Universidade de Aveiro 2016 Departamento de Biologia
MÓNICA
CATARINA
CASTRO
OLIVEIRA
Proteasome-proteins: are these putative targets for
basal-like breast cancer therapy with AAV-vectors?
Proteínas do proteossoma: possíveis alvos
terapêuticos com vectores AAV no cancro da mama
do tipo basal?
DECLARAÇÃO
Declaro que este relatório é integralmente da minha autoria, estando
devidamente referenciadas as fontes e obras consultadas, bem como
identificadas de modo claro as citações dessas obras. Não contém, por isso,
qualquer tipo de plágio quer de textos publicados, qualquer que seja o meio
dessa publicação, incluindo meios eletrónicos, quer de trabalhos académicos.
Universidade de Aveiro 2016 Departamento de Biologia
MÓNICA
CATARINA
CASTRO
OLIVEIRA
Proteasome-proteins: are these putative targets for
basal-like breast cancer therapy with AAV-vectors?
Proteínas do proteossoma: possíveis alvos
terapêuticos com vectores AAV no cancro da mama
do tipo basal?
Dissertação apresentada à Universidade de Aveiro para cumprimento dos requisitos necessários à obtenção do grau de Mestre em Biologia Molecular e Celular, realizada sob a orientação científica da Professora Doutora Maria de Lourdes Gomes Pereira, Professora Associada com Agregação do
Departamento de Biologia da Universidade de Aveiro e da Doutora Joana Cancela de Amorim Falcão Paredes, Investigadora Doutorada do Instituto de Patologia e Imunologia Molecular da Universidade do Porto (IPATIMUP) /Instituto de Investigação e Inovação em Saúde (I3S).
This work was supported by FCT grant PTDC/BBB-BIO/1240/2012.
"The journey of discovery does not necessarily require finding new landscapes but observing with different eyes" Marcel Proust
o júri
presidente Prof. Doutora Maria Helena Abreu Silva
Professora auxiliar do Departamento de Biologia da Universidade de Aveiro
vogais Prof. Doutora Maria de Fátima Moutinho Gärtner (Arguente)
Professora catedrática no Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto
Doutora Joana Cancela de Amorim Falcão Paredes (Orientadora)
Investigadora Principal do Instituto de Patologia e Imunologia Molecular da Universidade do Porto (IPATIMUP)/Instituto de Investigação e Inovação em Saúde (I3S)
agradecimentos
Devemos sempreprimeiro lugar, agradecer àqueles que são a razão de tudo isto ser possível, os muito a muita gente e formalismos á parte, gostaria de, em meus Pais. Que tanto batalharam e se sacrificaram para que eu pudesse chegar aqui e pudesse ser o que sou. Pela vida, coragem, paciência,preocupação, dedicação, por todos os ensinamentos e apoio incansável sem nunca me impedirem de atingir os meus objetivos. Meras palavras não são capazes de exprimir a gratidão que sinto. E ao meu irmão, pelo carinho e mimos que muitas vezes me dá e que tanto faz por mim.
Este é o culminar de um ano de trabalho na qual estiveram envolvidas pessoas incríveis sem as quais nada disto teria sido atingível, a essas não poderi a deixar de estender o meu enorme agradecimento e demonstrar o meu apreço. À minha orientadora Joana Paredes, por me ter concedido esta grande oportunidade e por tantas outras. Pela sua simpatia e disponibilidade, pela confiança, por me ter amparado e permitido que eu pudesse crescer em vários aspetos no seu grupo, por ser um exemplo a seguir neste mundo tão
competitivo e me ter inspirado a ser mais e melhor.
À Ana que foi um excelente pilar e uma ajuda crucial neste percurso, que me deu as bases e ensinamentos necessários. Por ser um exemplo de força e persistência e, sem saber, me ter feito compreender que tudo é possível com uma boa gestão do nosso tempo.
Como não poderia deixar de ser, agradecer à MAMA TEAM que foi o meu berço nesta grande caminhada. Ao André que sempre se demonstrou disponível para me ensinar, pelo carinho, motivação e apoio em vários
momentos. À Bárbara, por ter puxado por mim e por vezes me levar ao limite, mas acima de tudo pelo que aprendi e cresci com ela. Ao Polónia pela
paciência e tempo disponibilizado em todas as lâminas que observou comigo e todos os ensinamentos que me transmitiu. À Rita, que em tão pouco tempo conseguiu ser tanto. À Rita Nobre, pela aprendizagem no início desta jornada. É com enorme orgulho que aprendi e cresci com todos vocês.
Ao gang do almoço, pela partilha, convívio e gargalhadas constantes que em certos momentos são exatamente o que precisámos.
Ao Nuno Mendes pelo acompanhamento, ensinamentos, pela incrível dedicação, carinho e disponibilidade. Á Raquel Seruca e ao fantástico grupo EPIC, á Regina, á Marta T. Pinto, a todos os que de alguma forma se cruzaram comigo e estiveram envolvidos neste percurso.
À Professora Maria de Lourdes, pela incrível ajuda, disponibilidade e prontidão no esclarecimento e resolução de problemas ou burocracias.
Ao Professor Sobrinho Simões que apesar de não se aperceber, o seu sorriso deu-me confiança logo no primeiro dia.
A todos os meus amigos, que acham que me tornei antissocial. Ao Fábio pelo apoio e força constantes, e não só. À Bibs por estar sempre presente. À Raquel por tornar as viagens de comboio mais fáceis e divertidas. Aos bests de Aveiro. A todos e aos restantes “Não abdiques. Leva a amizade a sério.”. A todas as lutadoras que são mães, amigas, filhas e que infelizmente perderam a batalha, a todos os casos que me motivaram a querer dar o meu contributo nesta área tão vasta. Sou uma gota neste imenso oceano (de tanto) por descobrir, e quero continuar a dar o meu contributo para que um dia essa batalha seja vencida por todos.
palavras-chave
Carcinoma da mama do tipo basal, proteínas do proteassoma, PSMA2, RNA de interferência, shRNA, terapia direcionada com AAV, terapia genética em cancro, vírus adeno-associados.resumo
O cancro da mama do tipo basal (BLBC) é um grupo de tumores muito agressivo associado a um mau prognóstico. De momento, não existe nenhum tratamento eficaz para o BLBC, uma vez que rapidamente adquiremresistência às terapias normalmente usadas. Assim, é urgente encontrar novas abordagens para tratar esta doença.
Com base em dados anteriores, o objetivo geral deste estudo foi avaliar se o PSMA2, uma proteína do proteassoma, seria um alvo putativo para a inibição para terapia em BLBC.
Desta forma, o primeiro objetivo específico foi avaliar o efeito anti-tumorigénico de vírus adeno-associados (AAV) capazes de entregar short hairpin RNAs (shRNA), anteriormente validados, capazes de inibir a expressão do PSMA2 em xenotransplantes de células BLBC em ratinho.
Para atingir esse objetivo, foram testados in vivo, vetores AAV2 com shRNAs para os genes PLK1 e PSMA2 para diferentes concentrações de partículas virais (2x1010, 2x109, 2x108 partículas virais/tumor), em que células
MDA-MB-468 BLBC foram injetadas na mama de ratinhos nude. Após cerca de um mês, foram realizadas injeções intratumorais com AAVs duas vezes por semana. A administração de AAV2-shPSMA2 resultou numa diminuição no crescimento do tumor sem toxicidade evidente, e este efeito foi mais significativo na concentração de 2x109 partículas virais/tumor.
O segundo objetivo específico foi analisar a expressão de PSMA2 em amostras humanas de cancro da mama, o que indica que há também uma importância clínica na inibição deste gene, uma vez que se mostrou estar associado a características menos favoráveis relacionadas com tumores da mama do tipo basal.
Em conclusão, embora ainda preliminar, os resultados obtidos abrem a possibilidade de direcionar uma terapia genética em BLBC usando vetores AAV recombinantes que entregam shRNAs para silenciar especificamente a expressão do gene PSMA2.
keywords
Basal-like breast cancer, proteasome proteins, PSMA2, RNA interference, shRNA, AAV targeted therapy, cancer gene therapy, adeno-associated virus.abstract
Basal-like breast cancer (BLBC) is an aggressive group of tumours associated to poor patient prognosis. Currently, there is no effective treatment for BLBC once they rapidly acquire resistance to standard therapies. For this reason, novel approaches to treat this disease are urgently needed.Based on previous data, the general goal of this study was to evaluate if PSMA2, a proteasome protein, was a putative target for inhibition in BLBC therapy.
In this way, the first specific aim was to evaluate the anti-tumorigenic effect of adeno-associated virus (AAV)-based vectors, that were able to deliver validated short hairpin RNAs (shRNAs) that inhibit the expression of PSMA2 in BLBC mouse xenografts. To achieve that aim, we have tested, in vivo, AAV2 vectors with shRNAs for the genes PLK1 and PSMA2 for different concentrations of viral particles (2x1010, 2x109, 2x108 VP/tumour),
MDA-MB-468 BLBC cells were injected into the mammary fat pad of nude mice and, after nearly one month, intratumoral injections with AAVs were performed twice a week. The delivery of AAV2-shPSMA2 resulted in a decrease in tumour growth with no obvious toxicity, and this effect was more significant at the concentration of 2x109 VP/mouse.
The second specific aim was to analyse the expression of PSMA2 in human breast cancer samples, which indicated that there is also a clinical importance in inhibiting this gene, once it showed to be associated with less favourable features that are linked to basal-like breast tumours.
In conclusion, although still preliminary, the results obtained open a possibility to direct a gene-based therapy in BLBC using recombinant AAVs that deliver shRNAs that specifically silence PSMA2 gene expression.
TABLE OF CONTENTS
Resumo………..………... XIII Abstract…...………... XV TABLE OF CONTENTS ... XVII TABLE LIST ... XIX FIGURE LIST... XXI ABBREVIATIONS ... XXIII
1. INTRODUCTION ... 1
1.1. Breast Cancer... 1
1.1.1.Statistics and Epidemiology... 1
1.1.2. Breast Cancer Molecular Subtypes ... 2
1.1.2.1. Luminal subtype ... 3
1.1.2.2. HER2-enriched subtype ... 4
1.1.2.3. Triple negative subtype ... 5
1.1.3. Basal-like breast cancer ... 6
1.1.4. Potential therapies to target BLBC ... 7
1.2. Cancer gene therapy ... 9
1.2.1. Adeno-associated virus and shRNA as potential cancer therapeutics ... 9
1.2.2. Proteasome as targets for BLBC therapy with AAV vectors ... 13
2. AIM... 17
3. MATERIAL AND METHODS ... 19
3.1. Cell lines and culture conditions... 19
3.2. Development of AAV2-shRNA vectors ... 19
3.3. In vivo experiments... 20
3.4. RNA extraction and quantitative RT-PCR... 21
3.5. Histological processing... 21
3.6. Immunohistochemistry ... 22
3.7. Human breast cancer samples ... 23
3.8. Semiquantitative evaluation of immunhohistochemistry for PSMA2 ... 24
3.9. Statistical analysis ... 24
4. RESULTS ... 25
4.1. The anti-tumorigenic activity of AAV2-based vectors delivering shRNA sequences targeting PSMA2 transcripts in an in vivo pre-clinical BLBC model... 25
4.2. Expression of PSMA2 in human breast cancer samples ... 38
4.2.1. Impact of PSMA2 gene expression in relapse-free survival of BC patients using KM-plotter ... 38
4.2.2. PSMA2 protein expression in a large series of human breast cancer samples ... 39
5. DISCUSSION ... 47
6. CONCLUSION AND FUTURE PERSPECTIVES... 59
7. REFERENCES ... 61
TABLE LIST
Table 1. Differences and current therapies in the different breast cancer subtypes ...7
Table 2. List of antibodies used and conditions of the immunohistochemical reactions performed in this study ... 23
Table 3. Number of animals used per group for in vivo evaluation of the anti -tumorigenic activity of shRNA expressed by AAV2 control vectors ... 26
Table 4. Number of animals used per group for in vivo evaluation of the anti -tumorigenic activity of shRNA expressed by AAV2 vectors for the selected genes (PLK1 and PSMA2) ... 29
Table 5. Immunohistochemical evaluation of PSMA2 protein expression ... 35
Table 6. Frequency of PSMA2 expression in an independent series of 467 invasive human breast carcinomas... 39
Table 7. Association between PSMA2 expression and breast cancer molecular subtypes and biological markers ... 42
Table 8. Association between PSMA2 expression and CSC markers... 43
Table 9. Association between PSMA2 expression and EMT markers... 44
FIGURE LIST
Figure 1. The top 10 leading types of cancer in 2012 ...1
Figure 2. Classification of breast cancer into distinct molecular subtypes ...3
Figure 3. Schematic representation of delivering systems of shRNA or siRNA into mammalian cells to promote RNA interference ... 12
Figure 4. Protein degradation via the ubiquitin proteasome pathway ... 13
Figure 5. A set of dependency genes in 17 human breast cancer cell lines ... 15
Figure 6. Experimental design used for in vivo evaluation of the anti-tumorigenic activity of shRNA expressed by AAV2 control vectors ... 25
Figure 7. Impact of shRNA expressed by AAV2 control vectors in tumour growth rate of BLBC xenografts of MDA-MB-468 cells ... 27
Figure 8. Experimental design used for the in vivo evaluation of the anti-tumorigenic activity of shRNA expressed by AAV2 vectors for the selected genes (PLK1 and PSMA2) ... 28
Figure 9. Impact of shRNA expressed by AAV2 vectors for the genes of interest (PLK1 and PSMA2) in the tumour growth rate of BLBC xenografts of MDA-MB-468 cells ... 30
Figure 10. Impact of shRNA expressed by AAV2 vectors for the genes of interest (PLK1 and PSMA2) in the fold increase in tumour growth of BLBC xenografts of MDA-MB-468 cells ... 31
Figure 11. H&E staining of primary MFP tumours ... 32
Figure 12. Vascular invasion analysis of MFP primary tumours ... 33
Figure 13. PSMA2 mRNA relative expression levels ... 34
Figure 14. Representative images of immunohistochemical staining of Ki-67 and cleaved-CASP3 ... 35
Figure 15. Immunohistochemical analysis of Ki-67 and cleaved-CASP3 ... 36
Figure 16. Experimental design used for in vivo evaluation of the anti-tumorigenic activity of AAV2-shCtr#2 vectors ... 37
Figure 17. Impact of shRNA expressed by an alternative AAV control vector in the tumour growth rate of BLBC xenografts of MDA-MB-468 cells... 38
Figure 18. Kaplan-Meier Plotter analysis showing PSMA2 mRNA expression ... 39
Figure 19. Representative image of PSMA2 cytoplasmic staining in a series of 467 i nvasive human breast carcinomas ... 40
Figure 20. Kaplan-Meier survival curves in a series of 467 human invasive breast carcinomas ... 41
ABBREVIATIONS
AAV Adeno-associated virus
ALDH1 Aldehyde dehydrogenase 1
AR Androgen receptor
BC Breast Cancer
BLBC Basal-like breast cancer
BRCA 1 Breast cancer 1
CAIX Carbonic anhydrase IX
CASP3 Caspase 3
CLDN Claudin
CSC Cancer stem cell
DFS Disease-free survival
DNA Deoxyribonucleic acid
DSB Double strand break
EGFR Epidermal growth factor receptor
EMT Epithelial-to-mesenchymal transition
ER Estrogen Receptor
GLUT1 Glucose transporter 1
HE Haematoxylin and Eosin
HER2 Human epidermal growth factor receptor 2
HIF1α Hypoxia-inducible factor 1α
IBET Institute of Experimental and Technological Biology
IHC Immunohistochemistry
ITR Inverted terminal repeats
OS Overall survival
PARP Poly (Adenosine diphosphate-ribose) polymerase
PBS Phosphate-Buffered Saline
PLK1 Polo-like kinase 1
PR Progesterone Receptor
PSMA2 Proteasome Subunit Alpha 2
RFS Relapse-free survival
RNAi Ribonucleic acid interference
shRNA Short hairpin ribonucleic acid
siRNA Small interfering ribonucleic acid
SSB Single strand break
1. INTRODUCTION
1.1. Breast Cancer
Breast cancer is an extraordinary complex disease originated due to genetic and epigenetic alterations occurring in epithelial cells of the breast (Polyak, 2011), being still a major health threat affecting millions of women in every part throughout the world (Boyle, 2012). Despite the improvements in medical tools to perform early detection and the advances in basic and clinical research, there is still the need to better understand the molecular basis that control this malignancy, in order to find more efficient therapeutic strategies (Smigal et al., 2006).
1.1.1. Statistics and Epidemiology
Breast cancer is by far one of the most frequently neoplasms diagnosed in women worldwide, accounting for 25% of all cancers. In 2012, it was estimated that breast cancer alone would be responsible for 1.67 million new cases diagnosed and 522.000 people would succumb from this disease. In women, this malignancy ranks first with the highest incidence rates in more developed countries and is considered the second most common cause of death by cancer, responsible for 15% of deaths worldwide (Figure 1A) (Ferlay et al., 2013).
Figure 1. The top 10 leading types of cancer in 2012. Estimates of incidence and mortality
rates in the World (A) and in Portugal (B), by gender. (Data from GLOBOCAN – IARC, Ferlay et
According to the GLOBOCAN project of the International Agency for Research on Cancer (IARC) (Ferlay et al., 2013), breast cancer is the most common form of cancer with the highest incidence rates in Portugal, with 6088 new cases in 2012 and the leading cause of female cancer related death (1570 deaths), along with colorectal cancer (Figure 1B).
Although breast cancer mortality rates have been decreasing over the years due to improved screening, non-Hispanic black women still have a higher probability of dying of this disease, while Caucasian women share the highest incidence rates (Bleyer & Welch, 2012; Siegel et al., 2016). In general, breast cancer is mainly found in women at an advanced age (≥70 years old) (Siegel et
al., 2016); however, when it occurs in younger women, it acquires features of
more aggressive forms of cancer, which are significantly associated with a worse prognosis (Gabriel & Domchek, 2010).
1.1.2. Breast Cancer Molecular Subtypes
Breast cancer is a highly heterogeneous disease, which implies further research to find targeted therapeutic approaches that would be able to overcome the aggressive nature of the different molecular subtypes (Hanahan & Weinberg, 2011; Polyak, 2011).
In the last two decades, technological development allowed to associate tumour heterogeneity to distinct gene expression profiles, enabling the molecular classification of breast cancer into distinct subgroups, reshaping not only the way these tumours are diagnosed and treated, as well as providing additional prognostic information (Polyak, 2011; Rakha & Reis-Filho, 2009). cDNA (complementary Deoxyribonucleic acid) microarray-based expression studies performed by Perou and colleagues (Perou et al., 2000), have identified for the first time, different breast cancer subtypes, that have been clustered according to their molecular characteristics (Sørlie et al., 2001). However, in order to translate this knowledge to the clinical routine, these different subgroups started to be distinguished based mainly on the protein expression of three molecular markers in tumour cells: both hormone receptors, the Estrogen Receptor (ER) and Progesterone Receptor (PR), and the human epi dermal growth factor receptor 2 (HER2) (Schnitt, 2010), allowing the classification into
hormone-receptor-positive or luminal subtype, the HER2-enriched subtype and the triple negative subtype (Nagini, 2016). This separation was crucial to determine which treatment would be more suitable for each patient and may present some advances regarding the identification of new therapeutic targets (Figure 2) (Eroles et al., 2012; Polyak, 2011; Schnitt, 2010).
Figure 2. Classification of breast cancer into distinct molecular subtypes. Breast cancer
molecular subtypes and their characteristics regarding the expression of receptors and their pathological features, such as histological grade, patient prognosis and treatment options (Adapted from Wong & Rebelo, 2012).
1.1.2.1. Luminal subtype
The luminal subtype is characterized by tumours that express ER and PR (Nagini, 2016), being subdivided into luminal A and luminal B subgroups (Weigelt et al., 2010).
Luminal A is the subtype that is more frequently found in patients and represents 50–60% of all the breast cancer cases (Yersal & Barutca, 2014). It is characterized by tumours that are usually HER2 negative, with low expression of the proliferation marker Ki-67, and with expression of cytokeratins 8/18 and 19, that are characteristic of the normal luminal epithelium of the breast.
Hormone Receptor+ and HER2- breast cancers tend to have a better patient
outcome, being significantly associated with higher survival rates and lower recurrence rates (Eroles et al., 2012; Inic et al., 2014; Yersal & Barutca, 2014).
Luminal B tumours have an increased expression of HER2 related genes. This subtype accounts for 15 to 20% of all breast cancers (Yersal & Barutca, 2014) and are clinically associated to worse patient prognosis and greater chance for local recurrence, once they have higher expression of proliferation genes, higher lymph node involvement and higher frequency of TP53 mutations than luminal A tumours (Inic et al., 2014; Prat & Perou, 2011).
Thus, while luminal A tumours are sensitive to current anti-hormonal therapy, such as tamoxifen, fulvestrant or aromatase inhibitors (Eroles et al., 2012), luminal B tumours are more resistant to these therapeutic approaches, being mainly responsive to neoadjuvant chemotherapy (Cheang et al., 2009).
1.1.2.2. HER2-enriched subtype
The HER2-enriched breast cancer subtype represents approximately 20-25% of all breast cancers cases (Toss & Cristofanilli, 2015). It is mainly characterized by the negative expression of hormone receptors (ER or PR) and strong expression of HER2. These tumours are usually associated to a high proliferation index, high histological grade and mutated p53 (Eroles et al., 2012; Toss & Cristofanilli, 2015; Yersal & Barutca, 2014).
The human epidermal growth factor receptor 2 (HER2), which is one of the members of the tyrosine kinase receptors family (Yersal & Barutca, 2014), is normally involved in the regulation of biological processes, such as cell growth, cell division and cell survival. When the HER2 gene is amplified, it works as an oncogenic driver of breast cancer, being associated with a more aggressive form of the disease and a poor clinical patient outcome (Iqbal & Iqbal, 2014).
Since these tumours are mainly negative for ER or PR, hormonal therapy is therefore inefficient. However, this subgroup can be controlled with anti -HER2 therapy, such as trastuzumab (Iqbal & Iqbal, 2014; Sadeghi et al., 2014). Trastuzumab was the first humanized monoclonal antibody to target the extracellular domain of the HER2 receptor, approved by the US Food and Drug Administration in 1998 (Wolff et al., 2007). It has been shown that Trastuzumab treatment was significantly efficient in improving overall patient survival, being therefore used as first line treatment in this specific molecular subtype (Mendes
Over time, with increased acquired chemoresistance, some other anti-HER2 targeted therapies came along, like lapatinib/neratinib/afatinib (all of them are tyrosine kinase inhibitors of HER2), pertuzumab (blocks HER2 dimerization) and ado-trastuzumab emtansine (trastuzumab linked to the cytotoxic agent mertansine (DM1)), that diverge in their mechanism of action, but all aim to target HER2 (Iqbal & Iqbal, 2014; Toss & Cristofanilli, 2015).
1.1.2.3. Triple negative subtype
Triple negative breast cancers (TNBC) are defined as a group of tumours that lack the expression of ER, PR and HER2, accounting for 15-20% of all diagnosed breast cancer cases (de Ruijter et al., 2011; Toss & Cristofanilli, 2015). TNBC are a heterogeneous group of tumours, which includes distinct molecular subtypes, such mesenchymal, luminal androgen receptor (AR), immunomodulatory, claudin-low, normal-like and basal-like (Chiorean et al., 2013; Lehmann et al., 2011; Toss & Cristofanilli, 2015).
The mesenchymal tumours are enriched in mesenchymal features and express genes involved in cell motility, growth, and cellular differentiation pathways. The AR subtype is characterized by an increased expression of genes involved in the AR signalling pathway, whereas the immunomodulatory subgroup is enriched in genes involved in immunological cellular processes, such as antigen processing and presentation and T-cell function (Chiorean et
al., 2013).
The claudin-low subgroup is characterized by low expression of genes involved in cell-cell adhesion and in tight junctions, such as claudins (CLDNs) 3, 4 and 7, E-cadherin and occludin. These are still highly enriched in cancer stem cells (CSC) and epithelial-to-mesenchymal transition (EMT) markers (Gerhard
et al., 2012; Toss & Cristofanilli, 2015; Yersal & Barutca, 2014). Interestingly, the
molecular subgroup called normal-like is not yet fully characterized; however it is defined by the expression of genes of the adipose tissue and resembles an expression pattern similar to fibroadenomas and normal breast tissue (Eroles et
al., 2012).
Finally, the last subtype is the basal-like and, although the majority of them have a triple negative phenotype, around 20% of basal-like tumours do not fall into the TNBC group (Anderson et al., 2014).
1.1.3. Basal-like breast cancer
Basal-like breast cancer (BLBC) is a molecular subgroup linked to a highly aggressive phenotype, with unfavourable patient prognosis and limited therapeutic options (Toss & Cristofanilli, 2015). As mentioned before, most of them are triple negative, lacking the expression of the therapeutic targets HER2 and ER/PR (de Ruijter et al., 2011).
BLBC correspond to 8 to 37% of all breast cancer cases (Yersal & Barutca, 2014), being more prevalent in patients with African origins and occurring at an early age (Badve et al., 2011). These are associated to shortest survival rates, mainly due to early relapse in the first five years after diagnosis (Bertucci et al., 2012).
Basal-like carcinomas typically express genes characteristic of the basal/myoepithelial layer of the normal mammary epithelium, including basal cytokeratins (5/6, 14, 17) (Badve et al., 2011), vimentin, P-cadherin (placental-cadherin) (Albergaria et al., 2011), nestin, CD44, and EGFR (epidermal growth factor receptor) (Choo & Nielsen, 2010; Toss & Cristofanilli, 2015). EGFR amplification was found in 23% of all BLBC cases and TP53 mutation is the most frequent alteration in this molecular subtype, being observed in 80% of the cases (Toss & Cristofanilli, 2015). Furthermore, it has also been shown that 20% of women with BLBC carry a germline mutation in BRCA1 (Breast Cancer 1) gene (Prat et al., 2014), in which its loss or silencing is also seen in sporadic BLBC (Engebraaten et al., 2013; Lord & Ashworth, 2008). BRCA1 is involved in the normal development of the mammary gland and is responsible for DNA repair and transcriptional regulation. It is widely known that the loss of function of BRCA1 predisposes the patient to a higher risk of developing a BLBC with increased aggressiveness and worse prognosis (Lord & Ashworth, 2008; Meric-Bernstam et al., 2013).
Histologically, BLBC tumours are poorly differentiated, associated to a larger tumour size, high grade lesions, high mitotic rate, extensive areas of necrosis, lymph node positivity and distinct morphological and cytological features. Moreover, they are more likely to metastasize to the lungs and brain (Anderson et al., 2014; Eroles et al., 2012), during the first 3 years of follow-up (Bertucci et al., 2012).
Currently there are no effective therapeutic options towards these tumours, which are mainly treated with chemotherapeutic regimens, for which they rapidly acquire resistance. Due to their poor clinical outcome, there is an urgent need to find alternative therapies against this type of tumours (Bertucci et al., 2012).
1.1.4. Potential therapies to target BLBC
The fundamental key to successfully treat breast cancer still depends on its early detection. However, finding new targets that lead to novel therapies to treat this disease has been one of the major efforts in breast cancer research (Engebraaten et al., 2013). In general, most therapies have been able to contain disease progression and some improvements and increasing survival rates have been observed for patients with good prognosis breast cancer subtypes (Mohamed et al., 2013).
Concerning BLBC, these remain a real challenge due to their lack of a single molecular target (Chiorean et al., 2013). Current therapeutic options (Table 1) used to treat these poor prognostic tumours are based essentially on surgery followed by adjuvant therapy, but these continue to lack the specificity needed towards cancer cells (Badve et al., 2011).
Table 1. Differences and current therapies in the different breast cancer subtypes
(Adapted from: Eroles et al., 2012).
Some cytotoxic agents, such as paclitaxel, doxorubicin, taxanes, anthracyclines and platinum-based compounds (cisplatin and carboplatin) interfere with BLBC cell growth and division (Shastry & Yardley, 2013).
However, although some tumours initially have a good response to these chemotherapy regimens, it is expected that patients will die from this disease within the following 5 years subsequently to therapy (Badve et al., 2011).
Several novel targeted agents are being explored and represent promising therapeutic strategies to treat TNBC/BLBC, including those targeting EGFR (erlotinib, panitumumab, cetuximab), PARP inhibitors, Src inhibitors, and angiogenesis inhibitors (like bevacizumab and sunitinib, that act by blocking the Vascular Endothelial Growth Factor - VEGF) (Anders & Carey, 2009; Nagini, 2016). From these, the ones that have been broadly studied and widely tested in clinical trials were the EGFR and PARP inhibitors.
Concerning EGFR, since it is a protein found to be overexpressed among 27 to 57% of BLBC, these patients would likely benefit from anti -EGFR therapies. Although these demonstrated low efficacy levels as single agents, the effects improved significantly when combined with cytotoxic agents (Anders & Carey, 2009; Shastry & Yardley, 2013).
In what concerns the inhibitors of Poly (Adenosine diphosphate-ribose) polymerases, generally known as PARPs, these are a new class of drugs that are currently under investigation to target tumours with DNA repair defects (Shastry & Yardley, 2013), such as BLBCs with dysfunctional BRCA1 function
(Prat et al., 2014).
PARP is involved in DNA repair, being important in the repair of single strand DNA breaks (SSBs) that occur within the cell by base excision repair (Murai et al., 2012). As a result of PARP inhibition, the unrepaired SSBs are converted into double-strand DNA breaks (DSBs) during replication. In a normal situation, these DSBs are repaired by the tumour suppressor BRCA1, via the homologous recombination pathway (Helleday, 2011; Lord & Ashworth, 2008; Murai et al., 2012). Thus, in breast carcinomas with BRCA1 mutations, these DSBs are not repaired resulting in their accumulation and leading to cell death. In this way, cells which lack a functional BRCA1 protein are sensitive to PARP inhibitors which prevent the PARP enzyme to repair the SSBs, being therefore lethal for cancer cells (Helleday, 2011; Murai et al., 2012).
PARP inhibitors, such as iniparib, olaparib or veliparib, are currently being tested to treat BLBC (Bertucci et al., 2012). Olaparib showed good response rates in phase I studies, but phase III clinical trials with iniparib does not
accomplish encouraging results to treat TNBC patients (Eroles et al., 2012). Even when these inhibitors were combined with chemotherapeutic agents, there was a significant improvement, but their efficacy is still uncertain (Shastry & Yardley, 2013).
1.2. Cancer gene therapy
The search for new cancer therapies has been growing over the last years, mainly due to increased cancer mortality rates, reduced efficacy of current therapeutic approaches and development of therapy resistance by cancer patients (Luo et al., 2015). Although still under study, gene therapy is a promising approach to treat several human diseases, including cancer (Wang et
al., 2015).
Gene therapy was intended to enable the delivery of genetic material into cells by the use of specific vectors, in order to repair abnormal gene function (Fujiwara, 2010).
As gene delivery systems, there is a wide spectrum of vectors that can be used, including non-viral, such as liposomes or nanoparticles, and the viral ones, namely retroviral, lentiviral or adenoviral vectors (Luo et al., 2015). Actually, concerning cancer research, virotherapy is clearly emerging (Kranzler
et al., 2009) and adenoviral vectors are presenting a promising potential in
delivering genes or drugs that promote anti-tumour effects (Liikanen et al., 2011).
1.2.1. Adeno-associated virus and shRNA as potential cancer therapeutics
Adeno-associated viruses (AAV) are small, non-enveloped single-stranded DNA (ssDNA) virus, which were discovered in 1965 and belong to the
Parvoviridae family (Luo et al., 2015, Samulski & Muzyczka, 2014). With a
diameter of about 20 nm (Grimm et al., 2005), these non-pathogenic viruses have been generating increasingly interest in viral gene therapy approaches due to their distinctive features. These include their wide range of tissue targets (Rogers et al., 2011), relatively strong clinical safety and efficacy profile, low
host immune response and toxicity, capacity to transduce dividing and quiescent cells and capacity to achieve long-term transgene expression (Grimm
et al., 2005; Wang et al., 2016).
A number of AAVs have been already genetically engineered to enhance therapeutic effects in cancer, by incorporating oncogene inhibitors or therapeutic genes with anti-tumour properties to be delivered towards cancer cells (Liikanen et al., 2011). These recombinant AAV (rAAV) have been successfully implemented for in vivo gene delivery and, among the most extensively studied AAV serotypes, is the AAV type 2 vector (AAV2). AAV2 is reported as the first vector used in pre-clinical studies for the treatment of a large number of diseases, including Leber’s congenital amaurosis (Hastie &
Samulski, 2015), haemophilia B, cystic fibrosis, Parkinson’s disease, cancer
and others (Hastie & Samulski, 2015; Samulski & Muzyczka, 2014).
AAV2 is a well-characterized serotype that achieves productive viral replication in the presence of a helper virus, most commonly the adenovirus (Samulski & Muzyczka, 2014). The AAV carries a ssDNA genome of about 4.7 kilobases within the capsid, that comprises two open reading frames (rep and
cap), flanked by inverted terminal repeats (ITRs) at the ends of the DNA strand,
which are essential cis-acting elements for viral replication and transgene packaging (Hastie & Samulski, 2015; Wang et al., 2015). The rep (replication) genes are critical for viral replication and the cap (capsid) gene encodes three structural viral capsid proteins (VP1, VP2 and VP3), being important in viral infection (Wang et al., 2016). Interestingly, it has been found that these sequences can be deleted in order to allow the packing of a foreign therapeutic gene sequence between the ITRs, which in turn will express the insert into the target cells (Borel et al., 2014; Kotterman & Schaffer, 2014; Sliva & Schnierle, 2010).
While the wild-type AAV has the ability to integrate in the chromosome 19 of
the human host (Deyle & Russel, 2009; Hastie & Samulski, 2015), these rAAVs
are engineered in a way that the viral genes are depleted, being no longer able
to integrate specifically into the host genome (Smith, 2008). Even though, rAAV DNA can persist in the form of episomal concatemers (Deyle & Russel, 2009;
Rogers et al., 2011), integration can still occur at very low frequencies (Hastie & Samulski, 2015; Wang et al., 2016).
To enter target cells, AAV2 relies on the recognition of cell membrane-associated heparan sulfate proteoglycan (HSPG) (e. g., galactose) or cell membrane receptors (e. g., integrins). After infection, its capsid enters the cell nucleus where it is of primordial importance the conversion of the viral ssDNA into a double-strand DNA for effective transgene transcription and expression (Samulski & Muzyczka, 2014).
AAV-mediated RNA interference (RNAi) is an attractive approach to silence the expression of a target gene in several diseases and holds a great promise as a therapeutic tool for cancer gene therapy (Shim & Kwon, 2010). RNAi is a post-transcriptional mechanism that plays an important role in gene expression regulation (Wilson & Doudna, 2013), by delivering exogenous small interfering RNA (siRNA) or short hairpin RNA (shRNA) into host cells to block protein synthesis by mediating the degradation of messenger RNA (mRNA) (Moore et
al., 2010; Perwitasari et al., 2013).
RNAi transfer vectors based on engineered AAVs have been widely explored and compared to other delivering systems, demonstrating a selective suppression of the expression of a single gene (Grimm et al., 2005). In order to achieve success in gene silencing, siRNAs or shRNAs have to be efficiently delivered into target cells, either by cell membrane permeabilization methods or by the use of viral vectors, respectively (Perwitasari et al., 2013). Delivering shRNA into the cell requires the transcription of the sequence in the cell nucleus, which is then recognized and processed by Drosha, followed by transportation to the cytoplasm by Exportin-5. Once in the cytoplasm, it is processed by Dicer into a siRNA (21–23 nucleotides), which is then incorporated into the RNA-induced silencing complex (RISC) that subsequently directs the guide siRNA, containing the exact complementary sequence, to the target mRNA, inducing the cleavage of the transcripts (Figure 3) (Dykxhoorn et
Figure 3. Schematic representation of delivering systems of shRNA or siRNA into mammalian cells to promote RNA interference. RNA silencing mediated by siRNA, using
transfection or electroporation methods, or delivering shRNA by AAV or lentiviral vectors. After transcription in the nucleus, shRNAs are processed and exported to the cytoplasm for further processing and incorporated into the RISC complex to induce gene silencing. Lentiviral vectors delivers transgenes that are stably integrated into the host genome, while AAV genome persist as episomes and rarely result in genome integration, being more appropriate for transient gene knockdown (Adapted from Perwitasari et al., 2013).
The first experiment reported regarding the use of AAV2 vectors expressing shRNA successfully inhibited p53 and caspase 8 expression (Grimm et al., 2005). However, although AAV-mediated gene delivery systems prove to be putatively successful for a wide variety of diseases due to their long-term expression, they also present real challenges for in vivo gene delivery. To achieve an effective therapy, it is mandatory to improve some limitations of these delivery systems, either by reducing their toxicity and the immune responses developed against these vectors (Wang et al., 2016) or by specifically improving their tropism towards cancer cells. A possibility to achieve tissue specificity is by altering AAVs natural wide tissue tropism through the modification of the viral capsid proteins, incorporating peptides into the AAV
capsid surface that will alter its interactions with other cellular receptors and, in this way, target the vector to specific cell types (Samulski & Muzyczka, 2014).
1.2.2. Proteasome as targets for BLBC therapy with AAV vectors The 26S proteasome is a large multicatalytic proteinase complex, which is responsible for maintaining homeostasis and plays a vital role in regulating intracellular proteins that cells depend upon for division and survival. It consists of an ubiquitin system conjugated with the proteasome and is present both in the cytoplasm and in the nucleus of all mammalian cells (Crawford et al., 2011). Ubiquitination is the first process to occur, in which the proteins are targeted for elimination with ubiquitin molecules. Thus, the 19S subunit of the proteasome complex recognizes the proteins with ubiquitin marks, removes the polyubiquitin chain and then directs the protein for hydrolysis that occurs in the 20S subunit, the catalytic core of the 26S proteasome (see Figure 4) (Bhattacharyya et al., 2014; Goldberg, 2007).
Figure 4. Protein degradation via the ubiquitin proteasome pathway. The 26S proteasomal
degradation of cellular proteins is complemented with the attachment of a polyubiquitin chain to the target proteins by E1, E2 and E3 enzymes. It is inside the 20S core that polyubiquitylated proteins will be degraded into smaller peptides. (Adapted from: Kopan & Ilagan, 2004).
Actually, in cancer cells, the proteasome is dysregulated and is responsible for eliminating proteins essential in cell regulation, for example degrading tumour suppressor proteins or DNA repair enzymes, which ultimately leads to cancer progression. Some tumours are dependent on the proteasome to resist
to therapies, by mediating the degradation of the NF-κB inhibitor, which leads to the activation of the NF-κB signaling pathway that drives uncontrolled proliferation of cancer cells and resistance to chemotherapeutic agents (Rastogi & Mishra, 2012). A widely known proteasome inhibitor, bortezomib, has demonstrated anti-tumour potential towards many cancer types and has already been approved to treat myeloma disease (Crawford et al., 2011; Rastogi & Mishra, 2012). Based on all these features, the proteasome became a good target for inhibition and a promising approach in anticancer therapies (Rastogi & Mishra, 2012).
Previous studies performed by Petrocca and Lieberman (Petrocca et al., 2013), using genome-wide siRNA screen, allowed the identification of genes whose expression was differentially required for the survival of basal-like breast cancer cells. In order to do that, BPLER cells (which closely resemble highly aggressive and poorly responsive human basal-like breast cancer cells) were compared to HMLER cells (representing less aggressive tumours) (Ince et al., 2007). The authors identified 154 genes as selectively required for BPLER cell survival and, from this list of genes, a subset of 23 were identified as highly selective BPLER dependency genes (Figure 5). Although this small subset of genes was related to different signalling pathways, seven were specifically
connected to proteasome machinery. Interestingly, based on the
overrepresentation of proteasome-related genes, Petrocca showed that proteasome inhibitors are selectively lethal for TNBC cell lines in vitro and in
vivo, and proposed that RNAi-based drugs would be a good approach as
targets to treat this disease (Petrocca et al., 2013). Thus, rAAV vectors engineered to transport shRNAs to target proteasome proteins, would be a great therapeutic tool to target basal-like breast cancer cells.
Figure 5. A set of dependency genes in 17 human breast cancer cell lines. Identification of
dependency genes needed for the survival of TNBCs. The screening of the dependency genes in human breast cancer cell lines was performed by siRNA knockdown in 7 basal -A (red), 6 luminal (blue), 3 basal-B (green) and 1 unclassified (purple) cell lines (Adapted from Petrocca et
2. AIM
The work developed under this Master thesis has been included in a large
collaborative project funded by FCT (PTDC/BBB-BIO/1240/2012, “Combining
siRNA and AAV therapy approaches to target human basal-like breast cancer: from vector development to anti-tumour efficacy evaluation”), which involved the
group of Prof. Paula Alves (Principal Investigator, from Institute of Experimental and Technological Biology (IBET), Oeiras, Portugal), the group of Prof. Joana Paredes (from Institute of Molecular Pathology and Immunology at the University of Porto (IPATIMUP), Porto, Portugal) and the group of Prof. Judy Lieberman (Harvard Medical School, Boston, USA).
In 2013, Judy Lieberman’s laboratory has identified a set of genes required for BLBC cell survival, by the use of a genome-wide siRNA lethality screen (Petrocca et al., 2013). Interestingly, it was observed that in BLBC cell lines the proteasome genes were highly represented hits. Also, compared to other breast cancer cell lines, the BLBC cells were selectively sensitive to proteasome
inhibitors. Moreover, proteasome inhibition decreased basal-like tumour growth
and was effective against tumour-initiating cells and metastasis in vivo.
Thus, the main goal of the project was to combine the development of engineered AAV vectors that would be able to infect specifically BLBC cells, together with the capacity that they have to transport and deliver shRNAs to silence the expression of genes on which BLBC depend to survive, such as the ones of the proteasome.
Thus, as can be seen in Annex I, IBET’s group has designed and produced 11 pAAV2-shRNA constructs, coding GFP as transgene: 6 constructs coding specific shRNA sequences targeting the highly selective BLBC survival genes
PSMA1, PSMA2, PSMB4, MCL1 and NCD80, identified in Judy Lieberman’s
Lab (Petrocca et al., 2013); 2 constructs lacking homology to the human transcriptome to be used as negative controls; and 2 constructs with sequences targeting the Polo-like kinase-1 (PLK1), essential for the survival of all breast cancer cell lines tested. shRNA expression and knockdown efficiency of the different plasmid constructs were first determined in a transient transfection assay in HEK293T cells.
The purification of the different AAV2-shRNA vectors was then performed, and just 8 different AAV2-shRNA vectors were obtained with high purity and high titers: 2 negative scramble controls, 1 PLK1 control, and 5 AAV2-shRNA vectors targeting 4 different BLBC survival genes). To validate their transduction efficiency in BLBC cells, 3 different cell lines (MDA-MB-468, HCC1954 and HCC1187) were transduced with increasing multiplicity of infection (MOI) of the different purified AAV2-shRNA vectors. Interestingly, transduction of MDA-MB-468 cells with AAV2-shPSMB4, AAV2-shPSMA2 and AAV2-shPSMA1 strongly decreased their cell viability. However, only AAV2-shPLK1 and AAV2-shPSMA2 transduction resulted in increased apoptosis of these cells. Thus, since AAV2-shPSMA2 showed the higher knockdown efficiency in both cell lines, as well as decreased MDA-MB-468 cell survival and induced apoptosis (2 fold), this viral vector has been selected to be tested in an in vivo pre-clinical model, by IPATIMUP’s group.
Thus, based on this preliminary data, the main aim of this thesis, at IPATIMUP, was to validate, in vivo, if PSMA2 could be a putative target for BLBC therapy.
To achieve that, two specific and experimental tasks have been designed:
1) To evaluate the anti-tumorigenic activity of AAV2-based vectors delivering shRNA sequences targeting PSMA2 transcripts in an in vivo pre-clinical BLBC model.
Within this task, the aim was to directly inject AAV2-shPSMA2 vectors, engineered by IBET, using MDA-MB-468 basal-like breast xenografts, in order to evaluate their anti-tumour efficacy and cytotoxicity.
2) To evaluate the expression of PSMA2 in a large series of human breast cancer samples.
The aim of this task was to evaluate the expression of PSMA2 in a series of 467 human invasive breast carcinomas, in order to investigate if there was an enrichment of its expression in basal-like breast cancer lesions, as well as to evaluate its association with clinicopathological features and patient prognosis.
3. MATERIAL AND METHODS
3.1. Cell lines and culture conditions
The human basal-like breast cancer cell line MDA-MB-468 from the
American Type Culture Collection (HTB-132™ ATCC®, Manassas, Virginia,
USA) was maintained as a monolayer culture in Dulbecco's Modified Eagle
Medium (DMEM, Gibco®, Life Technologies), supplemented with 10% of fetal
bovine serum (FBS, Thermo Scientific, USA) and 1% of penicillin/streptomycin (Gibco®, Life Technologies). Cells were grown in a humidified incubator at 37°C
with an atmosphere of 5% carbon dioxide (CO2). Cells were left to grow until
confluence and then washed with Phosphate-Buffered Saline (PBS, Fisher Scientific, USA) and exposed to trypsin (0.05% Trypsin-EDTA 1x, Gibco®, Life Technologies) for 10 minutes. The enzyme activity was stopped with DMEM supplemented medium. The cells were counted and centrifuged at 1200 rpm for 5 min and the pellet was resuspended in medium without supplements.
3.2. Development of AAV2-shRNA vectors
AAV2-shRNA plasmid vectors were kindly provided by IBET, where these were designed and validated for knockdown efficiency. The following vectors have been acquired: one expressing a scramble shRNA#1-Ctr (lacking homology to the human transcriptome, to be used as a negative control), one expressing a shRNA-PLK1 (sequence targeting the Polo-like kinase-1 (PLK1), essential for the survival of all breast cancer cell lines) and one expressing shRNA-PSMA2 (targeting the Proteasome subunit type-2 (PSMA2) gene, essential for BLBC survival) (Petrocca et al., 2013). These vectors simultaneously express the shRNA and a GFP reporter gene for monitoring infection. Knockdown efficiency and cell survival was evaluated after infection (see Annex I for detailed information).
3.3. In vivo experiments
Female N:NIH(s)II:nu/nu nude mice were used to evaluate the anti-tumorigenic effect of AAV2 vectors. The animals were housed at the I3S Animal House under a specific pathogen free (SPF) environment, with controlled conditions of temperature (between 20-24ºC) and humidity (45-65%), and with 15 to 20 air changes per hour under 12h light - 12h dark cycle. Animals were maintained in type II-L polycarbonate cages with 4-5 females per cage, with food and water provided ad libitum.
All animal experiments and euthanasia were carried out in accordance to the European Guidelines for the Care and Use of Laboratory Animals, directive 2010/63/UE and decreto-lei n. º 113/2013 (Portugal).
In order to produce orthotopic primary tumours, human MDA-MB-468 breast
cancer cells (2×106 cells/100μL medium without supplements) were
subcutaneously inoculated into the lower mammary fat pad of female nude mice, with 6-8 weeks of age, using a 25 gauge syringe needle.
Mice were weighted weekly and primary tumours growth was evaluated by measuring tumours with calipers two times a week. Tumours were measured in two perpendicular dimensions and the volume was estimated using the formula
[volume = (width)2 × (length)/2] for approximating the volume (mm3) of an
ellipsoid.
When tumours reached 100-150 mm3, mice were randomly separated into
different groups, with approximate average tumour volume. AAV2-shRNA
intratumoral injections (50μl) were performed twice a week during 3-4 weeks.
Animals were euthanized 50 to 55 days after tumour cell inoculation.
Mice were monitored for their health and behavioural condition. Clinical signs associated with tumour progression were considered for monitoring animal's wellbeing. Score sheets for experimental procedures with bilateral tumours were reviewed, as well as humane endpoints.
The follow-up of the animals was performed under the criteria established at the
i3S Animal House. In terms of tumour size, these could not exceed 2000mm3
or, in case of bilateral tumours, the sum of the tumours could not exceed
In this specific study, tumours developed ulcers due to inner characteristics of the inoculated breast cancer cells. Mice bearing ulcerated tumours were closely monitored and the endpoints were taken into consideration. Disinfection and size of the ulcer were criteria that were permanently taken into account.
As soon as animals started to develop signs of illness, such as loss of 20% of body weight, ulcerated tumours exceeding 50% of the surface area of the
tumours or a total tumours volume that exceeded 2000mm3, these were
euthanized.
Animals were anesthetized with isoflurane (IsoFlo, Abbott) and cervical displacement was performed to euthanize the animal.
At necropsy, mammary fat pad tumours and livers were collected from each mouse for further analysis. The tumours were preserved in the following way: ½ of the tumours was snap frozen in liquid nitrogen for further RNA extraction and analysis, the other ½ was preserved in 10% buffered formalin solution (Bio-Optica, Italy) for histological and immunohistochemical analysis. Macroscopic metastases were searched, particularly in lungs and livers.
3.4. RNA extraction and quantitative RT-PCR
Tumours preserved in liquid nitrogen were homogenized with a Tissue Ruptor (Qiagen) and RNA was isolated using RNeasy Mini Kit (Ref. 74106, Qiagen, USA). Quantitative Real-Time PCR (qRT-PCR) reaction and analysis
were performed at IBET using the LightCycler® 480 SYBR Green I Master Mix
(Roche Life Science) system, in order to evaluate the efficiency of gene transfer
in vivo. cDNA was synthesized with Advantage® RT-for-PCR Kit (Clontech
Laboratories, USA) from RNA samples of mice tumours treated with PBS, shCtr and shPSMA2 AAV2 vectors, using human specific primers to evaluate gene silencing. Relative PSMA2 mRNA expression was compared with 3 housekeeping genes (HPRT1, RPL22 and GAPDH), which were used to normalize cDNA quantity, individually.
3.5. Histological processing
Part of the tumour excised from each animal was placed into cassettes correctly identified and fixed in 10% buffered formalin solution and then
embedded in paraffin, in order to allow histological characterization. The histological processing and Haematoxilin & Eosin (H&E) staining of livers and tumours was performed at the Histopathology Unit of I3S. For histological examination, formalin-fixed paraffin-embedded tumours and liver samples were cut in 4µm thick sections that were used for H&E staining and immunohistological characterization.
3.6. Immunohistochemistry
Slides were dewaxed with clear-rite (Thermo Scientific™ Richard-Allan Scientific™, USA) and rehydrated in a series of decreasing concentrations of ethanol solutions (100%, 100%, 70%) to tap water. Heat-induced epitope retrieval was carried out in citrate buffer (pH 6, AP-9003-500, Thermo
ScientificTM, USA), in a 98ºC water bath, for 30 minutes. Slides were left to cool
down for 30 minutes at room temperature. After washing in PBS, endogenous peroxidase activity was blocked with 3% hydrogen peroxide (GPC8054-E, Atom Scientific, UK) in methanol (M/400/FP21, Fisher Scientific, UK), for 10 minutes. Non-specific blocking was performed for 10 minutes with Ultra Vision Protein
Block (TA-125-PBQ, Thermo ScientifcTM, USA). Primary antibodies, listed in
Table 2, were diluted in Antibody diluent OP Quanto (TA-125-ADQ, Thermo
ScientifcTM) and incubated 1h at room temperature and then labelled with DAKO
RealTM EnvisionTM HRP Rabbit/Mouse secondary antibody (K5007, Dako) for 30
minutes. Color development was achieved with a chromogenic substrate
system 3,3’-diaminobenzidine tetrahydrochloride (DAB, Dako REALTM
EnVisionTM, DAKO) for 7 minutes. Tissues were counterstained with Gill’s
haematoxylin III (Bio-Optica, Italy) for 1 minute and then immersed in tap water for 2 minutes. Sections were dehydrated in a series of increasing concentrations of ethanol solutions (70%, 100%, 100%), clarified in clear-rite and then mounted permanently with mounting medium (Ref. 4112, Thermo Scientific™ Richard-Allan Scientific™, USA) for visualization under the microscope.
Paraffin sections of normal liver and testis were included as positive controls for PSMA2 staining. MDA-MB-468 xenograft tumours were used as positive controls for Ki-67 and cleaved-caspase3 (CASP3) staining.
H&E stained sections were analysed by a breast cancer pathologist, to evaluate histopathological features, such as inflammation, vascular invasion and extension of necrosis. Livers were also analysed to evaluate hepatotoxicity. Immunostained slides were analysed to evaluate cell proliferation (IHC for
Ki-67, % Ki-67+ cells), apoptotic index (IHC for cleaved-caspase3, % of CASP3+
cells), PSMA2 protein expression (IHC for PSMA2, score used described below in section 3.8).
The following primary antibodies were used for immunohistological analysis:
Table 2. List ofantibodies used and conditions of the immunohistochemical reactions performed in this study.
Primary Antibody
Retrieval Detection
Antibody Clone Supplier Dilution Incubation
Time (min) PSMA2 HPA00 8188 Sigma 1:350 60 Citrate Buffer (40 min) HRP-Polymer* Ki-67 SP6 Cell Marque 1:300 60 Citrate Buffer (40 min) HRP-Polymer* Cleaved-Caspase3 (Asp175) (D35E) Cell Signalling 1:150 60 Citrate Buffer (40 min) HRP-Polymer*
*HRP-Polymer (Horseradish peroxidase – polymer)
3.7. Human breast cancer samples
A series of 467 human invasive breast carcinomas were kindly provided by Dr. Jorge Cameselle-Teijeiro (Pathology Department of Hospital Xeral-Cíes, Vigo, Spain), diagnosed between 1978 and 1992. This series was fully characterized for clinical and pathological features, such as patient age at diagnosis, tumour size, histological grade, and lymph node invasion, as well as data concerning disease-free and overall patient survival. The expression of key molecular markers were already available in this series, such as ER, PR, HER2, which allow us to determine the molecular subtype of each tumour and other markers such as Ki-67, CD44/CD24, ALDH1, CD49f, CLDNs 3, 4 and 7, E-cadherin (E-cad), P-E-cadherin (P-cad), Vimentin, HIF1α, CAIX and GLUT1.
Patient follow-up information was available for 455 cases, ranging from a minimum of 1 to a maximum of 120 months after the diagnosis. The disease-free survival (DFS) was defined as the interval between the diagnosis to the date of breast-cancer-derived relapse, whereas overall survival (OS) was considered as the number of months from the diagnosis to the disease-related death.
3.8. Semiquantitative evaluation of immunhohistochemistry for PSMA2 For PSMA2 evaluation in human breast cancer samples, the cytoplasmic staining intensity was evaluated on a scale of 0 to 3 (0, negative; 1, weak; 2, moderate; 3, strong) and extension was registered as the percentage of positive cells for PSMA2: 0, 0% of immunoreactive cells; 1, <5% of immunoreactive cells; 2, 5–50% of immunoreactive cells; and 3, >50% of immunoreactive cells. Some samples could not be assessed due to core falling or lack of tumours. Concerning the immunohistochemical PSMA2 evaluation in MDA-MB-468 tumours xenografts, we decided to score in a different way, since this cell line is already positive for this marker. Cases scored with 0 were considered negative, cases scored with 1 and 2 were considered to have low expression, the ones with 3 and 4 were considered to have moderate expression and cases with scores of 5 and 6 were considered to have high PSMA2 expression.
3.9. Statistical analysis
Statistical analysis and graphical representation of tumours volume, and fold increase in tumours volume was performed using the Graphpad 6 (Prism®) software. The statistical difference between mice groups was determined by Student’s t-test, where it was considered statistically different when p<0.05. Statistical analysis for immunohistochemistry results were performed by SPSS
statistics 17.0 software (SPSS Inc., USA). X2 test and contingency tables were
used to determine associations between groups and one-way ANOVA followed by Tukey’s test was used to compare all groups to each other. The results were
4. RESULTS
4.1. The anti-tumorigenic activity of AAV2-based vectors delivering shRNA sequences targeting PSMA2 transcripts in an in vivo pre-clinical BLBC model
1st Experiment.
Impact of shRNA expressed by AAV2 control vectors in tumour growth An initial study was performed in order to evaluate the tumorigenic and hepatotoxic effect of the shRNA expressed by AAV2 control vectors. For that AAV2-shCtr#1 (negative control) vector was tested for different multiplicities of
infection (MOIs). As shown in Figure 6 and in Table 3, 2x1010, 2x109 and 2x108
(also referred to as 2E10, 2E9 and 2E8, respectively) viral particles (VP)/tumour, were compared with PBS (that resembles normal tumour growth), to determine the optimal viral vector dosage and to assess the tumour growth rate of BLBC xenografts of MDA-MB-468 cells in response to these vectors (3 to 4 mice were used per group, n=15).
Figure 6. Experimental design used for in vivo evaluation of the anti-tumorigenic activity of shRNA expressed by AAV2 control vectors. Mice were inoculated with MDA-MB-468, and
at day 0 AAV intratumoral administrations were performed for the concentrations of 2E10, 2E9 and 2E8 VP/tumour of AAV2-shCtr vectors.
