Desatinib as an Option to Treat P-cadherin Overexpressing Poor Prognosis Breast Cancer

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João Filipe Urze de Almeida

Dasatinib as an option to treat P-cadherin overexpressing poor

prognosis breast cancer

Dissertação de Candidatura ao grau de Mestre em Oncologia, Especialização em Oncologia Molecular, submetida ao Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto.

Orientadora - Joana Paredes, PhD,

Investigadora Principal no grupo Cancer Genetics Instituto de Patologia e Imunologia Molecular da Universidade do Porto

Coorientadora - Ana Sofia Ribeiro, PhD

Pós-Doutorada no grupo Cancer Genetics Instituto de Patologia e Imunologia Molecular da Universidade do Porto

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III Urze-Almeida, J. (2015). Dasatinib as an option to treat P-cadherin overexpressing poor prognosis breast cancer. (Master thesis). Instituto de Ciências Biomédicas Abel Salazar, ICBAS, Porto.

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V

Agradecimentos

Gostaria em primeiro lugar de deixar um especial agradecimento às minhas orientadoras Ana Ribeiro e Joana Paredes. Muito obrigado pela dedicação que ofereceste, disponibilidade e atenção que dispensaste e paciência que tiveste Ana e obrigado por esta oportunidade e suporte que me deste, Joana. Tenho também de deixar o meu obrigado aos meus colegas do grupo Cancer Genetics, futuro EPIC, por toda a ajuda laboratorial que me ofereceram e pelo saber que me transmitiram. Obrigado Rita, Babi, André, Madalena e Mónica. Agradeço também ao Polónia por ter dispensado tempo para ver todas as lâminas de histologia e imunohistoquímica que advieram do projeto.

Em segundo quero agradecer à minha família. À minha mãe e aos meus primos que me apoiaram durante todo o mestrado e durante esta fase da minha vida. Um muito obrigado.

Em especial ao pessoal do biotério/IPATIMUP Animal House. Ao Nuno Mendes por me ter ensinado e inspirado a trabalhar com animais e por toda a orientação, opiniões e colaboração na solução de dúvidas. Também não podia deixar de referir a Dona Conceição e a Patrícia que me acolheram da melhor forma possível no biotério.

Resta agradecer à Laço por ter financiado o projeto do estudo do Dasatinib em tumores da mama com elevada expressão de caderina-P (Bolsa Laço 2014).

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VII

Abstract

The treatment of triple-negative breast cancers (TNBC) is particularly challenging, since these tumors lack validated molecular targets. The basal-like subgroup, characterized by the overexpression of basal markers, like cytokeratin 5 and P-cadherin, is the most common among TNBC. P-cadherin is overexpressed in more than 50% of TNBC, being significantly associated with poor patient survival. In vitro, the overexpression of this cell-cell adhesion molecule increases cell-cell invasion capacity, cell-cell migration, confers stem cell-cell properties and promotes a tumorigenic behavior. Some of these properties might be due to p120ctn, which translocation from the membrane to the cytoplasm is involved in cell’s motility and invasiveness. However, till now, there are no drugs targeting P-cadherin. Thus, targeting its intracellular pathway can be an interesting therapeutic alternative. Our group has recently described a downstream signaling activated by P-cadherin expression - Src family kinase (SFK), which activation is highly associated to cancer progression, responsible for potentiating proliferation, migration, and survival of neoplastic cells, in part through p120ctn activation. Interestingly, unpublished data showed that treatment of P-cadherin-overexpressing breast cancer cells with Dasatinib, a FDA-approved SFK inhibitor, promoted a significant decrease in cell migration and invasion. Additionally, Dasatinib induced the membrane stabilization of the E-cadherin/p120ctn complex, promoting an epithelial-like phenotype.

Taking these observations into account, the aim of this thesis was to evaluate the in vivo effect of Dasatinib in the treatment of cadherin-overexpressing TNBC. For that, three P-cadherin overexpressing breast cancer models (SUM149PT, BT20 and MDA-MB-468) were inoculated in the mammary fat pad (Model 1) or in the tail vein (Model 2) of female nude mice. The purpose was to evaluate the effect of Dasatinib in mammary tumor growth, metastization, and mice survival. SFK inhibition, as well as cadherin/catenin complex alterations, were also analyzed in the primary tumor by immunohistochemistry.

Despite a modest inhibition in the Src protein activation residue (Tyr416) in the mammary tumors, there was a clear influence of Dasatinib in treated mice, since there was a significant increase in their overall survival. Moreover, a significant inhibition was observed in the tumor growth rate in Dasatinib treated animals inoculated with MDA-MB-468 cells, showing also decreased vascular invasion, as well as a reduction of p120ctn cytoplasmic levels. In what concerns metastization, no significant differences were observed, although the Dasatinib treated group showed a delay in the metastization process.

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VIII Our results point to a possible and important effect of Dasatinib in promoting an anti-tumorigenic and anti-invasive behavior of P-cadherin highly aggressive TNBC cells, opening a new therapeutic window of opportunity for the treatment of P-cadherin overexpressing highly aggressive breast tumours.

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IX

Resumo

O tratamento do cancro da mama triplo negativo é particularmente difícil devido à inexistência de alvos moleculares. O subgrupo de carcinomas do tipo basal, assim caracterizado por apresentar elevada sobre-expressão de marcadores basais, como a citoqueratina 5 e a caderina-P, é o subtipo mais comum de cancro da mama triplo negativo. De facto, a caderina-P encontra-se sobre-expressa em 50% dos cancros da mama triplo-negativos, associando-se a uma baixa sobrevida destes pacientes. In vitro, verificámos que a presença desta molécula de adesão, em células de cancro da mama, promove propriedades estaminais, invasão e migração celular. Parte destas propriedades podem ser devido à destabilização da p120ctn, uma proteína da família das cateninas, que se liga às caderinas, e cuja ativação está envolvida em processos de invasão e motilidade celular. Até ao momento não existem fármacos que tenham como alvo a caderina-P, sendo por isso necessário encontrar alternativas terapêuticas que afetem as vias de sinalização ativadas pela caderina-P. Recentemente, o nosso grupo demonstrou que a ativação da via da família da Src (SFK) é promovida pela expressão de caderina-P. A SFK é uma via de sinalização altamente associada aos processos de tumorigénese, sendo responsável por potenciar a proliferação, migração e sobrevivência das células neoplásicas, através da interação com a p120ctn.

Curiosamente, resultados preliminares do nosso grupo demonstram que a expressão de caderina-P ativa a via de sinalização da SFK. O tratamento de células de cancro da mama com Dasatinib, que é um inibidor da SFK já aprovado pela FDA, promove uma diminuição significativa na migração e invasão de células com sobre-expressão de caderina-P. Além disso, o tratamento com Dasatinib induz a estabilização do complexo caderina-E/p120ctn na membrana, promovendo um fenótipo do tipo epitelial.

Tendo isto em consideração, o objetivo desta tese foi avaliar o efeito do Dasatinib, em modelos animais, como alterativa para o tratamento de tumores triplo-negativos que sobre-expressam caderina-P. Como tal, recorremos a três modelos in vivo, nos quais células de cancro da mama (SUM149PT, BT20 e MDA-MB-468) com sobre-expressão de caderina-P foram inoculadas na mama (Modelo 1) ou na veia caudal (Modelo 2) de fêmeas de ratinhos imunodeficientes. O objetivo foi assim avaliar o efeito do Dasatinib no crescimento dos tumores mamários, na metastização, e na sobrevida dos animais tratados. Avaliou-se ainda, por IHC, a inibição de SFK assim como as alterações do complexo caderina/catenina nos tumores primários.

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X No geral, apesar de observarmos uma inibição modesta da SFK nos tumores primários, verificou-se um efeito claro na sobrevida dos ratinhos tratados com Dasatinib, o que levou a um aumento da taxa de sobrevivência. Observámos também alterações no tamanho do tumor (MDA-MB-468), uma vez que os ratinhos tratados com Dasatinib apresentavam um crescimento mais lento dos tumores primários, com diminuição de invasão vascular, assim como uma redução da p120ctn no citoplasma. Em termos de metastização, não se observaram diferenças significativas, apesar dos resultados sugerirem que o processo de metastização parece ser retardado nos animais tratados com Dasatinib.

Os nossos resultados apontam para um possível e importante efeito anti-tumorigénico e anti-invasivo do Dasatinib em células agressivas de cancro da mama triplo-negativas, com sobreexpressão de caderina-P, criando novas oportunidades terapêuticas para o tratamento destes tumores altamente agressivos e de mau prognóstico.

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List of Figures

Figure 1: Breast normal architecture. ... 25

Figure 2: Classification systems of Breast Cancer. ... 29

Figure 3: Molecular classification of breast cancer and survival rates... 30

Figure 4: P-cadherin structure and cellular localization... 33

Figure 5: Src interactions in cytoskeleton regulation and cell’s transformation. ... 37

Figure 6: Spontaneous Model for Tumour and Metastasis Formation scheme... 49

Figure 7: Experimental Model for Metastasis Evaluation scheme. ... 50

Figure 8: Percentage of Weight for the Spontaneous Model for Tumor and Metastasis Formation.. ... 62

Figure 9: Primary tumor growth in every week of the Spontaneous Model for Tumor and Metastasis Formation. ... 65

Figure 10: Examples of primary tumor macroscopic analysis from the Spontaneous Model in Tumor and Metastasis Formation. ... 66

Figure 11: Growth rate of recurrences and metastasis versus primary tumor. ... 67

Figure 12: H&E staining of primary tumors in the Spontaneous Model in Tumor and Metastasis Formation. ... 70

Figure 13: H&E staining for recurrence and metastasis for the Spontaneous Model in Tumour and Metastasis Formation. ... 71

Figure 14: Vascular invasion in primary tumours for the Spontaneous Model for Tumours and Metastasis Formation. ... 72

Figure 15: Lungs with infection characteristic histology. ... 73

Figure 16: Kaplan-Meier curves representing overall survival of Control group versus Dasatinib group. ... 75

Figure 17: Overall protein expression. ... 77

Figure 18: Immunohistochemical analysis of Src Tyr416 profile. ... 78

Figure 19: Immunohistochemical analysis of p120ctn profile. ... 79

Figure 20: Immunohistochemical analysis of P-cadherin profile.. ... 80

Figure 21: Immunohistochemical analysis of E-cadherin profile. ... 81

Figure 22: p120ctn protein expression and Src correlation. ... 82

Figure 23: Weight variation in the Experimental Model for Metastasis Evaluation.. ... 87

Figure 24: In vitro xenograft culture of spontaneous Model in Tumor and Metastasis Formation. ...123

Figure 25: SUM149PT mammary fat pad tumour IHC analysis. ...125

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List of Tables

Table 1: Resume table of used animal models and experimental designs. ... 52 Table 2: List of used antibodies for IHC study. ... 55 Table 3: Mice with recurrences in the Spontaneous Model for Tumor and Metastasis

Formation.. ... 68

Table 4: Metastasis found in the Spontaneous Model for Tumor and Metastasis

Formation. ... 69

Table 5: Median Survival Time of Different cell line in the Spontaneous Model for Tumor

and Metastasis Formation. ... 76

Table 6: Resume analysis of the Spontaneous Model for Tumor and Metastasis

Formation. ... 83

Table 7: Lung metastasis in the Experimental Model on Metastasis Evaluation.. ... 88 Table 8: Dasatinib in vivo previous works. Table obtained in order to stipulate the used

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XIX

Abbreviation List

AFAP1 Actin Filament-Associated Protein 1

BCC Breast Cancer Cell

BCR-ABL Breakpoint Cluster Region - Abelson murine leukemia viral oncogene

βctn β Catenin

BL Basal Like

CBD Catenin Binding Domain

CDKs Cyclin Dependent Kinases

CIS Carcinoma in situ

CK5/6 Cytokeratin 5/6

CLB Catenin Lysis Buffer

CML Chronic Myeloid Leukemia

CYP3A1 Cytochrome P450 3A1

DAB 3,3’ - diaminobenzidine DFS Disease Free Survival

DMEM Dulbecco’s Modified Eagle Medium DMSO Dimethyl sulfoxide

E-cadherin Epithelial Cadherin

EC Extracellular domain

ECM Extracellular Matrix

EDTA Ethylendiamine Tetraacetic Acid

EGFR Epidermal Growth Factor Receptor

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XX EphA2 Ephrin type A receptor 2

ERα Estrogen Receptor α FAK Focal Adhesion Kinase

FBS Fetal Bovine Serum

FDA Food and Drug Administration

γctn γ Catenin

HBSS Hank’s Balanced Salt Solution H&E Hematoxylin & Eosin

HER2 Human Epidermal Growth Factor Receptor 2

HR Hormone Receptor

IC50 Half Maximum Inhibitory Concentration

ID Intracellular Domain

IHC Immunohistochemistry

JMD Juxtamembrane Domain

MAPK Mitogen-Activated Protein Kinase

MFP Mammary Fat Pad

MMPs Matrix Metalloproteinases

MST Median Survival Time

ON Overnight

OS Overall Survival

P-cadherin Placental Cadherin

p63 Protein 63

p120ctn p120 Catenin

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XXI PBS Phosphate-Buffered Saline

PDGFR Platelet-Derived Growth Factor Receptor

Ph-ALL Philadelphia chromosome-positive Acute Lymphoblastic Leukemia

PI3K Phosphoinositide 3-Kinase

PR Progesterone Receptor

SFK Src Family Kinase

TD Transmembrane Domain

TDLU Terminal Duct Lobular Unit

TNBC Triple Negative Breast Cancer

TNM Tumor Nodule Metastasis System

TV Tail Vein

Tyr416 Tyrosine 416

Tyr527 Tyrosine 527

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Chapter I - Introduction

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1. Breast Cancer

Considered a major public health problem in the world, breast cancer tops the five most frequent cancers in woman, and age seems to be a crucial risk factor, with more than 50% of patients being 65 years or older and 30% having more than 70 years (Elomrani et al., 2015).

1.1. Normal breast

The human breast is composed of fat tissue and mammary gland tissue, externally surrounded by skin and internally by fascia and chest muscles. The normal gland has two layers of cells. The internal cuboid cells, called luminal cells, are in direct contact with the lumen of the gland and are responsible for milk production. The external ones are called myoepithelial cells and are in direct contact with the internal luminal cells and the basal membrane (Figure 1). These cells are responsible for the contraction and forced milk secretion by the luminal cell to the lumen of the gland when in lactation.The mammary gland starts at the nipple forming lactiferous ducts that end at the terminal duct lobular unit (TDLU), where the milk is produced (Macias et al., 2012). The initiation of breast cancer is due to transforming (genetic and epigenetic) events in a single epithelial cell.

A B

Pandey et al., 2011 Polyak, 2007

Figure 1: Breast normal architecture. A) Transversal cut of the mammary gland, where it is possible to

observe the interior luminal cells surrounded by the myoepithelial cells and basal membrane. B) Distinction between duct and lobule in the TDLU. At green and pink are represented the myoepitheli al cells and putative progenitors, respectively, at grey and blue are represented the luminal cells and corresponding progenitors, respectively. Adapted from Pandey et al., 2011 and Polyak, 2007.

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1.2. Breast Cancer Classification Systems

The natural history of breast cancer involves progression through defined pathological and clinical stages, starting with ductal hyperproliferation, with subsequent evolution into in situ and invasive carcinomas, and finally into metastatic disease (Kumar et al., 2013). While there has been a dramatic improvement in our ability to detect early-stage disease, our understanding of tumor progression factors and our ability to selectively interfere with them lag far behind (Bleyer et al., 2012).

Different classification systems have been created and applied in order to proper characterize breast cancer. The purpose for classification is to select the best treatment, as different treatments exists and can only be effective in a selective type of breast cancer. So, cancers that are similar, are grouped together, mainly in histopathological type and grade, staging, and molecular expression (Figure 2) (Viale, 2012).

Histological type

Despite different histologies, the majority of breast cancers derives from epithelial cells (mostly luminal progenitors) of the ducts or lobules of the mammary gland, being classified as mammary ductal carcinoma or mammary lobular carcinoma, respectively. When a tumor is present within the normal epithelium, without rupture of the basal membrane and no invasion of the surrounding tissue, it is denominated carcinoma in situ (CIS). In contrast, invasive carcinoma invades the surrounding tissue. Also, peripheral and/or lymphatic and vascular space invasion is considered for proper classification, known as a sign of aggressiveness (Viale, 2012). Concerning invasive breast cancer, Invasive Ductal Carcinoma accounts for 50% to 80% and Invasive Lobular Carcinoma represents 15% to 5%. Although these are the most common types, 10% to 25% consist of more rare histological subtypes, like mucinous, apocrine, metaplastic, medullary, micropapillary, neuroendocrine, pleomorphic lobular and mixed lobular-ductal carcinoma (Dreyer et al., 2013).

Differentiation

When assessing tumor differentiation, the tumor can be graded by the difference it presents from the normal breast tissue. When normal cells develop, they differentiate in shape and form in order to function as part of an organ. In the breast carcinogenesis process, the cells lose their differentiation by passing from a hyperplastic state to in situ neoplasia and ending in an invasive stage, becoming disorganized, with alterations in cell

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27 polarization, uncontrolled divisions and abnormal nuclei (Kumar et al., 2013). Interestingly, invasive tumors show loss of the myoepithelial cell layer, which seem to present a tumor suppressive role (Pandey et al., 2011). In breast cancer, the pathologist evaluates the maintenance of tubule formation, nuclear pleomorphism and number of mitosis. Depending on the subtype, cancers are then described as well differentiated (low-grade), moderately differentiated (intermediate-grade) and poorly differentiated (high-grade). Poorly differentiated cancers are associated with a worse patient prognosis (Malhotra et al., 2010).

Staging

Staging is done in order to determine how widespread a cancer is when it is diagnosed. Cancer stage is based on four characteristics, such as tumor size, if the cancer is invasive or not, if it has spread to lymph nodes or if it has metastasized to distant sites. This parameter is usually expressed in a scale from 0 to IV, where 0 describes a non-invasive cancer, I to III indicates dissemination through the breast and lymph nodes, and IV describes an invasive cancer that has spread beyond original tissue and lymph nodes, through other parts of the body. The most usual staging system is the TNM, which discriminates between tumor size (T), lymph node involvement (N) and metastasis (M) (Senkus et al., 2013).

Molecular expression

Over the last three decades, the routine determination of Estrogen Receptor  (ERα) and other key protein, such as Human Epidermal Growth Factor Receptor 2 (HER2), has provided evidence that breast cancer can have several distinct molecular subtypes. In fact, nowadays, these two markers are used in the routine practice, and their positive expression helps to define the treatment that should be given to breast cancer patients (Prat et al., 2013).

Gene expression profile

This classification was first introduced in 2000 by Perou and co-workers. They uncovered similarities and differences in gene expression among the analyzed tumors. By analyzing 8102 human genes in 62 breast tumors, in a microarray analysis, they were able to divide breast tumors into specific molecular subtypes, linking their gene expression profile to

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28 clinical outcome and response to therapy of breast cancer patients. In this way, four major groups were identified by molecular characterization, being defined as Luminal, HER2 Overexpression, Basal-Like and Normal-Like types (Perou et al., 2000).

1.3. Molecular subtypes of Breast Cancer

In the last years, several groups have been able to distinguish molecularly different subtypes of breast cancer. As expected, the majority of the studies generally separated the tumor samples into those that were clinically described as ERα positive and those that were ERα negative (Prat et al., 2013).

Combining the gene expression profile data with the clinical opportunity for treatment, and based on Hormone Receptors (HR) (ERα and/or Progesterone Receptor (PR)) and HER2 expression, it is possible to subdivide this complex molecular taxonomy in three major subtypes with distinct biology, clinical outcome and response to therapy:

1) ER Positive/Luminal tumors; 2) HER2 overexpressing tumors; 3) Triple-negative tumors.

ER



Positive/ Luminal tumors

The Luminal tumors are characterized by the relatively high expression of luminal markers, such as HR, ERα and/or PR, and the epithelial cytokeratins 8 and 18. Still, these can be divided in two categories, classified as A and B. This division is due to different clusters of expressed genes between them, as well as with different prognosis outcome.

Luminal A is the most common subtype and is associated with a less aggressive phenotype, in comparing with luminal B subtype, which shows increased proliferation rates, higher grade and poor prognosis (Inic et al., 2014). Actually, the proliferation marker – ki67 – is the common used strategy to differentiate both subtypes by immunohistochemistry (IHC) (Viale, 2012). Additionally, Luminal B appears to express lower amounts of ERα associated genes and there is also a small percentage (around 10%) that expresses HER2 (the so called triple positive breast cancer) (Tran et al., 2011).

ER Positive/Luminal tumors are clinically treated with targeted therapy anti-ER expression and/or activity, generally presenting a good prognosis for breast cancer patients (Yamamoto-Ibusuki et al., 2015).

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Figure 2: Classification systems of Breast Cancer. The scheme represents the four currently used

classifications based on histopathology (dividing in situ from invasion), molecular, differentiation and stage. Engstrom et al., 2015; Peddi, et al., 2012; Dreyer et al., 2013; Marshall, 2014.

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HER2 Overexpressing tumors

HER2 overexpression itself represents another type of molecular characterization, which comprises 15% to 20% of all human breast cancers. These tumors do not express HR associated genes, instead they show amplification of the HER2 gene, with consequent overexpression of the HER2 protein. This protein is a tyrosine kinase receptor that mediates critical signaling functions in normal and malignant breast epithelial cells (Salmon et al. 2011). This type of tumors are generally associated with high grade, increased growth rate, early metastasis and decreased rates of survival (Verma et al., 2012).

HER2 overexpressing tumors are clinically treated with targeted therapy monoclonal antibodies against HER2, showing good clinical responses to the treatment and improving the survival of breast cancer patients (Dent et al., 2013).

Triple-negative tumors

For last, Triple Negative Breast Cancer (TNBC) are those that lack the expression of HR and HER2 protein. They represent around 15% (Figure 3) of all breast cancers and are usually associated with high grade tumors and show a poor patient prognosis. Because TNBC do not present the expression of these molecules, there is no current approved target or hormone therapy for these type of tumors (Murawa et al., 2014).

Figure 3: Molecular classification of breast cancer and survi val rates. A) Molecular distribution of

breast cancer with luminal type presenting the majority and TNBC the minority, with only around 15% of the breast cancer cases. B) Survival Kaplan-Meier curves of Luminal types A (light blue), B with HER2 (green) and B without HER2 (dark blue), HER2 overexpression (pink), and TNBC divided in Basal-Like (yellow) and the other known subtypes (red). TNBC along with HER2 overexpression present the worst survival. Survival curve adapted from Engstrom et al., 2013

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2. Triple Negative Breast Cancer

TNBC forms a class of cancer that fail to express HR, ER and PR, and HER2. This subgroup is majorly constituted by the Basal-Like breast cancer subtype, previously defined by gene expression profile, accounting for 70% of all TNBC. The remaining 30% comprises five less represented subgroups (Peddi et al., 2012):

1) Claudine-Low subgroup, that lacks the expression of claudin proteins and shows high expression of epithelial-mesenchymal transition (EMT) and stem cell markers; 2) Immunomodulatory subgroup, that shows high expression of genes involved in cell

signaling and immune response;

3) Mesenchymal subgroup, showing the expression of genes that codify for proteins involved in motility and in the interaction with the extracellular matrix (ECM); 4) Mesenchymal Stem-Like subgroup, similar to the previous, but with an increased

expression of proteins involved in growth signaling and in stemness;

5) Luminal Androgen Receptor subgroup, showing high expression of genes involved in the hormonally regulated pathways, although do not have ER and PR expression (Peddi et al., 2012)

Basal-like tumors

Basal-Like (BL) is the most common subgroup within TNBC, which makes it a great target in scientific studies. It is distinct from other TNBC subgroups since it expresses proteins associated to myoepithelial cells of the breast, like cytokeratin 5/6 (CK5/6), 14, and 17, protein 63 (p63), P-cadherin, vimentin and Epidermal Growth Factor Receptor (EGFR) (Rakha et al., 2007).

BL-TNBC present a current challenge for the scientific community, since finding a proper target systemic therapy has been difficult (Rakha et al., 2009). Presenting some of the worst cancer prognosis, patients can only count with surgery and chemotherapy, as even radiotherapy does not show significant effects (Niwin’ska et al., 2010).

BL-TNBC is more prevalent in younger patients and in African-American and Hispanic descendants.Also the premenopausal status, increased parity, high histological grade and tumors size, appear to be risk factors for this subtype of breast cancer (Anderson et al., 2014; Boyle, 2012).

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32 Histologically, BL-TNBC are infiltrating ductal carcinomas with solid growth patterns. These tumors tend to metastasize to lungs and brain and with less frequency to the bone. Brain metastasis give the worst prognosis, with a median survival time (MST) of 2.9 to 4.9 months after diagnosis (Pogoda et al., 2013; Niwin’ska et al., 2010). BL-TNBC patients present the poorer distant metastasis-free survival, disease-free survival (DFS), and overall survival (OS), showing a 5-year survival rate of 14% in non-Hispanic black women with late-stage (Boyle, 2012; Haffty et al., 2006; Foulkes et al., 2010).

BL-TNBC tumors present an initial good response to chemotherapy, particularly with anthracycline and taxanes (Ismail-Khan et al., 2010). The adjuvant chemotherapies regimens are still being determined; nevertheless, the current treatment to manage BL-TNBC is the use of chemotherapy based on targeting DNA repair complexes (with platinum compounds and taxanes), target p53 (with taxanes), affect cells proliferation (by the use of anthracycline regimens) and clinical trials in target therapy (Wahba et al., 2015).

Recently, there are many studies being made in order to find new therapeutic targets or drugs that will improve the survival of BL-TNBC. Currently, pre-clinical and clinical trials are being tested against antiangiogenic agents, targets of EGFR, Poly ADP Ribose Polymerase (PARP) inhibitors, as well as inhibitors of Phosphoinositide 3-Kinase (PI3K), Src and Cyclin Dependent Kinases (CDKs) (Mohamed et al., 2013).

3. P-cadherin

Modifications of cell-cell interactions occur during carcinogenesis, where the disruption of cell-cell contacts is one of the key events for tumor progression. In breast cancer progression, the main classical cadherins which are usually altered are both Epithelial Cadherin (E-cadherin) and Placental Cadherin (P-cadherin) (Paredes et al., 2012).

3.1. Classical cadherins

Classical cadherins are cell-cell adhesion transmembrane glycoproteins, highly associated with neoplastic processes, as their loss or gain are involved in motility, invasion and metastatic capacity of breast cancer cells. They are localized at the cells membrane surface and show homophilic binding to cadherins from adjacent cells, forming cell-cell junction of the epithelial cells, in a calcium dependent way (Rossetti et al., 2015).

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33 These molecules are mainly composed by three main domains:

1) Extracellular domain (EC) divided in five subunits (EC1 – EC5), responsible for the cadherin identification, and where calcium ions (Ca2+) bind. Calcium is necessary for the process, as its absence inhibits the normal conformation of the molecule. EC1 is responsible for adhesion properties;

2) Transmembrane domain (TD) that connects the extracellular domain with the interior of the cell;

3) Intercellular domain (ID) composed by the juxtamembrane domain (JMD) and the catenin biding domain (CBD), responsible for the binding to catenins (Paredes et al., 2012).

Catenins are the main proteins that bind to cadherins in cell’s ID and help in the process of adhesion. Their interaction with the ID is crucial for the cadherin stabilization and proper function (Figure 4). Also, these are responsible for the interaction of cadherins with cell’s actin cytoskeleton. The main catenins are β-catenin (βctn), γ-catenin (γctn) and p120catenin (p120ctn). Both βctn and γctn are regulated by the CBD, by tyrosine kinases and by transcription factors, and are involved in tissue organization. p120ctn is the catenin that is in direct interaction with the JMD, and is involved in various intercellular processes, such as tyrosine kinase phosphorylation, cadherin trafficking, cell’s stability, adhesive capacities and cell motility (Paredes et al., 2004 & Hengel et al., 2007).

Figure 4: P-cadherin structure and cellular localization. P-cadherin is present at the

membrane surface, presenting an extracellular domain composed by five EC repeats, being EC1 responsible for the adhesion with homologue molecules. Ca2+ is essential for the molecule conformation. The intracellular domain is composed by the JMD, where p120ctn binds, and by CBD, where βctn binds. βctn interacts with α-catenin, which interacts with the actin cytoskeleton. Adapted from Albergaria et al., 2011.

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3.2. Cadherins in Cancer

The majority of the studies implicating cadherins in carcinogenesis have been focused on E-cadherin, because it is the major cadherin expressed by epithelial cells. In vitro and in vivo studies showed that inhibiting E-cadherin function turned non-invasive epithelial and polarized cells into invasive cells and is now generally accepted that E-cadherin is the main suppressor of epithelial tumor invasion. Indeed, decreased or loss of E-cadherin expression and/or function has also been observed during progression of most human carcinomas (Kowalski et al.,2003; Canel et al., 2013), being associated to tumors with an increased infiltrative pattern of growth, including sporadic and hereditary diffuse gastric and lobular breast cancers(Engstrom et al., 2015; Oliveira et al., 2009).

The role played by P-cadherin in carcinogenesis is still a matter of debate. In contrast with what has been observed for E-cadherin, P-cadherin seems to be upregulated in several solid tumors, including breast, prostate, colon, pancreatic and bladder cancer (Imai et al., 2008).

3.3. P-Cadherin in Breast Cancer

Being one of the main focus of our group, P-cadherin is a cell-cell adhesion molecule that has been found to be overexpressed in around 30% to 50% of invasive breast carcinomas, most of them in BL-TNBC. In normal epithelial tissues, like breast or skin, P-cadherin is confined to the basal layer, being co-localized with E-P-cadherin expression (Paredes et al., 2007; Paredes et al., 2012). When overexpressed in breast cancer, it’s associated with worse prognosis and shorter survivals rates. The tumors tend to present a high histological grade and local and distant metastasis are frequently associated, which indicates its potential for inducing invasion. According to Ribeiro et al., P-cadherin is responsible for the cell secretion of matrix metalloproteinases (MMPs), enzymes responsible for the degradation of most ECM components, like the ones constituting the basal membrane, potentiating cell invasion. Also, MMPs appear to cleave the P-cadherin extracellular domain, generating a pro-invasive soluble fragment (Ribeiro et al., 2010). Moreover, P-cadherin expression has also been associated with stem cell properties of breast cancer cells (BCC), inducing increased self-renewal ability, cell growth and resistance to radiation in vitro, and in vivo tumorigenic ability (Vieira et al., 2012; Ribeiro et al., 2015).

Interestingly, our group proved that this P-cadherin role in breast cancer cells is dependent of its co-expression with E-cadherin. Probably this effect is dependent on

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35 altered interactions of both molecules at the cell’s membrane, since tumor cells just expressing one of the cadherins present tighter cell-to-cell adhesion (Ribeiro et al., 2013).

Additionally, co-expression of both cadherins seems to interfere with the cellular localization of catenins, namely with p120ctn. As previously mentioned, p120ctn is associated with cell motility, promoting signaling pathways that are implicated in cancer cell invasion. Ribeiro et al., showed that the presence of both cadherins, besides demonstrating an aberrant cell behavior, tend to compete in the catenin membrane localization. P-cadherin presence destabilizes the E-cadherin/p120ctn complex, increasing the presence of p120ctn in the cytoplasm (Ribeiro et al., 2013). Once in the cytoplasm, p120ctn inhibits RhoA and activates Rac1 and Cdc42, which are responsible for the cytoskeleton polymerization and by the induction of migration and motility (Paredes et al., 2008). However, besides interfering with these GTPases from the Rho family, p120ctn also is associated with activation of Src tyrosine kinase family (Vieira et al., 2014). These kinases are responsible for the phosphorylation of p120ctn, promoting its delocalization from the membrane to the cytoplasm, inducing all the mentioned tumorigenic effects (Xiao et al., 2007; Dohn et al., 2009).

4. Src signaling pathways

4.1. Src

Src is a member of the Src Family Protein Kinases (SFK), a group of pleiotropic non-receptor tyrosine kinase proteins involved in many cellular processes (Zhang et al., 2012). Other members include Yes, Fyn, Fgr, Yrk, Lyn, Blk, Hck and Lck, accounting for a total of 9 members that form the SFK group. Src is the most known and famous, as it was the first proto-oncogene to be discovered. Normally involved in processes related to adhesion, when activated is known to potentiate tumor cell proliferation and survival, along with angiogenesis and invasion (Finn, 2008). Src is known to be involved in many types of cancers, including sarcomas (Chen et al., 2015), colon (Brunton et al., 2005), lung (Zhang et al., 2007), pancreas (Yezhelyev et al., 2004), prostate (Saito et al., 2010) and breast (Zhang et al., 2013). Due to its diverse tumorigenic effects, Src is considered an interesting drug target for preventing tumor progression, and multiple are the studies in several cancer types that demonstrate the benefit of Src inhibition, in vivo and in vitro (Chatzizacharias et al., 2012).

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36 As a tyrosine kinase, Src activity is regulated by the structural changes that occur in the protein, implied by phosphorylation and dephosphorylation of the tyrosine kinase residues, Tyrosine 527 (Tyr527) and Tyrosine 416 (Tyr416). The phosphorylation of Tyr527 reduces Src activity, while its dephosphorylation increases the kinase activity. As for Tyr416, it appears to present an autophosphorylation activity responsible for kinase activation (Zhang et al., 2012; Finn, 2008).

Functionally, SFKs regulate intracellular signaling pathways activating transmembrane growth factors and cytokine receptors, like Vascular Endothelial Growth Factor Receptor (VEGFR), HER2, and EGFR (Irby et al., 2000).

4.2. Src and cell-cell adhesion

In cancer cells, Src is responsible for alterations at cadherins junctions and cell motility, through the interaction with p120ctn, leading to weak interactions (Zhang et al., 2012). Both tyrosine domains, Tyr527 and Tyr416, are known to activate p120ctn (Owens et al., 2000). Besides p120ctn, Src regulates p85-cortactin and p110/actin filament associated protein 1 (p110/AFAP1), known to be involved in invadopodia and podosomes formation and cell contractility. Furthermore, it can also activate p130cas and p125/focal adhesion kinase (p125/FAK), known to be involved in cell adhesion and survival by integrin mediated processes (Figure 5). The constitutive phosphorylation of p120ctn by Src is responsible for the decrease in E-cadherin expression in breast cancer (Dohn et al., 2009; Reynolds et al., 2014).

More specifically in breast cancer, particularly in TNBC, both cytoplasmic and membrane Src expression has been detected. Interestingly, around 74% of TNBC show active Src with membrane expression, suggesting that it can be interacting with p120ctn (Tryfonopoulos et al., 2011). Recently, our group has shown for the first time a link between P-cadherin expression and Src activation, although the mechanism is not fully understood. This link seems to be promoted by two different ways. By one hand, Vieira et al, demonstrated that P-cadherin facilitates the adhesion of cells to extracellular matrix components through the laminin receptor α6β4 integrin, which in turns is associated with the activation of Src and FAK pathway in a P-cadherin dependent way (Vieira et al., 2014). Another alternative is through p120ctn activation, in P-cadherin positive BCC. Our group has described that BL-TNBC show co-expression of both E-cadherin and P-cadherin (Ribeiro et al., 2013), with cytoplasmic accumulation of p120ctn (Paredes et al., 2008), presenting a poor patient survival. In fact, P-cadherin protein interferes with E-cadherin function, without affecting its expression and by disrupting the cell-cell adhesion

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37 E-cadherin/p120ctn complex at the cell membrane. In this way, E-cadherin is not stabilized at the cell membrane, and p120ctn delocalizes to the cytoplasm affecting the cytoskeleton polymerization and consequent cell migration and motility (as previously referred).

In a robust theory, P-cadherin activation of Src by interaction with α6β4 laminin may give the trigger, along with other many stimulus, for the loss of E-cadherin/p120ctn complex and its cytoplasmic accumulation, which may lead to cell migration and invasion.

4.3. Src family Kinase inhibitors

Assumed the role of SFKs in growth, proliferation, invasion, angiogenesis and metastasis, it is clear the importance of targeting these kinases in order to inhibit their function. Blocking Src activation may slow disease progression and play an important role in adjuvant setting to prevent recurrence and metastasis. This inhibition can also reduce the development of bone metastasis and the associated pain (Creedon et al., 2012).

Figure 5:Src interactions in cytoskeleton regulation and cell’s transformation. It’s represented the interaction

of Src with four substrates, p120ctn in the cadherin complex, p85-cortactin that regulates pseudopods formation, p130cas/p125FAK complex crucial in integrin’s connection to the adhesion matrix (basal membrane), and p110-AFAP1 involved in actin filament regulation. Src is a key factor in cells adhesion and locomotion. Adapted from Reynolds et al., 2014.

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38 However, mutations in Src are not the main mechanism of SFK activation in human cancers; consequently, inhibiting a single target of Src may be unsuccessful. Concerning selectivity, cellular potency and possible therapeutic application, Src kinase inhibitors have appeared as the most successful therapeutic agents to date. Numerous classes of low-molecular-weight compounds that are ATP-competitive inhibitors of Src-mediated tyrosine phosphorylation have been described (Summy et al., 2003; Chatzizacharias et al., 2012; Irby et al., 2000). Some of these inhibitors even achieve a moderate to high selectivity within the Src family (Creedon et al., 2012).

One of the most interesting ones is Dasatinib (Sprycel, BMS354825, Bristol-Myers Squibb Oncology), which is a highly potent, ATP-competitive kinase inhibitor with antiproliferative activity (Gnoni et al., 2011). Initially, it was developed as a breakpoint cluster region – Abelson murine leukemia viral oncogene (BCR-ABL) kinase inhibitor, which is the responsible for the presence of the Philadelphia chromosome in chronic myeloid leukemia (CML). This was the first and only currently known Food and Drug Administration (FDA) approved SFK/ABL dual inhibitor for the treatment of CML and Philadelphia chromosome-positive Acute Lymphoblastic Leukemia (Ph-ALL) (Breccia et al., 2013). Currently Dasatinib is used as second line treatment in this disease, for imatinib-resistance or intolerance CML and Ph-ALL (Gnoni et al., 2011). Besides that, other functions were attributed to Dasatinib, being able to inhibit SFKs, but it also inhibits Ephrin type A receptor 2 (EphA2), platelet derived growth factor receptor (PDGFR) and c-KIT (Frangou et al., 2014). Beyond SFKs, it also binds to other tyrosine kinases, such as the mitogen-activated protein kinases (MAPK) (Sawyers et al., 1999). Other Src inhibitors, currently in clinical trials for cancer therapeutics, are Saracatinib (AZD0530, AstraZeneca) and Bosutinib (SKI-606, Wyeth) (Zhang et al., 2012; Chen et al., 2015).

Since Dasatinib has been shown to inhibit SFK activity in epithelial cell lines, current clinical trials have been trying to use it in the treatment of solid tumors. It is not clear which is the mechanism that will be more relevant in clinical applications, because it may have several effects on migration and invasion, as well as on inhibiting proliferation (Reynolds et al., 2014; Gnoni et al., 2011). The inhibitor potential of Dasatinib against SFKs (half maximum inhibitory concentration (IC50) = 0.5 nmol/L) is greater than against BCR-ABL (IC50 = 1 nmol/L) (Breccia et al., 2013).

A major phase I trial in 2009, published by Demetri and co-workers, was elaborated in order to determine the maximum tolerated dose in advanced solid tumors, by a dose-escalation study. It revealed that Dasatinib was well tolerated by the patients, with limited observed toxicity, mostly included in grade 1 or 2. A dose of 70 mg to 120 mg was recommended twice daily for further studies (Demetri et al., 2009). Another clinical trial

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39 phase I, published in 2010 by Johnson et al., defined that a maximum dose of 180 mg once daily was well tolerated, even with co-administration of a Cytochrome P450 3A4 (CYP3A4) inhibitor, the known responsible for primary Dasatinib metabolization (Johnson et al., 2010).

These results encouraged phase II clinical trials, some of which were done in breast cancer. Mayer and co-workers, published in 2011, a manuscript describing Dasatinib effect against advanced HR positive and HER2-overexpression advanced breast cancer. They concluded that the drug alone showed limited activity at a maximum dose of 100 mg twice a day (Mayer et al., 2011).

4.4. SFK inhibition in TNBC

In vitro studies have shown that, within all breast cancer cell lines, BL-TNBC cells showed higher sensitivity to Dasatinib treatment (Finn et al., 2007; Karim et al., 2013), since it leads to a complete inhibition of SFK activation.

Recently, it has been published a pre-clinical study focused on the effect of drug repositioning in different pathways activation using a pre-clinical model of TNBC metastization to lungs, brain and bone. Interestingly, Dasatinib appeared as one of the promising drugs for brain metastasis, one of the most common sites of TNBC metastasis. Dasatinib treatment led to a decrease in brain colonization of BCC, with decreased proliferation and anti-tumor activity (Zhao et al., 2013).

Nevertheless, clinical trials involving Dasatinib for the treatment of breast cancer patients (especially to BL-TNBC) showed limited activity, indicating that the mechanism of response to this pharmacological agent is not fully understood (Finn et al., 2011). The major problem may be due to poor selection of patients that will in fact benefit from this therapy, since TNBC comprises a very heterogeneous group of tumors. Therefore, there is the need of additional biomarkers linked to SFK signaling pathway that may constitute a new tool for therapeutic eligibility. Our preliminary data suggests that P-cadherin could be one of those potential markers, since it is upstream of Src, promoting the activation of this pathway, which is associated with a more aggressive phenotype and poor patient survival.

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41

Chapter II - Aims

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43 A primary goal in breast cancer research is to find specific biomarkers/pathways in poor prognostic BL-TNBC, which could have an important clinical impact for their effective treatment.

Although P-cadherin seems to be an interesting therapeutic target for BL-TNBC, to date, no FDA-approved drugs directly targeting P-cadherin exist, a limitation that creates the need to identify alternatives to impair P-cadherin-dependent signaling. Taking our results into account, and based on promising preclinical evidence on the relationship between SFK and solid cancers, our aim was to evaluate, in vivo, the impact of Dasatinib treatment in

TNBC progression, as possible target therapy for P-cadherin-overexpressing breast carcinomas.

In order to achieve this goal, two distinct models were performed:

Model 1: The Spontaneous Model for Tumor and Metastasis Formation; Model 2: The Experimental Model for Metastasis Evaluation.

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45

Chapter III - Materials and Methods

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47

1. Cell lines for inoculation and culture conditions

Human breast cancer cell lines were obtained as follows: BT20 and MDA-MB-468 were acquired from American Type Culture Collection (Manassas, VA, USA). SUM149PT was provided by Dr. Stephen Ethier (University of Michigan, USA). MDA-MB-231.Br.HER2+ were kindly given by Dr. Patricia Steeg (National Cancer Institute, USA) (Palmieri et al., 2007). Cell cultures were maintained in incubators at 37ºC and 5% CO2. BT20 and MDA-MB-468 were cultured in Dulbecco’s Modified Eagle Media (DMEM) (Gibco®), supplemented with 10% fetal bovine serum (FBS). SUM149PT cells were cultured in a 50%/50% mixture of Ham’s F12 media and DMEM, supplemented with 5% FBS, 10 μg/mL insulin and 2,5 μg/mL hydrocortisone. All media where supplemented with 1% mixture of 100 I.U./mL penicillin and 100 μg/mL streptomycin.

For preparing cells to inoculate in the mammary fat pad and tail vein of the female mice, cells were detached with Tripsin-EDTA (Gibco®) and counted in automatic counter TC20TM (Bio-Rad Laboratories, Inc). A cell suspension was prepared in medium without any supplementation until further use.

2. Drug preparation

Dasatinib was purchased from Sequoia Research Products (Pangbourne, Berks, UK) as a white to pale yellow crystalline powder, being then diluted with dimethyl sulfoxide (DMSO) (Sigma®) to a final concentration of 100 mg/ml and stored at -20ºC.

Doses of Dasatinib to be administered were prepared weekly by dilution in citrate buffer 80 mM (pH=3.1). The citrate buffer was prepared as follows: sodium citrate powder was diluted in citric acid solution 1M, obtained by dissolving citric acid in deionized water, to a final concentration of 80nM and with pH=3.1. The citrate buffer solution was made every three weeks, filtered in flow chamber and kept at 4ºC.

Every week, daily doses of Dasatinib were prepared to a final concentration of 10 mg/kg, in order to treat mice of both experimental models. There was an initial period of two weeks in the Spontaneous Model for Tumor and Metastasis Formation, in which the dose was doubled (20 mg/Kg) for proper acclimatization purposes.

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48

3. Animal model and experimental design

Animal experiments were carried out in accordance with the Guidelines for the Care and Use of Laboratory Animals, directive 86/609/EEC. All animals used were female in order to have the biological environment proper for breast cancer studies. Female N:NIH(s)II:nu/nu nude mice, maintained and housed at IPATIMUP-Animal House, were inoculated with human breast cancer cell lines, between five to eight weeks of age. Before initiating the treatment, all mice were randomly separated into Control and Dasatinib treated groups. Four to five mice were kept per cage with food and water ad libitum.

Two models were followed:

1) The Spontaneous Model for Tumor and Metastasis Formation consists in the inoculation of breast cancer cells in the mammary fat pad of nude mice. With tumor development is possible to understand if the drug reaches the local and if it acts in the tumor growth. After primary tumor surgery, the mice’s life is preserved in order to perform survival studies (Figure 6).

2) The Experimental Model for Metastasis Evaluation consists in the inoculation of breast cancer cells in the tail vein of nude mice. This type of study permits to access if breast cancer cells are capable of colonizing organs that are more exposed to the blood stream, namely lungs (Figure 7).

Both experimental models are well known in the scientific community for breast cancer studies (Padua et al., 2008; Chen et al., 2011; Oskarsson et al., 2011; Fridman et al., 2012; Kocatürk et al., 2015).

3.1. Orthotopic mammary fat pad inoculation

For the Spontaneous Model for Tumor and Metastasis Formation, 100 μL containing 2x106 viable P-cadherin overexpressing BCC (BT20, SUM149PT and MDA-MB-468) were inoculated into the mammary fat pad, with 1 mL syringe and a 25G needle in the left abdominal mammary gland of the 4th nipple pair. For a proper inoculation, mice underwent anesthesia with isoflurane (IsoFlo, Esteve), in order to be place in a temporary state of numbness just for inoculation. Toe-pinprick was used for proper confirmation of mouse numbness.

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49 A total of seventeen nude mice were inoculated for the SUM149PT breast cancer cell line (n=17). A total of fifteen nude mice were inoculated for the BT20 breast cancer cell line (n=15). And a total of thirteen nude mice were inoculated for the MDA -MB-468 breast cancer cell lines (n=13).

When tumors were palpable mice were randomly distributed into treatment and control groups, presenting each group equal average tumor volume before initiation of treatment. Half of the mice started the oral daily treatment with Dasatinib, whereas the other half started to be treated just with the drug vehicle (DMSO diluted in citrate buffer). Mice were examined daily and weighted and tumor volume was estimated for the mammary fat pad every week. Tumors were measured in two perpendicular dimensions and the volume was estimated by the formula [volume = (length) x (width)2/2] for approximating the volume of a ellipsoid. Primary tumors were removed by surgery, whenever these reach an average volume of 700 mm3 (BT20), 1000 mm3 (SUM149PT) and 600 mm3 (MDA-MB-468). After primary tumor removal, mice were maintained for possible recurrences.

3.2. Primary tumors and recurrence surgery

Whenever tumors reached the desired volume, surgery was performed to remove them. Not all tumors were excised at the same time. Animal were anaesthetized with a mixture of

Figure 6: Spontaneous Model for Tumour and Metastasis Formation scheme. Mice are inoculated at day

0 with 2x106 cells and treatment starts when nodules are palpable, time at which mice are randomly distributed. After, primary tumour is removed when the estimated condition are satisfied and mice life is preserved with treatment maintenance. When mice revealed signs of disease or inoperable recurrences, euthanasia is done.

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50 75 mg/Kg ketamine (IMALGENE® 1000) with 0.5 mg/Kg medetomidine hydrochloride (Medetor®). Ketamine and medetomidine induce muscle relaxation, deep sedation and loss of locomotion. The anesthesia was prepared and administered to the right lower half abdominal region by intraperitoneal injection with 1 mL syringe and 25G needle. The volume administered would have in consideration the animal body weight.

Mice were placed in a thermic pillow, and tumor removal was made by rupture of the skin with a scissor, without peritoneal rupture except when signs of invasion were seen. Tumor was divided in 3 parts as follows: 1/3 to snap freeze in liquid nitrogen (for further RNA studies), fix 1/3 in formaldehyde (for further histologic characterization) and 1/3 to tumor xenograft dissociation (for in vitro cell culture studies). Wound was closed with absorbent suture.

After suture, mice were injected with 1-2.5 mg/Kg Antipamezol (Reversor®) to reverse the anesthesia effect and with 20-40 mg/Kg of Tramodol (Tramal®,) to diminish post-surgical pain. Mice were controlled till they started waking from anesthesia.

3.3. Tail vein inoculation

For Lung Experimental Model of Metastasis Evaluation, different cell concentrations of viable P-cadherin overexpressing BCC (BT20 and MDA-MB-468) and a known highly metastatic BCC (MDA-MB-231.Br.HER2+) were intravenously inoculated in the tail vein, with 1 mL syringe and a 25G needle. Mice were properly restrained. Due to high mortality given to inoculation difficulties, doses were reduced along the experiment from 2x106 cells to 5x105 cells, in the following order:

 MDA-MB-231.Br.HER2+: started with 2x106

cells and reduced to 1x106 cells:  MDA-MB-468: inoculated with 1x106

cells;  BT20: started with 1x106

cells and reduced to 5x105 cells.

Figure 7: Experimental Model for Metastasis Evaluation scheme. Mice are inoculated and treatment begins right after inoculation. The mice are kept until they present signs of disease or reach the defined time-point, time when lungs are obtained

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51 A total of ten nude mice were inoculated for the MDA-MB-231.Br.HER2+ breast cancer cell line (n=10). A total of eleven nude mice were inoculated for the BT20 breast cancer cell line (n=11). And a total of sixteen nude mice were inoculated for the MDA-MB-468 breast cancer cell lines (n=16).

Dasatinib was orally administered already in the first day, to a final concentration of 10 mg/kg, during 5 days per week (control mice have been treated with the drug vehicle for the same time). Mice were examined daily and weighted two times a week to control possible weight losses. Mice were euthanized when there were signs of disease, such as prolonged accelerated breath and loss of more than 20% of the weight, or still other clinical sign of pain and stress.

In the MDA-MB-231.BR.HER2+ there were successfully inoculated four mice with the dose of 2x106 cell per 100 µL and six with 1x106 cell per 50 µL. As for BT20 four mice were inoculated with the dose of 1x106 cells per 100 µL and seven with 5x105 cells per 50 µL.

A limit time-point for euthanasia was defined, after previous optimization, as follows:

 70 days for MDA-MB-231.Br.HER2+ inoculated mice;  100 days for BT20 inoculated mice;

 120 days for MDA-MB-468 inoculated mice.

Lungs were then fixed, processed for routine light microscopy and histologically examined in hematoxylin and eosin (H&E) staining.

3.4. Euthanasia

Animal euthanasia was carried out in accordance with the directive nº 113/2013. Whenever mice presented signs of disease, such as prolonged accelerated breath, abdominal ascites or weight loss of 20% of body fat (Human Endpoints), euthanasia was performed. Mice were put dormant with Isoflurane (IsoFlo, Estive) in a 50 ml falcon tube. After dormant state confirmation, with toe-pinprick, cervical dislocation was performed. The necropsy started with a skin incision from the urogenital zone to the neck of the mouse. Search for lymph nodes from inguinal area and axillary area was performed. After, abdominal cavity was opened by peritoneal membrane incision and visceral examination was done to search for possible metastasis. Tumors found and liver were removed for further studies. Then, for thoracic examination and lung extraction, ribs were cut from the side to get a full exposure of the thoracic cavity. Lungs were removed and screened for macroscopic metastasis and perfused with neutral buffered, formalin solution 10% (Sigma-Aldrich®). After, brain was

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52 accessed by head decapitation and exposure by scalp removal, being laid in neutral buffered formalin solution 10% fixation for 7 days. Remains were rejected if no macroscopic metastasis were observed.

For Model 1: Spontaneous Model in Tumor and Metastasis Formation, mice were euthanized as they start to present signs of illness, without a defined finishing date for the experiment. In relation to Model 2: Experimental Model for Metastasis Evaluation, mice from both Control and Dasatinib groups were euthanized at the determined time.

For all experimental models, metastasis localization and tumor size was done at the time of euthanasia. Histopathological features were late carefully assessed only in lungs. Due to the reduced size, metastasis were impossible to uncover for RNA studies and in vitro studies. When no observed metastasis were seen, fixation in formalin solution was performed for further histological analysis of lungs.

Table 1: Resume table of used animal models and experimental designs.

Cell Lines

Animals (n)

Inoculation Dose Place of Inoculation

Control Dasatinib

Spontaneous Model for Tumor and Metastasis Formation

SUM149 PT 9 8 2x106 cells

Mammary Fat Pad

BT20 7 8 2x106 cells

MDA-MB-468 6 6 2x106_cells Experimental Model on Metastasis Evaluation

BT20 7 6 1x106 cells to 5x105 cells

Tail Vein

MDA-MB-468 8 8 1x106 cells

MDA-MB-231.Br.HER2+ 5 5 2x10

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4. Xenograft culture

4.1. Collagenase dissociation

After tumor removal, a small part of the tumor was extracted for tumor dissociation. Tumors were preserved in simple media with 2% penicillin/streptomycin. In quarantine flow chamber, xenograft tumor tissue was placed in a cell culture plate (10 cm) for dissociation. Tissues were minced in very fine pieces using two sterile scalpels. Minced tissues were transferred for a conical 50 mL tube and 10-15 mL Collagenase solution was added.

Collagenase solution was made by dilution of 100 U/mg of Collagenase Type I (EDM Millipore Corporation, Temecula, CA, USA) lyophilized enzyme (8 mg) in 15 mL of Hank’s Balanced Salt Solution (HBSS) media with 5% penicillin / streptomycin.

After, 50 mL conical tube was placed in 37ºC water bath with constant pipette every 15 minutes. In total of 45 minutes, a double volume of complete media was added and sample was sieved through BD cell strainer tube (40 μM), passing the suspension to a new 50 mL tube. After, centrifugation was done in 5804 R refrigerated benchtop centrifuge (Eppendorf), at 1200 rpm for 5 minutes at 4ºC, for fully collagenase inactivation. Supernatant was discarded and two washes were made with HBSS at 1200 rpm for 5 minutes at 4ºC. After, cells were resuspended in complete culture media and placed in cell culture flask.

Cells were followed for several days. If cell culture presented signs of contamination by bacterial or fungi, culture was discarded. If cross contamination with fibroblast was observed, cells were maintained in culture until fibroblast population decreased. If none of the postulated conditions were observed, after 80% to 90% confluency, cells were trypsinized for cell cryopreservation and for cell lysate.

4.2. Cell protein lysates

For cell lysates, cell culture showed around 50 to 60% confluence. Cells were initially washed in Phosphate-Buffered Saline (PBS). Catenin Lysis Buffer (CLB) with protease inhibitor (1:7) and phosphatase inhibitor (1:100) was added to the cell flask. CLB was previously prepared by addition of 1% Triton-x 100 and NP-40 in PBS. After addition of CLB to the cells, the flask remained at 4ºC for 10 minutes, after which it was scrapped with a policeman and protein extracts were recovered to an eppendorf. Cell lysates were resuspended, vortexed, centrifuged at 14 000 rpm, for 10 minutes at 4ºC, and supernatant