more pronounced in responder PDX. The combination of Everolimus with chemotherapy was tested in one PDX and resulted in increased efficacy.In conclusion, in this work we showed that Vandetanib and Everolimus could be effective in treating TNBC. Further investigations are still needed to validate response related biomarkers.

Docetaxel is a chemotherapeutic agent in the taxane group of drugs which is commonly used in the first line setting for metastatic hormone receptor negative breast cancer. We present a case of a 46 year old female who was diagnosed with de novo triple negative metastatic breast carcinoma, and has had an extended progression free survival (PFS) of almost 5 years on first line single agent treatment with Docetaxel. 46 year old female presented with a large left breast mass as well as axillary mass which revealed grade 3 invasive ductal carcinoma of breast on biopsy of both sites. Tumor was estrogen and progesterone receptor negative. Pathology showed discordance in HER2 testing between FISH and IHC, however on repeat testing, HER2 was confirmed to be negative. PET/CT scan for staging revealed large left sided pleural effusion and abnormal soft tissue in the lower anterior and posterior chest on the left concerning for pleural metastases. Patient underwent CT guided biopsy of left lower pleural space which was consistent with metastatic adenocarcinoma with breast primary. She was started on first line single agent chemotherapy with Docetaxel 100mg/m2 every 3 weeks. Tumor markers were non-contributory to assess disease response. Repeat systemic imaging in 3 months showed excellent partial response with decrease in size of breast mass, conglomerate axillary lymph nodes as well as pleural based metastatic foci. Patient had grade 1 neuropathy secondary to Docetaxel which was tolerable. Patient also had significant fatigue with warranted dose reduction by 20% after 6 months. She also demonstrated other adverse effects of Docetaxel such as nail dystrophy and mild blepharitis which were also tolerable. Patient showed good tolerance to chemotherapy, with intermittent treatment holidays. CT scans continued to demonstrate good response with stable size of breast and lung masses. After two years of stable disease and fair tolerance (after completing 34 cycles), chemo regimen was changed to every 4 weeks per patient’s wish. She was also started on Gabapentin for chemotherapy related neuropathy. At the end of 4 years, patient had completed 55 cycles of agent Docetaxel, maintaining ECOG of 1, with grade 2 neuropathy controlled with gabapentin. Patient is currently 56 months out from her initial diagnosis of metastatic triple negative breast cancer and follow-up scans continue to show stable disease. She has developed profound fatigue after several months of treatment. Patient has also faced challenges with fluid retention secondary to Docetaxel. Although her performance status remains fair, patient is contemplating changing frequency of chemotherapy to every 5 or every 6 weeks. Triple negative breast cancer is an aggressive disease with limited options of treatment with chemotherapy agents and no role for endocrine therapy or HER2 targeted treatment options. Docetaxel has shown to have median survival ranging between 10.1 to 14.7 months depending on the dose. Our patient has so far shown extended PFS of 56 months, with single agent Docetaxel in first line setting which surpasses current national averages.