HOXB genes function in Breast Cancer

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IN ST IT U T O D E C IÊ N C IA S B IO M ÉD IC A S A B EL S A LA Z A R

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HOXB

genes function in breast cancer

Mafalda Araújo Pereira

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2018

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2018

MESTRADO ONCOLOGIA

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i

Mafalda Araújo Pereira

HOXB genes function in breast cancer

Dissertação de Candidatura ao grau de

Mestre

em

Oncologia

Molecular

submetida ao Instituto de Ciências

Biomédicas de Abel Salazar da

Universidade do Porto

Orientadora Doutora Carla Renata Gonçalves de

Freitas

Categoria Investigadora Auxiliar

Afiliação

Grupo “Cell Growth and Differentiation”

Instituto de Biologia Molecular e Celular

Instituto de Investigação e Inovação em

Saúde- Universidade do Porto

Co-orientador Doutor Pedro Nuno Simões Rodrigues

Categoria Professor Associado

Afiliação Departamento de Produção Aquática

Instituto de Ciências Biomédicas Abel

Salazar - Universidade do Porto

Vice-Reitor

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ii

ACKNOWLEDGEMENTS

Everyday throughout the making of this master’s thesis I have learned something new. I have grown personally and professionally, a fact that would not have been possible without the support of several people.

My sincere thanks to Professor Carmen Jerónimo, Diretor of the Master Course in Oncology for accepting my application in the course and for collaborating in the project, allowing us to further complement and improve our research.

To the VDR group at i3s, thank you all for welcoming me as one of your own, for tolerating my presence and helping me in any way you could. To Joana Teixeira for allowing me to have a peek inside the bioinformatics point of view, your help was essential in this project. I am keeping my fingers crossed to further new discoveries with you. To my fellow musketeers, Fábio Júnio and Marta Duque for putting up with me nights in a row, challenging me with stimulating discussions and overall making me a better person in both science and life. To Nuno Padrão for indulging with me in the silliest things inside and

outside the lab, do not dare to ever lose that spark you carry with you. I would also like to

thank every member of the CGD group for helping me in my several existential crises in the lab, it has been lovely working alongside you. To Ana Paço for the great work previously developed in the group, for introducing me to the world of cell culture and for being always available to help me whenever I needed it. To Simone Bessa for teaching me so many incredible new things and for being an amazing partner despite my non-stop questioning. When you first arrived at the group I was in absolute awe, somehow, I am still in awe today. You are incredibly smart and kind I am extremely proud to call you my friend.

I thank my co-supervisor Dr. Pedro Rodrigues for the continuous encouragement throughout my academic career which for me has been a privilege. Likewise, to my supervisor Dr. Renata Freitas, to whom there are no words to express enough gratitude. You have accompanied me for years now and guided me with so much patience and motivation that I am going to be forever grateful. You have given me immense new opportunities and tools to develop the exact kind of career I want. It has been an honour having you as my mentor. I sincerely hope I have been up to the expectations, and if not, I will promise to work harder. Thank you!

Last but not least, I would like to thank my friends back home and family, in particular my parents. You are my backbone and this accomplishment would not had been possible without your relentless support. Thank you for everything you have sacrificed for your kids and I am very sorry for my grumpy moods on the weekends. I hope I have made you proud. This is one is for you!

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v

RESUMO

Introdução: Os genes HOX codificam fatores de transcrição fundamentais para o desenvolvimento embrionário, afetando processos como: proliferação celular, apoptose, diferenciação, mobilidade e sinalização. Dito isto, não é surpreendente o envolvimento de proteínas HOX num contexto oncológico, onde estes processos estão deveras alterados. O cancro da mama é o cancro mais comum e mais letal entre as mulheres em todo o mundo. Curiosamente, 27 genes HOX foram encontrados como desregulados nesta patologia, com uma tendência para estarem sobreexpressos, tais como alguns genes do “cluster” HOXB. No entanto, permanece desconhecido, o impacto direto da desregulação dos HOX na maquinaria molecular por trás do início e da progressão do cancro. Resultados preliminares do nosso laboratório apontam para um envolvimento do HOXB7 no controlo indireto epigenético da CDH1, interferindo na expressão de uma DNA metiltransferase, DNMT3B.

Objetivos: O principal objetivo desta dissertação de mestrado foi explorar o impacto da desregulação dos genes HOXB nos mecanismos moleculares envolvidos no desenvolvimento do cancro da mama in vitro e em biópsias de tumores.

Materiais e Métodos: Começamos por realizar um ensaio de agregação nas células MDA-MB-231 transfectadas com siHOXB7 para explorar o impacto do HOXB7 na desregulação da expressão da CDH1 e DNMT3B. De seguida, caracterizamos a expressão do HOXB7 e dos seus alvos putativos em quatro linhas celulares, que representam os distintos subtipos de cancro da mama, e em biópsias de tumores por qPCR e western blot. De igual modo, determinamos também os níveis de expressão de mRNA do HOXB8 in vitro e em biópsias por qPCR. Além disso, conseguimos testar a melhor sequência de siRNA para realizar um decréscimo transiente na expressão do HOXB8 nas células MDA-MB-231. Analisamos também por qPCR a expressão de um gene novo em cancro da mama, COMMD7, em células e biópsias.

Resultados e Discussão: A partir do ensaio de agregação pressupomos que o silenciamento do HOXB7 nas células MDA-MB-231 pode não ser suficiente para modificar a função da proteína CDH1, uma vez que não se formaram agregados. No entanto, com o perfil basal dos níveis de expressão de mRNA de HOXB7, CDH1 and DNMT3B, é nos possível identificar outras linhas celulares de cancro da mama como melhores pretendentes para continuar a estudar a nossa hipótese. Os nossos resultados de expressão de HOXB7 em tecidos de tumor contradizem não só os nossos resultados in

vitro como estudos prévios. Em contrapartida as análises relativas ao HOXB8 estão de

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vi diferenciar a expressão dos nossos alvos por tecido normal e cada subtipo de cancro da mama aumentando assim a especificidade no estudo. Análises in silico de dados de ChIP-seq de uma linha de cancro da mama permitiram-nos explorar uma ligação não direta entre o HOXB7 e o promotor da DNMT3B. No entanto, através destas análises descobrimos um sítio putativo de ligação no COMMD7, um gene que representa a sequência codificante mais próxima da DNMT3B e é primeiramente reportado por nós em cancro da cama, por estudos in vitro e em biópsias.

Conclusões e perspetivas futuras: Como última observação os nossos estudos contribuíram para uma melhor compreensão dos mecanismos moleculares por trás dos genes HOXB, in vitro e em tecidos malignos, fazendo com que fosse possível explorar através de futuras análises no laboratório a função dos genes HOXB e o novo oncogene

COMMD7, em cancro da mama. Este projeto habilitou-nos a explorar no futuro a

desregulação dos HOX considerando o envolvimento de um novo alvo, tendo em contra a variabilidade e a heterogeneidade do cancro da mama.

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vii

ABSTRACT

Introduction: HOX genes encode transcription factors crucial for embryonic development, affecting cell proliferation, apoptosis, differentiation, motility and signalling. Therefore, is not surprising an involvement of HOX proteins in an oncological context, where these cellular processes are severely affected. Breast cancer is the most common and deadliest cancer among women worldwide. Interestingly, 27 HOX genes have been found to be deregulated in this malignancy and most have a tendency for overexpression such as those from the

HOXB cluster. However, it remains unknown the direct impact of these HOX deregulation

in the molecular networks behind cancer initiation and progression. Preliminary results of our lab point to an involvement of HOXB7 in the indirect epigenetic control of CDH1, interfering with the expression of a DNA methyltransferase, DNMT3B.

Aims: The major goal of this master dissertation was to investigate the impact of HOXB genes deregulation in the molecular networks involved in breast cancer progression in vitro and in biopsies from patients.

Materials and Methods: We started by performing an aggregation assay of MDA-MB-231 transfected with siHOXB7 to explore the impact of HOXB7 downregulation in CDH1 and

DNMT3B expression. Then we characterized HOXB7 and its putative downstream targets

in four cell lines, which depict each breast cancer subtype, and in biopsies from patients by qPCR and western blot. Likewise, HOXB8 mRNA expression levels were also described in

vitro and in tumour tissues by qPCR. Moreover, we were able to test the best sequence of

siRNA to perform a transient knockdown of HOXB8 in MDA-MB-231 cells. Furthermore,

COMMD7, a novel gene in breast cancer was also described as altered in cells and biopsies

by qPCR.

Results and Discussion: From the aggregation assay we postulate that the silencing of

HOXB7 in MDA-MB-231 cells might be insufficient to modify the function of CDH1 proteins

due to the lack of aggregates formation. However, with the basal profile of HOXB7, CDH1

and DNMT3B mRNA expression levels now we were able to identify other breast cancer

cell lines as better candidates to pursue our work hypothesis. HOXB7 expression in tumour tissues contradict our in vitro results and some previous reports. On the other hand, HOXB8 analyses seems to be in accordance with the results obtained for HOXB7. Nonetheless, here we distinguished our target expressions within each breast cancer subtype and normal breast tissues, increasing specificity in our studies. In silico analyses using ChIP-Seq data from a particular breast cancer cell line lead us to identify a non-direct binding between HOXB7 and DNMT3B promotor. However, we discovered through these analyses that HOXB7 has a putative binding site in COMMD7, a gene which represents the closest coding

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viii sequence in the vicinity of DNMT3B and first reported by us in breast cancer, through in

vitro and tumour biopsies analyses.

Conclusions and future perspectives: As a final remark, our work has contributed to a deeper understanding to the molecular machinery behind HOXB genes in vitro and tumour tissues and made the essential preliminary sets for future experiments in the lab regarding the function of HOXB genes and also the novel oncogene in breast cancer, COMMD7. Thus, with this project we are now able to pursue further studies taking account the variability and heterogeneity of breast cancer, while having also unravelled a possible new target involved in HOX molecular network in breast cancer.

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ix

LIST OF ABBREVIATIONS

AI – Aromatase inhibitor BrCa – Breast cancer

CDH1 – E-cadherin gene

CDH1 – E-cadherin protein

ChIP – Chromatin immunoprecipitation

COMMD7 – COpper Metabolism MURR1 Domain-containing 7 CpG – Cytosine-phosphate-Guanine

CSC – Cancer stem cell CTC – Circulating tumour cells DCIS – Ductal carcinoma in situ LCIS – Lobular carcinoma in situ

DMEM – Dulbecco's Modified Eagle Medium DNA – Deoxyribonucleic acid

DNMT - DNA methyltransferase

DNMT3B - DNA methyltransferase 3 beta EGF – Epidermal growth factor

EGFR - Epidermal growth factor receptor EMT - Epithelial–mesenchymal transition ER – Estrogen receptor

FBS – Fetal Bovine Serum FW – Forward

H3K27ac – Histone H3 lysine 27 acetylation

HER2 – Human epidermal growth factor 2 receptor HOX – Homeobox

HOXB7 - Homeobox B7 HOXB8 - Homeobox B8 IC – Invasive carcinoma

IDC – Invasive ductal carcinoma ILC – Invasive lobular carcinoma MBD – Mammographic breast density MRI – Magnetic resonance imaging mRNA – Messenger ribonucleic acid

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x NBr – Normal breast

NF-κB - Nuclear factor kappa B NHEJ – Non-homolgous end-joining

OTCD – Oncogerminative theory of cancer development OC – Oncogerminative cells

O/N – Over-Night

PBS – Phosphate-Buffered Saline PCR – Polymerase Chain Reaction PR - Progesterone Receptor qPCR – Quantitative real-time PCR RPM – Rotations per minute

RNA – Ribonucleic acid

RT-PCR – Reverse transcriptase PCR RV – Reverse

siC- – Negative control siRNA siRNA – Small interfering RNA

siHOXB7 – Small interfering RNA specific for HOXB7 siHOXB8 – Small interfering RNA specific for HOXB8

TALE – Three amino acid loop extension

TAM – Tamoxifen

TF – Transcription factor

TNBC – Triple negative breast cancer TNM – Tumour-Node-Metastases TP53 – Tumour protein 53

WHO – World Health Organization WT – Wild Type

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xi

FIGURES INDEX

Figure 1: Incidence (A) and Mortality (B) associated to the 5 most common cancers in European women (2012). Each portion of the pie chart represents the percentage of the total number of cases or deaths. Adapted from 1_{... 2}

Figure 2: BrCa incidence and mortality standardized by age, distributed by area and country in Europe 2012. Adapted from 1

. ... 3

Figure 3: Diagram representing risk factors and preventions for BrCa. Pyramid on the left shows the five most important risk factors: aging, family history, reproductive factors, estrogen levels and life habits. Forms of BrCa prevention are indicated in the right: screening (mammography and MRI), chemoprevention (with SERMs and AIs) and biological prevention (herceptin and pertuzumab). PD1/PDL1 inhibitors are also presented as promising immunotherapy drugs used in BrCa treatment. Adapted from9_{. ... 7}

Figure 4: Diagram representing the natural history of most BrCas. DCIS. A-B: The cancer process stars with abnormal proliferation of the epithelial cells of ducts or lobules. C-D: Over time, normal cells generated by extra-proliferation (hyperplasia) accumulate abnormalities (atypical hyperplasia). E: Eventually these cells become invasive and start to expand to other parts of the body. Adapted from44_{. ... 8}

Figure 5: Histopathological subtypes of BrCa. Adapted from 42

. ... 9

Figure 6: Diagram representing aberrant HOX genes expression in various solid tumour types. Adapted from 91_{. ...16}

Figure 7: HOXB7 role in pathways critical to initiation and progression of solid cancers.

Adapted from103_{. ...19}

Figure 8: Worflow followed by our team (CGD group/i3s) to study HOXB7 function in BrCa cell lines. ...21 Figure 9: Working hypothesis of HOXB7 function in cancer. ...23 Figure 10: HOXB7 mRNA expression levels by qPCR in MDA-MB-231 cells. Y-axis depicts 2-ΔΔCT values and X-axis indicates the three time points evaluated post-transfection. HOXB7 expression of siC- and siHOXB7 conditions are compared to WT cells of each time point post-transfection. Colour bars indicate each of the cellular conditions tested: non-manipulated cells (WT); negative control siRNA transfected cells (siC-); HOXB7 siRNA transfected cells (siHOXB7). Note stronger silencing of HOXB7 at 48h and 72h post-transfection. ...37

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xii Figure 11: Representative panel of siHOXB7-transfected MDA-MB-231 cells aggregation assay (5x objective). Three conditions for MDA-MB-231 cells are depicted: non-manipulated (WT), transfected with a negative control (siC-) and siRNA for HOXB7 (siHOXB7). MCF-7 cells used as a positive control of aggregation. The lines in the panel show each condition without (-MB2) and with (+MB2) addition of MB2 antibody. Note the lack of aggregates formation in MDA-MB-231 WT, siC- and siHOXB7 cells. ...38 Figure 12: CDH1 mRNA expression levels by qPCR in MDA-MB-231 cells. Y-axis depicts 2-ΔΔCT values and X-axis indicates the three time points evaluated post-transfection. CDH1 expression of siC- and siHOXB7 conditions are compared to WT cells of each time point post-transfection. Colour bars indicate each of the cellular conditions tested: non-manipulated cells (WT); negative control siRNA transfected cells (siC-); HOXB7 siRNA transfected cells (siHOXB7). Note higher levels of CDH1 expression in siHOXB7 condition. ...39 Figure 13: DNMT3B mRNA expression levels by qPCR in MDA-MB-231 cells. Y-axis depicts 2-ΔΔCT values and X-axis indicates the three time points evaluated post-transfection.

DNMT3B expression of siC- and siHOXB7 conditions are compared to WT cells of each

time point post-transfection. Colour bars indicate each of the cellular conditions tested: non-manipulated cells (WT); negative siRNA transfected cells (siC-); HOXB7 siRNA transfected cells (siHOXB7). No significant alterations between DNMT3B expression were observed. ...40 Figure 14: Representative 1.2 % agarose gel for RNA quality assessment. 1: BT -474; 2: SKBR3; 3: MDA-MB-231; 4: MCF-7. Note intact RNA in all cell lines indicated by the clear visibility of the 18S and 28S ribosomal RNA bands. ...41 Figure 15: Representative 2 % agarose gel for cDNA synthesis validation from RNA of NBr tissues. 1: 1Kb Ladder; 2: MDA-MB-231 3: Negative control. ...41 Figure 16: HOXB7 basal mRNA expression levels analysed by qPCR. The differences in expression of BrCa cells (MCF-7; BT-474; SKBR3; MDA-MB-231) were compared to MCF10A cells. HOXB7 is significantly overexpressed in all BrCa cells. The * denotes p-value < 0.05, the ** denotes p-p-value < 0.01 by unpaired T test with Welch's correction. Y-axis depicts 2-ΔCT values of HOXB7 expression. ...42 Figure 17: Box-plots of mRNA expression levels of HOXB7 in human BrCa tissues analysed by qPCR. A. NBr (n=11) depicts significant higher levels of HOXB7 than BrCa (n=109) tissues. B. HOXB7 in Triple Negative BrCa is significantly downregulated when compared to NBr Tissue (Luminal A (n=33), Luminal B (n=33), HER2+ (n=10), Triple Negative (n=33)). Outliers were identified by the SPSS software and indicated with º when

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xiii considered as “Mild Values” and with * when considered as “Extreme Values”. Y-axis depicts 2-ΔCT values of HOXB7 expression. ...44 Figure 18: CDH1 basal mRNA expression levels analysed by qPCR. The differences in expression of BrCa cells (MCF-7; BT-474; SKBR3; MDA-MB-231) were compared to MCF10A normal cells. MCF-7 and BT-474 cells overexpress CDH1. The * denotes p-value < 0.05 by unpaired T test with Welch's correction. Y-axis depicts 2-ΔCT values of CDH1 expression. ...45 Figure 19: CDH1 basal protein expression level in cells by western blotting. PCS-600-010, MCF-7 and BT-474 present high levels of CDH1 protein. Note the absence of protein expression in SKBR3 and MDA-MB-231 cells. ...46 Figure 20: Box-plots of mRNA expression levels of CDH1 in human BrCa tissues analysed by qPCR. A. No significant differences were found in CDH1 expression between NBr (n=11) and BrCa (n=109) tissues. B. CDH1 expression between NBr (n=11) and each subtype of BrCa shown no significant differences (Luminal A (n=33), Luminal B (n=33), HER2+ (n=10), Triple Negative (n=33)). Outliers were identified by the SPSS software and indicated with º when considered as “Mild Values” and with * when considered as “Extreme Values”. Y-axis depicts 2-ΔCT values of CDH1 expression. ...47 Figure 21: DNMT3B basal mRNA expression levels analysed by qPCR. The differences in expression of BrCa cells (MCF-7; BT-474; SKBR3; MDA-MB-231) were compared to MCF10A normal cells. DNMT3B is significantly overexpressed in all BrCa cells. The * denotes p-value < 0.05 and the ** denotes p-value < 0.01 by unpaired T test with Welch's correction. Y-axis depicts 2-ΔCT values of DNMT3B expression. ...48 Figure 22: DNMT3B basal protein expression level in cells by western blotting. MCF-7, BT-474 and MDA-MB-231 have high DNMT3B protein levels. PCS-600-010, MCF10A and SKBR present moderate expression levels of DNMT3B. ...49 Figure 23: Box-plots of mRNA expression levels of DNMT3B in human BrCa tissues analysed by qPCR. A. BrCa (n=109) depicted significant higher levels of DNMT3B than NBr (n=11) tissues. B. DNMT3B expression was significant upregulated in HER2+ when compared to NBr (n=11) and Luminal A tissues (Luminal A (n=33), Luminal B (n=33), HER2+ (n=10), Triple Negative (n=33)). Outliers were identified by the SPSS software and indicated with º when considered as “Mild Values” and with * when considered as “Extreme Values”. Y-axis depicts 2-ΔCT_{values of DNMT3B expression. ...50}

Figure 24: HOXB8 basal mRNA expression levels analysed by qPCR. The differences in expression of BrCa cells (MCF-7; BT-474; SKBR3; MDA-MB-231) were compared to MCF10A normal cells. HOXB8 is overexpressed in MCF-7, BT-474 and MDA-MB-231 cells.

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xiv The * denotes p-value < 0.05, the ** denotes p-value < 0.01 and the *** denotes p-value < 0.001 by unpaired T test with Welch's correction. Y-axis depicts 2-ΔCT values of HOXB8 expression. ...52 Figure 25: Box-plots of mRNA expression levels of HOXB8 in human BrCa tissues analysed by qPCR. A. NBr (n=11) depicted significant higher levels of HOXB8 than BrCa (n=109) tissues. B. HOXB8 expression was significant downregulated in Luminal A, Luminal B and Triple Negative BrCa when compared to NBr (n=11) tissues (Luminal A (n=33), Luminal B (n=33), HER2+ (n=10), Triple Negative (n=33)). Outliers were identified by the SPSS software and indicated with º when considered as “Mild Values” and with * when considered as “Extreme Values”. Y-axis depicts 2-ΔCT_{values of HOXB8 expression. ...53}

Figure 26: HOXB8 mRNA expression levels analysed by qPCR. Y-axis depicts 2-ΔΔCT values and X-axis depicts the several conditions tested. Note that the combination of sequences (SEQ) 1 and 2 presents promotes the highest HOXB8 silencing and the non-significant variation in the expression of non-transfected cells (WT), transfected with HiPerfect (HIP) and negative control siRNA (siC-). ...54 Figure 27: Human genomic landscape of COMMD7 and DNMT3B that includes an enriched region for HOXB7 binding site (red arrow). ENCODE data, showing the H3K27ac signal distribution that marks active enhancer/promoters and chromatin state segmentation data set. These results were generated by ENCODE consortium and are available on the Genome Browser at UCSC146_{. ...55}

Figure 28: COMMD7 basal mRNA expression levels analysed by qPCR. The differences in expression of BrCa cells (MCF-7; BT-474; SKBR3; MDA-MB-231) were compared to MCF10A normal cells. MCF-7, BT-474 and MDA-MB-231 cells have significantly higher levels of COMMD7. The * denotes p-value < 0.05, the ** denotes p-value < 0.01 and the *** denotes p-value < 0.001 by unpaired T test with Welch's correction. Y-axis depicts 2-ΔCT values of COMMD7 relative expression. ...56 Figure 29: Box-plots of mRNA expression levels of COMMD7 in human BrCa tissues analysed by qPCR. A. No significant differences were found in COMMD7 expression between NBr (n=11) and BrCa (n=109) tissues. B. COMMD7 expression was significant downregulated in Triple Negative BrCa when compared to Luminal A, Luminal B and NBr tissues (Luminal A (n=33), Luminal B (n=33), HER2+ (n=10), Triple Negative (n=33)). Outliers were identified by the SPSS software and indicated with º when considered as “Mild Values” and with * when considered as “Extreme Values”. Y-axis depicts 2-ΔCT_{values of}

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xv Figure 30: MDA-MB-231 DNA after sonication. Note in 2 and 3 the smear of fragmented DNA located between 1000 bp with a strong band close to 500bp. 1 – 200 bp ladder, 2 – 20 cycles of sonication, 3 – 32 cycles of sonication...58

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xvi

TABLES INDEX

Table 1: Workflow for early-staged BrCa diagnosis. Adapted from31_{. ... 6}

Table 2: Summary of molecular tests most commonly used for BrCa in current clinical practice. Adapted from 53_{. ...11}

Table 3: HOX genes involved in tumorigenesis and metastasis. Adapted from 66 - ...17

Table 4: Classification of BrCa cell lines. Adapted from 133_{. ...27}

Table 5: Components of the PCR reaction to validate the cDNA synthesis (*Qiagen Multiplex PCR kit; Qiagen, Hilden, Germany)...29

Table 6: PCR amplification protocol for β-Actin. Forward: GAGCACAGAGCCTCGCCTTT. Reverse: ACATGCCGGAGCCGTTGTC. Product size: 108 bp ...29

Table 7: HOXB7, HOXB8 and GAPDH primers sequence, product length and annealing temperature. HOXB7 primer sequence obtained from 134_{. HOXB8 and GAPDH primers} designed using primer3 online software. ...30

Table 8: Aggregation assay conditions. *MB2 – anti-CDH1 antibody, 1:50 dilution ...31

Table 9: Components of the Cell Lysis Buffer. ...33

Table 10: Components of the Nuclear Lysis Buffer. ...33

Table 11: BrCa subtypes and the respective p-values of CDH1 expression when compared with NBr Tissue by non-parametric Mann-Whitney U test. Note non-significances found for all molecular sub-types of BrCa indicated by p-values >0,05. ...46

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1

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2

1.1 Breast Cancer

1.1.1 Epidemiology

Breast Cancer (BrCa) is the most frequently occurring cancer in women and one of the most common cancers not only in Europe, as well as worldwide(Fig. 1.A) 1-3_{. BrCa is also the}

leading cause of death among women (Fig. 1.B) 1,4_{. In 40 European countries in 2012, the}

estimated age-adjusted annual incidence of BrCa was 94.2/100 000 and the mortality rates were 23.1/100 000 and it was by far the most commonly diagnosed cancer in women (Fig. 2) 1_{. On the other hand, male BrCa is quite rare and accounts for less than 1% of all cancers}

in men 5_.

The increasing incidence of BrCa in Europe has been frequently associated with known risk factors such as, low parity, age of first birth, use of post-menopause therapy, obesity, physical inactivity, among others 1,4_{. Nevertheless, the introduction of mammographic}

screening as well as the increased age of the women population may have also contributed to obtain higher values of BrCa incidence. In contrast, the implementation of cancer control measures, leading to early diagnosis and implementation of adjuvant therapies, have been contributing to revert the BrCa incidence and mortality rates 1,4_{. Although the specific}

contribution of each measure it is still debatable 6_{, Portugal and Spain provide good}

examples of the advantages of these same measures (Fig. 2). While the incidence of BrCa

A

INCIDENCE

B

MORTALITY

Figure 1: Incidence (A) and Mortality (B) associated to the 5 most common cancers in European

women (2012). Each portion of the pie chart represents the percentage of the total number of cases or deaths. Adapted from 1_.

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3 is close to the European average in these countries, they present the lowest mortality rates. Therefore, mortality associated to BrCa seems to be highly dependent on national health system policies 1_{. Overall, the studies performed since 1990 suggest that the risk of getting}

BrCa seems to be stabilizing in Europe whereas the death risk appears to be declining 7_.

According to Allemani and colleagues 8_{ten-year survival of BrCa in most European regions}

exceeds 70%, in which, 89% was for local, 62% for regional and 10% for metastatic disease.

1.1.2 Risk Factors

Identifying risk factors and their action mechanism is critical for cancer prevention. The five major risk factors involved in BrCa are aging, family history, reproductive factors, estrogen levels and life habits 9_{(Fig. 3).}_{Age has been pinpointed as the major risk factor for BrCa,} given that its incidence rises sharply throughout a woman’s lifetime 3,10_{. For example, 2 in}

each 3 invasive BrCa are found in women over 55 years old. However, when BrCa occurs at younger ages, before 40, the risk of relapse and death is considerably higher 11_{. Thus,}

many studies suggest that 40-45 years is the approximate threshold for prognosis with worse outcome of younger age women with BrCa 8,11_.

Figure 2: BrCa incidence and mortality standardized by age, distributed by area and country in

Europe 2012. Adapted from 1

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4 Although, older women have a higher BrCa incidence, it is still not known the explanation behind these disparities and whether they can be explained by a numerous events accumulated throughout a woman’s lifetime or by a one-time event set off with greater frequency in older instead of younger women 10_{. In addition, ethnicity also alters the age of}

BrCa risk 3,10_{. For example, American women of African descendance are more prone to}

develop BrCa in more aggressive and advanced stages before 40 years, contrarily to Caucasian women 10,12_.

Family history of BrCa can increase the development of this pathology especially among younger women 13_{. The risk for BrCa relates directly with the number of first-degree relatives}

with the disease. BrCa progression comprises numerous genetic alterations that can activate oncogenes and disturb the function of specific tumour progression genes 14_.

Therefore, additional DNA repair genes such as Tumour Protein p53 (TP53) 15_and

Checkpoint kinase 2 (CHEK2), both associated with Li-Fraumeni syndrome 16_and

Phosphatase and Tensin homolog (PTEN) also play a role in familial BrCa.

Hereditary in BrCa is associated to 5 to 10% of the diagnosed cases. The two major susceptibility genes involved in hereditary BrCa are BRCA1 and BRCA2. Mutations in these genes confer a BrCa risk that ranges from 43% to 85% 17_{. Several genetic linkage studies}

have tried, with little success so far, to identify novel or major BrCa susceptibility genes 18_,

suggesting that BrCa susceptibility is “polygenic”, meaning that BrCa susceptibility is granted by a large number of loci, each with a small effect on BrCa risk 19_{. In commercially}

available risk assessment panels, STK11, CDH1, PALB2, ATM, NBN and NF1 are also included as potential driver genetic alterations for BrCa 20_{. Breast tumours that arise due to}

genetically transmitted abnormalities, as BRCA1/BRCA2 and TP53 mutations, mainly belong to the younger age group (< 45 year age), displaying a more aggressive phenotype

3_{. Therefore, several genetic studies are trying to demonstrate the presence of specific}

genetic alterations in familial BrCa, so these same alterations can be suggested as measures to reduce BrCa risk and improve the early detection of the disease.

The personal history of breast lesions is also related with a higher risk of BrCa. Women diagnosed with these lesions, namely those with a history of proliferative breast disease with atypia (PBDA) have higher risk to develop carcinomas 21_{. Likewise, mammographic}

breast density (MBD) also influences BrCa risk and women with a denser breast tissue are at a higher risk of BrCa. A series of factors such as age, genetics, age and body mass index, pregnancy and menopause seem to affect MBD 22_.

High levels of endogenous estrogen exposure have also been described as BrCa risk factors and various studies have consistently demonstrated that the increased use of

(22)

post-5 menopause hormones are accompanied by a BrCa risk, since Estrogen expression dysregulation is involved not only in normal breast (NBr) formation as well in breast tumorigenic processes 23_{. Likewise, reproductive factors such as null parity, menarche in}

early ages and advanced-age menopause increase BrCa risk 3,4,2410)_{. On the other hand,}

increased parity, early age at birth of first child have a protective effect in BrCa risk, therefore, childbearing seems to have a dual effect 24_{. The effect of breastfeeding on BrCa}

risk is quite controversial but appears to be responsible for a BrCa reduced risk, although it tends to vary between the distinct BrCa subtypes 25,26_{. In addition, postmenopausal}

hormone therapy and postmenopausal obesity also increase BrCa risk27_.

Risk factors, related with unhealthy lifestyles such as alcohol consumption, smoking, lack of physical activity and high-fat diets are associated with higher rates of BrCa24_.

Notwithstanding, numerous reports have noticed a meaningful protective effect between an increased fiber intake and lower risk of BrCa. A mechanism that may explain this association is that a high fiber diet leads to more estrogen release therefore decreasing plasma estradiol concentrations. Furthermore, high levels of fecal fiber can disrupt gut estrogen absorption, reducing the total amount of estrogen in the body28_{. Moreover, research suggest that}

Vitamin D deficiency is also associated with BrCa risk in pre and post-menopausal women29_.

At last, ionizing radiation exposure has also been recognized as a risk factor for development of BrCa. Age at radiation exposure has been inversely associated with BrCa risk 30_.

1.1.3 Diagnosis and Screening

Diagnosis of BrCa is initially based on clinical examination, which includes bimanual palpation of the breasts and locoregional lymph nodes as well as assessment for distant metastases, in combination with imaging techniques (Table 1) 31_{. A first approach for}

diagnostic through imaging tools comprises bilateral mammography coupled with breast and regional lymph nodes ultrasound 32_{. However, in younger women (under the age of 35),}

mammograms have not been shown to be clinically effective in the evaluation of BrCa symptoms as a result of higher density in young breast tissue 33_{. Therefore, due to an}

increased sensitivity and specificity, magnetic resonance imaging (MRI) has become in daily practice, increasingly important in the detection and delineation of BrCa of specific cases (Fig.3) 31,34_{. BrCa evaluation can only be correctly determined by tissue sampling of}

the primary tumour and cytology/histology of the axillary nodes, with further pathological examination if involvement is suspected 31_{. Furthermore, a core needle biopsy is used for}

pathological diagnosis and the final classifications of the tumour is established by the World Health Organization (WHO) and the Tumour-Node-Metastases (TNM) staging system 35_.

(23)

6

Table 1: Workflow for early-staged BrCa diagnosis. Adapted from31_.

MRI: magnetic resonance imaging; ER: estrogen receptor; PR: progesterone receptor; HER2: human epidermal growth factor 2 receptor.

During therapy for metastatic disease, in addition to diagnostic imaging, personal history and physical examination of the patient, the tumour markers (TM) cancer antigen 15-3 (CA 15-3) and carcinoembryonic antigen (CEA) are commonly used as well 36_{. Although}

specificity of TMs between BrCa subtypes it is still poorly understood, BrCa specific survival is significantly worse for patients with elevated TMs 36_.

Several population-based mammography screening programmes have been established in Europe, in order to detect BrCa at a pre-clinical stage 37_{. Monitoring by mammography every}

2 years, in women with ages between 50 and 69 has shown the highest benefit in mortality reduction31_{. Moreover, an annual MRI is recommended in women with familial BrCa,}

alongside or alternating every half-year with mammography. This follow-up should start 10 years before age-at-diagnosis of the youngest case in the family 31_.

Assessment of general health status History

Menopausal status Physical examination Full blood count

Liver, renal and cardiac (in patients planned for anthracycline and/or trastuzumab treatment) function tests, alkaline phosphatase and calcium

Assessment of primary tumour Physical examination

Mammography Breast ultrasound Breast MRI

Core biopsy with pathology determination of histology, grade, ER, PR, HER2 and Ki67

Assessment of regional lymph nodes Physical examination Ultrasound

Ultrasound-guided biopsy if suspicious

Assessment of metastatic disease Physical examination

Other tests are not routinely recommended, unless locally advanced or when symptoms suggestive of metastases are present

(24)

7

1.1.4 Histopathology

BrCa is a highly cellular and molecular heterogenous pathology with distinct clinical outcomes and responsiveness to treatments 38,39_{. Clinically, the histopathological}

classification of BrCa is based on the diversity of the morphological characteristics of tumours 40_{. The WHO identified some 20 major tumour types as well as 18 minor subtypes}

in 2003, and currently this classification is adopted worldwide and essential for pathological diagnostic 40,41_.

The majority of BrCa arises from epithelial cells and can be subdivided into in situ and invasive carcinomas that are classified as ductal and lobular according to the site from which the tumour originated. In situ carcinomas are characterized by atypical epithelial proliferations confined to the ductal/lobular branches of the breast, without the ability to invade through the basement membrane 42_{. Although this type of carcinoma may lack the}

ability to invade the adjacent tissues it may subsequently develop and become invasive 42_.

Therefore, in situ carcinomas can be grouped in ductal carcinoma in situ (DCIS) or lobular carcinoma in situ (LCIS)41_{. However, it also common to see progression from DCIS to LCIS,}

which is designated as lobular cancerization and produce extensive lesions 42_{. Thus, DCIS}

is considered a pre-stage for the subsequent progression of lobular and invasive carcinomas (Fig. 4). Since 1983, the incidence of DCIS has been strikingly increasing due to the widespread screening mammography as well as increasing awareness of BrCa. DCIS

Figure 3: Diagram representing risk factors and preventions for BrCa. Pyramid on the left shows the

five most important risk factors: aging, family history, reproductive factors, estrogen levels and life

habits. Forms of BrCa prevention are indicated in the right: screening (mammography and MRI), chemoprevention (with SERMs and AIs) and biological prevention (herceptin and pertuzumab). PD1/PDL1 inhibitors are also presented as promising immunotherapy drugs used in BrCa treatment.

(25)

8 is responsible for 10-30% of all malignancies detected in current screening programs. Analyses of parameters such as nuclear atypia, intraluminal necrosis, mitotic activity and calcification, allows to classify the DCIS in Low-Grade, Intermediate-Grade and High- Grade, which directly proportional to the risk to become invasive (Fig. 5) 42_.

On the other hand, invasive carcinomas (IC) represent the most common lesion of BrCa and are characterized by having malignant abnormal ductal proliferation of neoplastic cells concomitant with stromal invasion 42_{. They can also be classified according with its site of}

origin as ductal (IDC) or lobular (ILC) (Fig. 4; Fig. 5) 41_{. The invasive ductal carcinoma (IDC)}

no specific type (NST) is the most common type of IC and represents 55% of all diagnosed BrCa 43_{. However, there are other types of IDC, with lower incidence: tubular, cribriform;}

mucinous (2%); medullary; papillary; micropapillary; apocrine; neuroendocrine; metaplastic; lipid-rich; secretory; oncocytic; odenoid cystic; acinic cell (Fig. 5). The invasive lobular carcinoma (ILC) Generally, they are divided in two major histopathological and biologically distinct classes: invasive ductal carcinomas (IDCs) not otherwise specified (NOS) that comprises the largest group of invasive BrCa accounting for up to 75% of all invasive carcinomas, and invasive lobular carcinoma (ILC) 42_{. ILC, represents 5-15% of invasive}

BrCa although its incidence appears to be increasing, notably in post-menopausal women, which may be partly related to hormone replacement treatment in this specific group of women 42_{. This type of BrCa presents 5 distinct histological variants: classic type;}

pleomorphic, histiocytoid, signet ring and tubulolobular (Fig. 5).44

Figure 4: Diagram representing the natural history of most BrCas. DCIS. A-B: The cancer process

stars with abnormal proliferation of the epithelial cells of ducts or lobules. C-D: Over time, normal cells generated by extra-proliferation (hyperplasia) accumulate abnormalities (atypical hyperplasia). E: Eventually these cells become invasive and start to expand to other parts of the body. Adapted

(26)

9

1.1.5 Prognostic and predictive factors

Prognostic factors related to morphological features, such as, lymph node status, size of tumour, histological type and grade have been established and are traditionally used for the assessment of BrCa patients 41_{. However, due to the limited prognosis and predictive power}

of the histopathological classification, gene expression profiling (GEP) studies have been developed for several years to find predictive biological markers that better correlate with the clinical outcome and treatment response. Thus, Estrogen Receptor (ER), Progesterone Receptor (PR) and Human Epidermal Growth Factor 2 Receptor (HER2) are predictive biological markers validated by immunohistochemical methods that are also conventionally used for patient prognosis and management alongside the traditional clinicopathological variables45_.

(27)

10 ER and PR are steroid receptors within the nucleus of breast cells, which bind directly to the DNA, changing its transcriptional activity. They exist in two main forms, ERα and ERβ and PRA and PRB, respectively 46_{. A cell is considered positive for the Estrogen Receptor}

(ER+) or for the Progesterone Receptor (PR+) if it expresses these receptors, allowing to receive signals from estrogen or progesterone that can promote growth. The combination of these steroid hormone receptors are very important and useful predictive factors. Approximately 75 % of all BrCa patients are ER+, in which 65 % and 80 % correspond to patients under and above 50 years respectively 47_{. ER+ tumours are generally more}

differentiated, less aggressive and associated with better prognosis than the ER negative (ER-) tumours 48_{. However, ER+ tumours have a higher risk of relapse than ER-}46_.

Moreover, ER- tumours have been associated with unlikeness of response to endocrine therapy, whereas, approximately 50% of the patients who are ER+ are responsive to anti-estrogen or aromatase inhibitors 45_{. The relapse risk has a tendency to be higher in tumours}

with the ER receptor 46_{. Furthermore, in patients harbouring ER+ tumours the presence of}

PR also has prognostic utility, since the low or absence of its expression is associated with a more proliferative and aggressive tumoral behaviour, poor prognosis and recurrence 49_.

Retrospective analyses of non-randomized studies suggest that tumours with both receptors are correlated with not only more favourable prognosis and endocrine response, as well as, favourable clinico-pathological parameters 48_.

HER2 gene is an oncogene belonging to the Epidermal Growth Factor (EGF) family that

encodes a 185-kDa transmembrane and is situated on the long arm of chromosome 17 50_.

Overexpression of HER2 is present in around 20-30 % of BrCa tumours thus allowing HER2 status to have prognostic and predictive relevance. In fact, when it is present it is correlated with increased aggressiveness of the disease, higher recurrence rate and reduced survival

50,51_{. The HER2 receptor can be clinically targeted with humanized monoclonal antibody}

Transtuzumab, therefore, its presence is a favourable response predictor due to improved

outcome in women with early BrCa when added to the chemotherapy regimens as an adjuvant therapy 49,50,52_{. In addition, HER2-positivity is also an indication of a good}

chemotherapy response to anthracyclines and taxanes’ therapy 49_.

Besides the above-mentioned biomarkers, a few others are being considered due to their biologically informative potential and clinical use. For instance, Ki67, a nuclear protein associated with proliferation markers has been linked to adverse outcome in patients with BrCa as well to prognostic utility 46,49_{. Likewise, cyclin D1 and cyclin E overexpression and}

amplification correlates with, not only poor prognosis, but also adverse endocrine response

49_{. Even though ER, PR and HER2 are still the only three mandatory biomarkers in all BrCa}

(28)

11 management in the last ten years due to their predictive/prognostic information. These multianalyte assays have also been mentioned as gene expression profiles since mostly of them are based on measure of mRNA levels for chosen genes53_{. Several tests in BrCa have}

been developed, including urokinase plasminogen activator (uPA)-PAI-1, Oncotype DX (Genomic Health Institute, CA, USA), MammaPrint (70-gene signature, Agendia, Amsterdam, Netherlands) and Prosigna BrcA assay (Table 2) that have been improving the prognosis and prediction of BrCa 46,49,53_{. Although these tests are objective, reproducible}

and precise, they cannot replace routine pathological evaluation and their use in clinical practice has remain limited for many healthcare systems due to high costs and technical difficulties49_.

Table 2: Summary of molecular tests most commonly used for BrCa in current clinical practice.

Adapted from 53_.

ER: estrogen receptor; PR: progesterone receptor; HER2: human epidermal growth factor 2 receptor.

1.6 Molecular subtypes

Perou and colleagues were the first to demonstrate with a landmark study the correlation between phenotypic diversity of breast tumours and gene expression patterns diversity 39_.

Since then, various GEP studies have been used to characterize the heterogeneity of BrCa. Following information from those studies, five intrinsic subtypes of BrCa have been proposed, presenting distinct biological features and clinical outcomes supported by the multi-analytic test PAM50 that allows significant prognostic and predictive value: Luminal A, Luminal B, HER2+, Basal-Like and Normal Breast-Like 39,54-56_.

The use of hierarchical clustering of GEP data allowed the identification of two major groups of BrCa tumours: ER+ and ER-. The ER+ tumours fall into the Luminal category, that are subdivided in Luminal A and B subtypes, and ER- tumours fall into the HER2+ and Basal subtypes. Molecular distinction between these tumour types is based on the expression of

Test Analyte Method Role in care ER, PR expression Protein Immunohistochemistry Subclassify tumour

Predict benefit of hormonal therapy

HER2 overexpression Protein Immunohistochemistry Subclassify tumour

Predict benefit of targeted therapy

HER2 amplification Tumour DNA Fluorescence in situ hybridization Subclassify tumour

Predict benefit of targeted therapy

Gene panel testing Tumour DNA Next-generation sequencing Identify predictive or prognostic mutations (actionable mutations)

MammaPrint Tumour RNA Microarray Predict recurrence

Select patients for chemotherapy

Oncotype DX Tumour RNA Reverse transcription-polymerase chain reaction

Predict recurrence

Prosigna BrCa assay Tumour RNA Microarray Predict recurrence

Germ line testing Nontumour RNA Sanger sequencing or next-generation sequencing

Identify genetic predisposition Counsel patients and relatives Guide screening and treatment

(29)

12 ER-related genes, that are more expressed in luminal A than in the luminal B subtype. In contrast, luminal B subtype has a higher expression of proliferative genes 56_{. In general,}

Luminal tumours are the most common subtype of BrCa and commonly have a good prognosis. However, luminal B tumours tend to present worse prognosis and be higher graded than luminal A 55,56_{. HER2+ tumours refer to those with not only HER2}

overexpression but also overexpression of other genes related to proliferation in the HER2 amplicon 38,56_{. A significant portion of this subtype, 40–80 %, have mutations related to the}

p53 gene 56_{and clinically, they have frequently an aggressive behaviour with a subsequent}

poorer prognosis 56_.

The high expression of common basal cytokeratins (CK5/6, CK17) and EGF receptors is characteristic of the basal-like subtype. However, the basal-like subtype is comprised almost entirely (80%) of Triple-Negative Breast Cancer (TNBC), which are tumours that lack ER, PR and HER2 expression 38,55,57_{. TNBC accounts for 10 – 20 % of all BrCa cases and}

affects with more frequency younger women and its prevalence is higher in African-American women as well 58_{. These tumours are associated with higher rates of distant}

recurrence, lower disease-specific survival and overall worse prognosis than women diagnosed with any other BrCa subtypes 58_{. Almost all women with metastatic TNBC die of}

their disease despite adjuvant chemotherapy and less than 30% survive 5 years 58_{. In}

addition, triple-negative tumours lack standard targeted therapy 56_.

A distinct group designated normal breast-like subtype was also identified and accounts for only 7.8 % of all BrCa but needs further investigation56_{. This group is characterized by}

normal breast tissue profiling and may represent contamination by the normal breast parenchyma alongside a low expression of genes that characterize luminal cells 38,56_.

However, this subgroup it is not considered in the daily practice.

Besides the classical immunohistochemistry markers, epigenetics data and information such as long non-coding RNA may provide novel insights on BrCa subtyping as well as on tumour classification and progression 56_.

(30)

13

1.2 HOXOME

1.2.1 The HOX family

The Homeobox (HOX) genes belong to one of the largest superfamilies of homeotic genes that were first identified in the early 1900s in the fruit fly, Drosophila melanogaster 59_{. They}

are a group of genes conserved throughout evolution that encode transcription factors (TF) which determine the development of different structures along the anterior–posterior (A/P) body axis during early embryonic development 60_{. These proteins share a highly}

evolutionarily conserved feature, which is a 60-amino-acid homeodomain in exon 2 as well as a less conserved Hexapeptide (HX) motif located N-terminal to the homeodomain 61_.

These domains are responsible for DNA-binding capacity and to specific recognition of binding sites, which can result in transcriptional activation or inhibition of their target genes

61_{. This gene family has been associated with cell-memory program of the embryonic cells}

being primarily responsible for cell pluripotency maintenance, cell fate determination and differentiation in multicellular organisms 62,63_.

In vertebrates, HOX genes are organized in four separate gene clusters, in which each cluster comprises a set of closely linked genes that often share enhancer regions. These clusters are named A (7p15), B (17q21.2), C (12q13) and D (2q31) and together contain the 39 HOX genes found in mammals64_{. Within these clusters, each gene is numbered from}

1 to 13, according to their relative position in the chromosome, although no cluster includes the full set of genes. The relative position inside the cluster also interferes in the co-factor interactions, specificity on the DNA binding potential and regulation of each member 64_.

In normal vertebrate development, the mechanisms involved in the definition of the HOX gene expression patterns, follow three basic principles. First, HOX expression along the A/P axis of the embryo correlates with the 3’–5’ order within the cluster, a phenomenon known as spatial collinearity 65_{. Thus, 3’ genes are expressed more anteriorly than 5’ genes} 66_{. Second, HOX gene expression occurs temporally according to their positions from 3’ to}

5’ within each cluster, designated as temporal collinearity 67_{. Third, HOX gene expression}

follows what is called as “posterior prevalence”, meaning that HOX genes positioned in a 5’ localization within the cluster present a dominant phenotype over the ones located more anteriorly (3’). In similarity with several other groups of TFs, HOX proteins have an exceptionally low half-life (10 hours), to most likely allow rapid transcriptional regulation 68_.

However, HOXA9 seems to be an exception since it is a relatively stable protein in HeLa cells (half-life of 26 hours) 69_.

(31)

14 The homeodomain of HOX proteins allows them to bind to the DNA of target genes as monomers or even heterodimers. Nevertheless, this homeodomain involves only a four base pair recognition sequence (TAAT/ATTA/TTAT/ATAA), causing a relatively non-specific binding. Therefore, HOX proteins require the help of cofactors to achieve high levels of regulatory specificity and affinity of DNA-binding to the target sites 70_{. The TALE (three}

amino acid loop extension) family is the HOX cofactors group better studied. In mammals, TALE proteins include PBX (PBX1, 2, 3, and 4), MEIS (MEIS1, 2, and 3) and PREP (PREP1 and 2). PBX family members bind to HOX proteins 1-11 71_{, whereas MEIS bind to HOX}

proteins 9-1372_{. Besides participating in the transcriptional regulation of HOX proteins target}

genes, these cofactors also have an important role in the post-translational regulation facilitating the entry of HOX proteins into the nucleus 73_.

Although HOX genes have been studied for several decades as developmental genes, as determinants of cell and tissue identity in embryonic development, they were recently associated with the renewal and differentiation of adult stem cells (SC) 60_{. For instance,}

HOXA expression has been linked to proliferation activity of adult hematopoietic stem and

progenitor cells (HSPCs) 74_{. Moreover, HOX genes also affect specification of distinct blood}

cell lineages and are involved in regulation of tissue identity during menstrual cycle and implantation 75,76_{. Furthermore, several solid and haematological malignancies have shown}

a strikingly dysregulation of HOX genes, being frequently overexpressed 77_.

1.2.2 HOX genes in cancer

The process of oncogenesis is characterized by alterations in cellular growth, differentiation and organization. These are basic mechanisms that are meticulously controlled and regulated during embryonic development and adult tissues. The Oncogerminative Theory of Cancer Development (OTCD) advocates that the deregulation of developmental genes is implied in cancer formation 67,78_{. This theory applies the basic rules of developmental}

biology to the tumour formation process. According to OTCD, tumour formation mimics early embryonic development. During neoplastic transformation, through the gain of mutations in several genes and epigenetic alterations, somatic cells are reprogrammed and become immortal, mimicking germline cells. These cells are then designated as Cancer Stem cells (CSCs) or Oncogerminative Cells (OCs). Thus, during tumour development the OC is thought to undergo biological pathways identical as those of a germline cell, when developing into a blastocyst.

The OTCD defends the existence of five stages of cancer development: 1) normal cell malignant development into OCs; 2) OCs reproduction; 3) formation of a multicellular spheroid (similar to blastocyst formation) including a diverse cell population in addition OCs;

(32)

15 4) oncospheroid vascularization and subsequent growth; 5) development of malignant tumours accompanied by disaggregation of OCs, their migration into the tissues of the organisms and development of metastases.

HOX functions during oncogenesis remain unclear. However, the great complexity of the HOX function in development seems to be reflected in oncogenesis and might be implicated in the transformations predicted by the OTCD. In cancer, HOX genes can have a role at cellular level and have been reported to regulate cell cycle and apoptosis 79_{and to promote}

proliferation 80_{. Nonetheless, HOX genes can also influence oncogenesis at the tumour}

level, by inducing angiogenesis 81_{, formation of metastasis}82_{and facilitating the resistance}

to radiation and drugs 83,84_{. These pro-oncogenic effects are concomitant with high levels of}

HOX gene expression, consequently being correlated with poorer prognosis and outcome

of the disease 77_{. In addition, the expression of many HOX genes are under the control of}

several molecules related to the development of cancer like retinoic acid (HOXA1, HOXB1,

HOXD4, HOXA5), vitamin D (HOXA10, HOXA11), testosterone (HOXA10, HOXA11),

progesterone (HOXA10, HOXA11) and estrogen (HOXA1, HOXA10, HOXB7, HOXB13,

HOXC10) 85_{. Moreover, several studies have focused on examining HOX genes expression}

and putative function in some cancer types. For instance, HOXA7 and HOXD13 have been described in lung cancer 86,87_{, HOXC4 and HOXC8 in prostate cancer}88_{, HOXB7 in ovarian}

cancer, HOXA10 in endometrial cancer 89_{and HOXB13 in prostate cancer}90_.

As in development, the function of these genes seems to be highly tissue specific. Thus, particular HOX genes may act as tumour suppressors or oncogenes, depending on tissue type and tumour site 73_{(Fig. 6). HOXB13, for example, functions as a tumour suppressor}90

required for normal organ development of prostate tissue91_{. However, when it is}

dysregulated in other tissue types, it becomes associated with tumorigenesis and more aggressive diseases 92_{. Another relevant gene is HOXA9, that is able to phenotypically alter}

normal cells into a malignant phenotype by immortalizing normal mice bone marrow cells when overexpressed93_{. The machinery behind why such identical TFs can have contrasting}

functions is still unknown, although it may be related to differential co-factor binding and for that reason differential regulation of target genes or specific epigenetic events60_.

HOX genes may also share similar oncogenic functions, making them potential therapeutic

targets 94_{. However, their opposing functionality vs. functional redundancy, in combination}

with low specificity to ligand binding sites, can potentially make very difficult to target HOX in cancer. A possible approach to address this issue is to target multiple sets of HOX genes in a way that singles out specific HOX functions as well, which could be achieved by the disruption of HOX proteins binding to specific co-factors 95_.

(33)

16 Abate-Shen in 2002 proposed three mechanisms that drive HOX gene dysregulation in cancer 67,96_{. The first is temporospatial dysregulation, i. e., in a tumour that arises from a}

specific tissue, HOX genes expression pattern is both spatial and temporally different from that in normal tissue. Events such as DNA hypermethylation and altered gene expression in HOX clusters are aberrant alterations that are commonly associated with cancer development 85_{. Another mechanism is gene dominance, in which higher HOX genes}

expression levels are observed than those normally detected in that tissue type. The last proposed mechanism is epigenetic dysregulation, for instance, through methylation and histone modification processes, resulting in HOX genes decreased expression or silencing in a tissue at a given time, when they should be normally expressed 67,96_{(Table 3).}

The discreet patterns of HOX gene expression observed in the distinct cancer types, reveal their potential to discriminate them before puncture of the primary tumour, for instance in circulating tumour cells (CTCs) 73_{. Moreover, HOX gene expression profiles could}

potentially act as a “fingerprint” of specific tumour types, thus allowing CTC detection without CTC isolation once it becomes apparent that there is a great degree of differential

HOX gene expression between mature blood cells and CTCs 97_.

Figure 6: Diagram representing aberrant HOX genes expression in various solid tumour types.

(34)

17 ALL: acute lymphoblastic leukaemia; AML: acute myeloid leukaemia; AR: androgen receptor; ER: estrogen receptor; FGF2: fibroblast growth factor 2; FLT3; fms-related tyrosine kinase 3: HGSIL: high grade squamous intraepithelial lesion; MLL: myeloid/lymphoid or mixed-lineage leukaemia; RARβ: retinoic acid receptor-β

1.2.3 HOXB cluster and BrCa

The mammary gland is an extraordinary organ regarding its development and functional differentiation. Unlike most mammalian organs that generally develop at embryonic stage with an almost linear progression towards functional maturity, the mammary gland’s development is primarily postpubertal. Furthermore, the mammalian development can also be described as a set of well-defined transitions, during which important developmental decisions are made for the differentiation, pattern formation and function of cells98_{. The}

characteristics above mentioned make this tissue a great model to study HOX genes function. Moreover, any developmental abnormalities in this organ can have several consequences, such as, lactational failure or even mammary cancer 98_{. Additionally, the}

expression of some HOX genes has been described to be sensitive to manipulation of

HOXB genes function in Breast Cancer

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HOXB

genes function in breast cancer

Mafalda Araújo Pereira

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2018

Mafalda Araújo Pereira

HOXB genes function in breast cancer

Dissertação de Candidatura ao grau de

Mestre

em

Oncologia

Molecular

submetida ao Instituto de Ciências

Biomédicas de Abel Salazar da

Universidade do Porto

Orientadora Doutora Carla Renata Gonçalves de

Freitas

Categoria Investigadora Auxiliar

Afiliação

Grupo “Cell Growth and Differentiation”

Instituto de Biologia Molecular e Celular

Instituto de Investigação e Inovação em

Saúde- Universidade do Porto

Co-orientador Doutor Pedro Nuno Simões Rodrigues

Categoria Professor Associado

Afiliação Departamento de Produção Aquática

Instituto de Ciências Biomédicas Abel

Salazar - Universidade do Porto

Vice-Reitor

TABLE OF CONTENTS

ACKNOWLEDGEMENTS

RESUMO

ABSTRACT

LIST OF ABBREVIATIONS

FIGURES INDEX