Targeting P53 Family in Cancer: Search for Inhibitors of the P53/P73 Interaction with Mdms

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M 2015

TARGETING P53 FAMILY IN CANCER: SEARCH FOR INHIBITORS OF THE P53/P73 INTERACTION WITH MDMS

HELENA ISABEL NOGUEIRA RAMOS ROCHA DISSERTAÇÃO DE MESTRADO APRESENTADA À FACULDADE DE ENGENHARIA DA UNIVERSIDADE DO PORTO EM BIOENGENHARIA

Integrated Master in Bioengineering - Molecular Biotechnology

Dissertation

Targeting p53 family in cancer: Search for inhibitors of the p53/p73 interaction with MDMs

Helena Isabel Nogueira Ramos Rocha

Supervisors Prof. Dr. Lucília Saraiva

Mariana Leão, PhD

Sara Moreira, MSc

Host Institution UCIBIO/REQUIMTE, Department of Biological Sciences, Microbiology Laboratory, Faculty of Pharmacy, University of Porto, Portugal

Porto, September 2015

ACKNOWLEDGMENTS

First of all I would like to thank my supervisor, Professor Lucília Saraiva, for the amazing opportunity to work in her research group. The dynamism, courage and the strength with which Prof. Lucília leads the group is indeed inspiring. Thank you for believing in me and in my abilities, for all the trust, encouragement and guidance these last months.

I would like to thank, with all my heart, my two co-supervisors Sara Gomes and Mariana Leão for all the valuable lessons, the patience and the constant support. Thank you for always be there for me, for the motivation and dedication. I will always be grateful for having the opportunity to have worked and learned so much with you.

I would also like to thank to my dear lab colleagues Joana Soares, Cláudia Bessa, Ana Sara Gomes and Liliana Raimundo for making me feel so welcome, for all the ideas, support, scientific discussions and friendship. Particularly, to Joana, my most sincere thanks. Even you were not supervising me, you always helped me and took time to listen all my doubts and questions, and to advise me the best possible way. Thank you for that.

Special thanks to my classmates, companions and, most of all, friends Maria João, Sarah, Diana, Catarina and Filipe. You certainly made this journey much easier and filled with many unforgettable moments. Thank you all for the true friendship.

Many thanks to my beloved parents. I wish I could one day return everything you have given me. Thank you for your unconditional support, love and patience.

And last, but certainly not least, a very special thanks to my boyfriend, companion and, above all, my best friend Nunes. This journey is also yours. Thank you very much just for being there when I needed, for listening to me, for advising me and supporting me all the time. Thank you for all the motivation and enthusiasm, and for believing in me even when I had doubts.

This work is funded by FEDER funds through the Operational Programme for Competitiveness Factors - COMPETE and by National Funds through FCT - Foundation for Science and Technology – under the UID/Multi/04378/2013 (UCIBIO/REQUIMTE) and the FCT project (PTDC/DTP-FTO/1981/2014).

ABSTRACT

The p53 family of transcription factors consists of three homologous proteins, p53, p63 and p73, with a central role in the control of cell differentiation, proliferation and death. The deregulation of this family plays a critical role in tumorigenesis and significantly affects tumor response to therapy. Therefore, this family is considered a key therapeutic target in cancer. The p53 family proteins are mainly inhibited by interaction with murine double minute (MDM) proteins, MDM2 and MDMX. The inhibition of these protein-protein interactions, for a full reactivation of p53 family proteins, represents a promising therapeutic anticancer strategy. Despite the vast number of inhibitors of the p53-MDM2 interaction already described, only few inhibitors of the p53-MDMX interaction and dual inhibitors of the p53-MDM2/MDMX interactions were reported to date. In addition, inhibitors of the p63 and p73 interaction with MDM2/MDMX are still missing. In the present work, two new activators of the p53 and p73 pathways were discovered, namely an inhibitor of the p73-MDM2 interaction (the xanthone LEM2), and a dual inhibitor of the p53-MDM2/MDMX interactions (the phenylalaninol-derived oxazolopyrrolidone lactam derivative JL-1). For these findings, the yeast Saccharomyces cerevisiae (without orthologues of p53 family proteins) greatly contributed as a simplified cell-based screening assay to search for potential inhibitors of the p53 and p73 interaction with MDMs. Herein, the molecular mechanisms of action of LEM2, as an inhibitor of the p73- MDM2 interaction, and of JL-1, as a dual inhibitor of the p53-MDM2/MDMX interactions, were confirmed in human tumor cell lines. Particularly, in a p53-null human tumor cell line, it is shown that LEM2 has a potent tumor growth inhibitory effect, higher than that obtained with nutlin-3a (the only known inhibitor of the p73-MDM2 interaction described to date). The disruption of the p73-MDM2 interaction by LEM2 was further confirmed by co- immunoprecipitation assays. In addition, LEM2 increased the protein levels of p73 and of its transcriptional targets (MDM2, Bax and p21), and induced cell cycle arrest, and apoptosis, with PARP cleavage, ROS generation and ∆ψm dissipation. Also, LEM2 did not show apparent in vitro genotoxic effects. On the other hand, using human colon adenocarcinoma HCT116 cell lines expressing wild-type p53 (HCT116 p53+/+) and its p53- null isogenic derivative (HCT116 p53-/-), it was shown that JL-1 exhibited a p53-dependent in vitro antitumor activity. In fact, JL-1 induced p53 stabilization, up-regulated p53 transcriptional targets (MDM2, MDMX, Bax, p21, and Puma), and induced cell cycle arrest and apoptosis, with PARP cleavage, in p53+/+, but not in p53-/-, HCT116 cells. The disruption of the p53-MDM2/MDMX interactions was further supported by co-immunoprecipitation

II assays, in HCT116 p53+/+ tumor cells. In addition, similar tumor cytotoxic effects were observed for JL-1 against MDM2-overexpressing osteosarcoma SJSA-1 and MDMX- overexpressing breast adenocarcinoma MCF-7 cell lines. JL-1 did not show apparent in vitro genotoxic effects and it was capable of inhibiting the migration of wt p53-expressing tumor cells. Importantly, JL-1 also showed promising in vivo antitumor properties against HCT116 p53+/+ tumor cells. Overall, this work provides a relevant contribution to the pharmacological knowledge of the p53 family proteins, with the identification of two new promising small-molecule activators of the p53 and p73 pathway. Besides their potential as anticancer agents, their scaffolds may open the way to new derivatives with improved pharmacological properties.

III

RESUMO

A família da p53 é constituída por três proteínas homólogas, a p53, p63 e a p73, que possuem um papel fundamental no controlo da diferenciação, proliferação e morte celular. A desregulação desta família apresenta um papel crítico na tumorigénese e afeta significativamente a resposta do tumor à terapia. Desta forma, esta família é considerada um alvo terapêutico chave no cancro. As proteínas da família da p53 são maioritariamente inibidas pela interação com as proteínas murine double minute (MDM), MDM2 e MDMX. A inibição destas interações proteína-proteína, para uma reativação mais eficaz das proteínas da família da p53, representa uma estratégica terapêutica anticancerígena promissora. Até à presente data, apesar do número elevado de inibidores da interação p53- MDM2 existentes, poucos são os inibidores da interação p53-MDMX e inibidores duais das interações p53-MDM2 e p53-MDMX descritos. Além disso, continuam por descobrir inibidores das interações entre p63 e p73 com as proteínas MDMs. No presente trabalho, foram descobertos dois novos ativadores das vias da p53 e da p73, nomeadamente, um inibidor da interação p73-MDM2 (a xantona LEM2), e um inibidor dual das interações p53-MDM2/X (uma oxazolopiperidona lactama derivada do fenilalaninol JL-1). Para estas descobertas, contribuiu a levedura Saccharomyces cerevisiae (que não possui ortólogos das proteínas da família da p53) como ensaio celular de screening simplificado para a pesquisa de potenciais inibidores das interações da p53 e p73 com as MDMs. Neste estudo, foram também confirmados, em linhas celulares tumorais humanas, os mecanismos moleculares de ação do LEM2, como inibidor da interação p73-MDM2, e do JL-1, como inibidor dual das interações p53-MDM2/X. Em particular, em linhas celulares tumorais humanas que não expressam a p53, foi demonstrado que o LEM2 tem um potente efeito inibidor de crescimento, superior ao obtido com a nutlina-3a (o único inibidor da interação p73-MDM2 descrito até à data). A inibição da interação p73-MDM2 pelo LEM2 foi confirmada por ensaios de co-imunoprecipitação. Além disso, o LEM2 promoveu o aumento dos níveis proteicos da p73 e dos seus alvos transcricionais (MDM2, Bax e p21), induziu a paragem do ciclo celular e apoptose, com clivagem de PARP, produção de espécies reativas do oxigénio e dissipação do potencial de membrana mitocondrial. Por outro lado, usando linhas celulares de adenocarcinoma do cólon HCT116, que expressam a forma nativa da p53 (HCT116 p53+/+) ou o seu derivado isogénico, que não expressa a p53 (HCT116 p53-/-), mostrou-se que o JL-1 exibiu uma atividade anti-tumoral in vitro dependente da p53. De facto, o JL-1 induziu a estabilização da p53, aumentou a expressão de alvos transcricionais da p53 (MDM2, MDMX, Bax, p21 e Puma), e induziu a paragem

IV do ciclo celular e apoptose, com clivagem de PARP, em células HCT116 p53+/+, mas não nas células p53-/-. A quebra das interações p53-MDM2/X foi sustentada por ensaios de co- imunoprecipitação, em células tumorais HCT116 p53+/+. Adicionalmente, foram observados efeitos tumorais citotóxicos similares do JL-1 em linhas celulares de osteossarcoma SJSA- 1, que sobre-expressam MDM2, e em linhas celulares do adenocarcinoma da mama MCF- 7, que sobre-expressam MDMX. O JL-1 não apresentou aparentes efeitos genotóxicos in vitro e foi capaz de inibir a invasão de células tumorais que expressavam a forma nativa da p53. Notavelmente, o JL-1 também apresentou propriedades anti-tumorais in vivo promissoras em células tumorais HCT116 p53+/+. De uma forma geral, este trabalho representa um contributo relevante para a farmacologia das proteínas da família da p53, através da identificação de duas novas pequenas moléculas inibidoras das vias da p53 e da p73. Além do potencial como agentes anticancerígenos, as suas estruturas podem abrir caminho a novos derivados com propriedades farmacológicas melhoradas.

CONTENTS

Acknowledgments ...... I Abstract ...... II Resumo ...... IV Contents ...... VI Abbreviations ...... IX 1. Introduction ...... 3 1.1. The p53 family proteins ...... 4 1.1.1. Structural features ...... 5 1.1.2. Biological functions ...... 7 Cell cycle progression control and DNA repair ...... 9 Apoptosis induction ...... 11 1.2. Endogenous modulators of p53 family proteins ...... 15 1.2.1. MDM2 and MDMX: protein structure ...... 16 1.2.2. p53 family-related biological functions of MDMs ...... 17 1.3. The p53 family in cancer targeted therapy ...... 19 1.4. Yeast as a cell model to study p53 family proteins ...... 24 1.5. Aims of research ...... 27 2. Materials and Methods ...... 31 2.1. Compounds ...... 31 2.2. Yeast Cells ...... 31 2.2.1. Yeast targeted screening assay ...... 31 2.2.2. Yeast cell cycle analysis ...... 31 2.3. Human tumor cell lines ...... 32 2.3.1. Human tumor cell lines and growth conditions ...... 32 2.3.2. Sulforhodamine B (SRB) assay...... 32 2.3.3. Analysis of cell cycle and apoptosis ...... 32 2.3.4. Western blot analysis ...... 33 2.3.5. Co-immunoprecipitation (co-IP) assay ...... 34

2.3.6. Analysis of mitochondrial membrane potential (Δψm) ...... 34 2.3.7. Analysis of reactive oxygen species (ROS) generation ...... 34 2.3.8. Genotoxicity studies by micronucleus assay ...... 34 2.3.9. In vitro migration assays ...... 35 2.3.10. In vivo experiments ...... 35 2.4. Yeast and human tumor cell lines ...... 36 2.4.1. Flow cytometric data acquisition and analysis ...... 36 2.4.2. Statistical analysis ...... 36 3. LEM2: a new small molecule inhibitor of the p73-MDM2 interaction ...... 39

3.1. Results ...... 40 3.1.1. LEM2 does not interfere with the p53-MDM2 interaction ...... 40 3.1.2. Identification of LEM2 as a potential inhibitor of the p73-MDM2 interaction using the yeast screening assay ...... 41 3.1.3. LEM2 activates the p73 pathway in human tumor cells through inhibition of the p73- MDM2 interaction ...... 43 3.1.4. LEM2 has no in vitro genotoxicity ...... 46 3.2. Discussion ...... 46 4. JL-1: a dual inhibitor of the p53 interaction with MDM2/MDMX ...... 51 4.1. Results ...... 52 4.1.1. JL-1 activates a p53-dependent pathway in wt p53-expressing human tumor cells...... 52 4.1.2. JL-1 has no in vitro genotoxicity ...... 56 4.1.3. JL-1 prevents the in vitro tumor cell migration ...... 57 4.1.3. JL-1 has potent in vivo antitumor activity ...... 57 4.2. Discussion ...... 58 5. General discussion and final remarks ...... 63 6. References ...... 67

LIST OF FIGURES

Figure 1 – Structural organization of p53 family proteins ...... 6 Figure 2 – Schematic representation of the biological functions of p53 family proteins...... 8 Figure 3 – General representation of the cell cycle regulation by p53 family proteins ...... 10 Figure 4 – Regulation of intrinsic and extrinsic apoptotic pathways by p53 ...... 12 Figure 5 – Schematic representation of MDM2 and MDMX domains ...... 16 Figure 6 – Regulation of p53 by its endogenous negative modulators MDM2 and MDMX...... 18 Figure 7 – Schematic representation of different yeast models frequently used to study p53...... 26 Figure 8 – Chemical structure of 3,4-dihydro-12-hydroxy-2,2-dimethyl-2H,6H-pyrano(3,2-b)xanthen- 6-one (LEM1)...... 39 Figure 9 – LEM2 did not disrupt the p53-MDM2 interaction in HCT116 p53+/+ cells ...... 41 Figure 10 – LEM2 reduces the inhibitory effect of MDM2 on p73 activity in yeast ...... 42 Figure 11 – LEM2 induces cell cycle arrest, apoptosis and PARP cleavage in HCT116 p53-/- cells...... 43

-/- Figure 12 – LEM2 induces ROS generation and ∆ψm dissipation in HCT116 p53 cells...... 44 Figure 13 – LEM2 increases the expression levels of p73 and up-regulates p73 target genes by blocking the p73 interaction with MDM2 in HCT116 p53-/- cells ...... 45 Figure 14 – LEM2 has no in vitro genotoxicity ...... 46 Figure 15 – JL-1 has in vitro antitumor activity and induces cell cycle arrest, apoptosis and PARP cleavage in p53+/+, but not in p53-/- HCT116 cells ...... 52 Figure 16 – JL-1 increases the expression levels of p53 and up-regulates p53 target genes by blocking the p53 interaction with MDM2/MDMX in p53+/+, but not in p53-/- HCT116 cells...... 53

VII

Figure 17 – JL-1 induces growth inhibition associated with cell cycle arrest and apoptosis in MDM2- and MDMX-overexpressing tumor cells through activation of the p53 pathway ...... 55 Figure 18 – JL-1 has no in vitro genotoxicity ...... 56 Figure 19 – JL-1 prevents the migration of HCT116 p53+/+ cells...... 57 Figure 20 – JL-1 has in vivo antitumor activity...... 58

LIST OF TABLES

Table 1 – Small molecules inhibitors of the p53-MDM2 interaction currently in clinical trials ...... 22

Table 2 – GI50 values obtained for LEM2 in human colon adenocarcinoma HCT116 tumor cell lines...... 40

VIII

ABBREVIATIONS

Apaf-1 – Apoptosis protease-activating factor 1 Bax – Bcl-2 associated X protein Bak – Bcl-2-antagonist/killer Bid – BH3 interacting-domain death agonist CAK – CDK-activating kinase Cdc25c – Cell division cycle 25 homolog c CDK – Cyclin-dependent kinase CFU – Colony-forming unit Co-IP – Co-immunoprecipitation Cyt c – Cytochrome c DBD – DNA-binding domain DED – Death effector domain DIABLO – Direct IAP-binding protein with low pI DISC – Death-inducing signalling complex DMSO – Dimethyl sulfoxide EndoG – Endonuclease G FADD – Fas-associated protein with death domain FASAY – Functional analysis of separated alleles GADD45 – Growth arrest and DNA-damage inducible protein

GI50 – Growth inhibition of 50% IAP – Inhibitor of apoptosis proteins MDM2 – Murine double minute 2 MDMX – Murine double minute X MN – Micronucleus MOMP – Mitochondrial outer membrane permeabilization mtCLIC – Mitochondrial chloride intracellular channel NES – Nuclear export signal NLS – Nuclear localization signal OD – Oligomerization domain

OD600 – Optical density at 600 nm Omi/HtrA2 – Serine protease high-temperature requirement protein A2 p53AIP1 – p53-regulated apoptosis inducing protein 1 p53RE – p53 responsive element PBS – Phosphate-buffered saline PI – Propidium iodide

PUMA – p53 upregulated modulator of apoptosis pRb – Retinoblastoma protein RE – Response element ROS – Reactive oxygen species SAM – Sterile-alpha motif SCF – Skp, Cullin, F-box containing complex SMAC – Second mitochondria-derived activator of caspases SRB – Sulforhodamine B TAD – Transactivation domain tBid – Truncated Bid TID – Transactivational inhibitory domain TNF – Tumour necrosis factor TRADD – TNF receptor-associated death domain TRAIL – Tumour necrosis factor-related apoptosis-inducing ligand wt – Wild-type Y2H – Yeast two-hybrid

I Introduction

1. INTRODUCTION

Cancer is one of the leading causes of morbidity and mortality worldwide, with approximately 14 million new cases and 8.2 million cancer-related deaths in 2012 [1]. According to the World Health Organization, the number of new cases will most likely increase about 70% over the next 20 years [1]. In fact, despite the increase in the relative survival rates due to improved diagnostic techniques, accuracy of prognosis and cancer treatments, the cancer incidence is increasing as a result of population aging and cancer- associated lifestyles [1]. Tumorigenesis is a multistep process, driving the progressive transformation of normal cells into malignant ones. These altered cells divide and grow in the presence of signals that normally inhibit cell growth, and develop new functional and structural characteristics [2]. In fact, the malignant transformation of a cell is attributed to a variety of genetic and epigenetic events, involving alterations in several oncogenes and tumor suppressor genes. The malignant phenotype is characterized by sustained proliferation, evasion of growth suppressors, resistance to cell death and replicative mortality, increased angiogenesis, and activation of invasion and metastasis [2, 3]. Although the complete sequence of events required for the development of human cancer is not completely understood, the most widely studied mechanisms are the inactivation of tumor suppressor genes and the activation of oncogenes. In fact, given their relevance for tumor initiation and progression, oncogenes and tumor suppressor genes have been thoroughly explored as valuable therapeutic targets [2]. Oncogenes typically encode proteins that stimulate cell proliferation with an inhibition of apoptosis, and are usually activated by mutation or gene fusion, by association with enhancer elements, or by amplification [4]. On the other hand, tumor suppressor genes typically act by inhibiting cell proliferation, through suppression of cell cycle progression and/or apoptosis promotion. Thus, when these genes partial or totally lose their functions, the cell can progress to cancer, commonly in combination with other genetic alterations. In many tumors, these tumor suppressor genes are lost or inactivated, thereby removing inhibitors of cell proliferation, and contributing to the abnormal proliferation of tumor cells [4]. Human cells present diverse tumor suppressor genes, being the TP53, which encodes the p53 protein, the most studied. Mutations in this gene lead to the loss of wild-type (wt) p53 activity, frequently detected in many different types of cancer. On the other hand, perturbations in the p53 signaling pathways are believed to be required for the development of most cancers, and evidences suggest that the reactivation of p53 function will have significant therapeutic benefits (reviewed in [5]).

1.1. THE P53 FAMILY PROTEINS

The p53 protein was discovered in 1979 as a nuclear protein that formed a complex with the SV40 tumor-virus oncoprotein, the large T-antigen [6, 7]. At this time, p53 was considered an oncogene. Only in 1989, p53 was established as a tumor suppressor since it was initially discovered as a mutant form, and wt p53 actually prevented malignant transformation of rat embryo fibroblasts cells by oncogenes [8]. Over the years, further observations demonstrated that, in fact, p53 has a tumor preventive activity. Indeed, several in vitro studies showed that the activity of p53 was commonly lost due to gene mutations or deletions in numerous cancer cell lines and tumors (reviewed in [9]). Also, the TP53 gene knockout mice demonstrated a high susceptibility to a variety of cancers, at a very young age (reviewed in [9]). Likewise, in humans, germline TP53 mutations are also associated with an increased cancer susceptibility and reduced age of onset, a condition called Li- Fraumeni syndrome (reviewed in [9]). It was also shown that p53 protein acts as an inducible sequence-specific DNA-binding transcription factor [10], controlling the transcription of numerous genes involved in different cellular processes in order to prevent the replication of damaged DNA or the proliferation of genetically altered cells. Due to this fundamental role in maintenance of genomic integrity, p53 has been called “the guardian of the genome” [11]. Almost two decades after the discovery of p53, two related homologs, TP63 [12] and TP73 [13] (encoding p63 and p73 proteins, respectively), were described. These three members – p53, p63 and p73 – constitute a family of transcription factors that share a high level of structural similarity, wherein p63 and p73 can transactivate p53 target genes (reviewed in [14]). However, their functional redundancy is limited, since the primary role of each p53 family member, determined by transgenic knockout mice models, shows that each protein has its own unique roles (reviewed in [14]). For instance, p63 is critical for the correct development of ectodermal-derived tissues, and p63 germline mutations are associated with a subset of ectodermal dysplasia syndromes [15]. On the other hand, p73 is involved in the regulation of neural and immune systems and, to date, no syndromes associated with p73 germline mutations have been identified [16]. Contrary to TP53, mutations in TP63 and TP73 genes are rare. Still, evidences showed that both p63 and p73 exert a tumor suppression function in many human tumors, even though their roles are highly complex mainly due to the existence of several isoforms in a same cellular context [17].

1.1.1. STRUCTURAL FEATURES

The TP53, TP63, and TP73 genes are located in chromosomes 17p13.1, 3q27-29, and 1p36.2-3, respectively [16]. The p53, p63 and p73 proteins respectively encoded by these genes present a high degree of structural homology, sharing three major domains: a transactivation domain (TAD), a DNA-binding domain (DBD), and an oligomerization domain (OD) (Figure 1A). The highest homology is found in the DBD (63% identity between p53 and p73, and 60% identity between p53 and p63) (reviewed in [16]), which explains why the three proteins can bind to the same DNA sequences and transactivate the same promoters. Regarding the OD, important for protein tetramerization, the amino acid identity between p53 and p63/p73 is less conserved (about 38%). This may explain why p63 and p73 are unable to form hetero-oligomeric complexes with wt p53 (reviewed in [16]. On the other hand, the OD homology between p63 and p73 is approximately 60%, what explains why these proteins are able to form heterotetramers, although with less efficiency than homotetramers [18]. Using different promoters and/or alternative splicing, TP53, TP63 and TP73 give rise to multiple isoforms, some of which have completely different functions [16]. In fact, the proteins with an N-terminal acidic TAD, termed TA isoforms (TAp63 and TAp73), result from transcription initiated at the P1 promoter; the N-terminal truncated proteins lacking TAD, called ΔN isoforms (ΔNp63 and ΔNp73), arise from transcription initiated at the P2 promoter [19]. Relating to TP53, it includes a P1’ promoter that gives rise to proteins containing the full TAD, a P1 promoter that produces proteins lacking the first 40 amino acids residues (Δ40p53), and a P2 promoter whose activation gives rise to proteins lacking the first 133 amino acids (Δ133p53) [16] (Figure 1B). The alternative splicing at the 3´end of p53, p63 and p73 transcripts gives rise to distinct ΔN and TA isoforms: eight splice variants for p53 (α, β, γ, δ, ε, ζ, Δp53 and ΔE6), five for p63 (α, β, γ, δ and ε), and nine for p73 (α, β, γ, δ, ε, θ, ζ, η and η1). Thus, it is noteworthy that the combination of alternative initiation of translation, alternative splicing, and alternative promoter usage can significantly increase protein diversity [16] (Figure 1B). Unlike p53, p63α and p73α isoforms possess an additional protein-protein interaction region at their C-terminus. It combines the sterile alpha motif (SAM), and the transcription inhibition domain (TID), implicated in lipid-membrane binding and repression of transcription through intra- or intermolecular associations with TAD (Figure 1A) [16, 17].

Figure 1 – Structural organization of p53 family proteins. (A) Structure of p53 family proteins. p53, p63 and p73 proteins consist of an amino-terminal transactivation domain (TAD), a central DNA- binding domain (DBD), and a carboxy-terminal oligomerization domain (OD). α-Isoforms of p63 and p73 encode also an additional sterile alpha motif (SAM) and a transactivation inhibitory domain (TID). (B) Architecture of human TP53, TP63 and TP73 genes. Transcription of p53 family genes is controlled by two promoters, P1 and P2. TP53 possesses an additional promoter, P1’. C-terminal alternative mRNA splicing leads to the generation of different isoforms of each protein. Numbered boxes indicate exons. C-terminal splicing events for all p53 family members are indicated by dotted lines and Greek letter designation (Adapted from [16]).

1.1.2. BIOLOGICAL FUNCTIONS

The p53 family proteins have similar, yet non-overlapping functions. As previously addressed, although these proteins are quite similar in structural organization, subtle differences influence their ability to interact with other proteins (co-activators, co-repressors or other regulatory proteins), and to recognize different DNA sequences with the subsequent transactivation of specific target genes [20]. Therefore, the molecular flexibility conferred by the structural complexity among these proteins governs the functional diversity within the family [19]. At physiological conditions, p53 is maintained at low levels by continuous ubiquitination and subsequent degradation by the proteasome [21]. However, upon exposure to different stress signals, including DNA damage and oncogenic stress, p53 undergoes post-translational modifications that lead to its stabilization and activation [22]. Once activated, p53 accumulates in the nucleus, where it acts as a transcription factor, coordinating a program that eventually leads to cell cycle arrest, senescence or cell death, which greatly depends on the level of cellular compromise [22, 23]. However, the assortment of activities attributed to this protein is wider. In fact, distinct studies revealed that p53 is also involved in other important cellular processes, including cell metabolism, autophagy, DNA repair and in the antioxidant response to increased reactive oxygen species (ROS) levels (reviewed in [22]). As mentioned before, although both p63 and p73 share many functional properties with p53, cooperating with it in the regulation of tumorigenesis, these proteins have unique roles in differentiation and development of unstressed cells (reviewed in [19]). Full-length and truncated p63/p73 isoforms generally exhibit opposite biological functions, yet their activities may depend on the cellular context (reviewed in [19]). Similarly to p53, TAp63 and TAp73 are also tumor suppressors due to their p53-like ability to induce cell cycle arrest and apoptosis (Figure 2). This similarity can be explained, at least in part, by transactivation of an overlapping set of target genes (reviewed in [19, 24]). Indeed, although the identification of specific p63 and p73 targets, TAp63 and TAp73 were shown to interact with p53-response elements (RE) in reporter and gel-shift assays and in chromatin immunoprecipitation experiments (reviewed in [24]). In contrast, ΔNp63 and ΔNp73 isoforms are potent dominant-negative inhibitors of TA isoforms and of p53, therefore acting as oncoproteins (reviewed in [16, 24]). It has been proposed that this ΔN transdominance effect can occur by two non-mutually exclusive mechanisms: i) ΔN isoforms can compete with TA isoforms for their target gene promoters, preventing TA-mediated transcriptional activity; ii) ΔN isoforms inhibit TA isoforms through formation of transcriptionally inactive heterocomplexes [24-26].

Figure 2 – Schematic representation of the biological functions of p53 family proteins. p53, p63 and p73 are sequence specific transcription factors. When activated by different stress conditions, these proteins regulate the transcription of an assortment of genes involved in different cellular processes with an important role in tumorigenesis such as cell cycle arrest, apoptosis, senescence and DNA repair.

It has been shown that the ΔNp73 levels can be regulated by p53 and TAp73 through direct activation of the P2 promoter of p73 [25, 27, 28]. It was also demonstrated that p53 can induce ΔNp63 expression [29]. In this way, auto-regulatory feedback loops are normally created in order to control the TA/ΔN ratio, and consequently, the expression levels of p53 family target genes, which may determine cell fate [19]. Consequently, an imbalance between TA and ΔN isoforms may lead to tumor development [19]. Accumulating studies showed that patients with cancers overexpressing oncogenic ΔN isoforms present poor prognosis and higher chemotherapeutic failure rates (reviewed in [16]). Despite that, due to the existence of additional transactivation domains, ΔN isoforms may be also transcriptionally active [30-32], being involved in cell cycle and apoptosis regulation in a TA- independent manner (reviewed in [24]). Despite the involvement of p53 family proteins in a vast number of cellular processes, in the present thesis, a particular attention will be given to their involvement in cell cycle, DNA repair and apoptosis. In fact, the primary role of p53 family proteins as tumor suppressors is to block cell cycle progression until DNA damage is repaired, or to induce apoptosis or senescence to remove irreparably damaged or malignant cells (reviewed in [33]).

Cell cycle progression control and DNA repair

The cell cycle deregulation is a common feature of human tumor cells and results in unscheduled cell proliferation, associated with genomic and chromosomal instability [34]. As tumor suppressors, the p53 family proteins exert pivotal functions in cell cycle progression, since they regulate G1/S and G2/M cell cycle checkpoints [20]. Cell cycle consists of two consecutive periods, mainly characterized by DNA replication followed by its division into two separate daughter cells. Cell division occurs during the mitosis (M) phase, which is preceded by a preparative stage, or interphase, that includes Gap 1 (G1), Gap 2 (G2) and synthesis (S) phases (Figure 3). The transition between the cell cycle phases occurs in an orderly fashion, being regulated by different cellular proteins. In order to prevent the transmission of damaged genetic material to daughter cells, appropriate progression through each phase of cell cycle is monitored by a series of checkpoints (reviewed in [34]). These cell cycle checkpoints are regulated by a family of serine/threonine protein kinases, the cyclin-dependent kinases (Cdk), which are activated by direct binding to their corresponding regulatory cyclin subunits. Cdk levels are usually constant throughout the cell cycle, whereas cyclins levels oscillate in synchrony with the cell cycle. Different types of cyclins are produced during the progression through the various phases of the cycle. Thus, the formation of different cyclin-Cdk complexes occurs periodically and according to the point of cell cycle, which in turn set off different cell cycle events (reviewed in [34]). The entry into the G1 cell cycle phase is governed by the formation and activation of the cyclin D-Cdk4/6 complexes (Figure 3). The activation of these complexes leads to partial phosphorylation of retinoblastoma protein (pRb), which remains bound to the E2F transcription factor. During the late G1 phase, the expression of E-type cyclins leads to cyclin E-Cdk2 complex formation, with subsequent hyperphosphorylation of pRb and the E2F release. Free E2F is then able to instigate the expression of genes required for S- phase entry. Under stress conditions, G1 arrest is induced through the inhibition of cyclin- Cdk complexes by Cdk inhibitors, such as p21 and p57/Kip2 (reviewed in [34]) (Figure 3). Once activated upon different stress signals, all the three members of the p53 family induce the transcription of p21, which binds to cyclin D-Cdk4/6 and cyclin E-Cdk2 complexes, halting cell cycle progression and DNA replication, and allowing for DNA repair (reviewed in [20]) (Figure 3). Both TAp63 and TAp73 can also induce G1 cell cycle arrest through transcriptional up-regulation of p57/Kip2 [29]. p53 also promotes the up-regulation of other proteins that contribute to G1 arrest, namely the human cell division control protein 4b (hCDC4b) (Figure 3), a F-box protein and component of the Skp, Cullin, F-box (SCF)

ubiquitin ligase complex, that targets cyclin E for ubiquitin-mediated degradation (reviewed in [20]).

Figure 3 – General representation of the cell cycle regulation by p53 family proteins. p53 family proteins induce cell cycle arrest by inhibiting G1/S or G2/M transition, regulating the expression of several proteins. CAK: cdk-activating kinase; Cdk: cyclin-dependent kinase; GADD45: growth arrest and DNA-damage inducible protein; Rb: retinoblastoma protein; Cdc25c: cell division cycle 25 homolog C; hCDC4b: human cell division control protein 4b (Adapted from [20]).

The G2/M checkpoint centers on the activation of the cyclin B1-Cdk1 complex (Figure 3). The p53 family proteins also affect this checkpoint by transcriptionally modulating the expression of multiple targets such as the cell division cycle 25 homolog C (Cdc25c), the scaffold protein 14-3-3σ, p21 and growth arrest and DNA damage inducible protein (GADD45) (Figure 3). Concerning to p21, although being the major mediator of G1 arrest, it also participates in G2 arrest through association with the cyclin B1-Cdk1 complex, consequently inhibiting its phosphorylation by Cdk activating kinase (CAK). In turn, GADD45, the first p53 target gene to be identified, associates with Cdk1, preventing its binding to cyclin B1 and subsequently, inhibiting its kinase activity. Both p53 and p73 induce the expression of 14-3-3σ, which is involved in G2 arrest by physically separating the cyclin B1-Cdk1 complex from its target proteins (reviewed in [20, 34]) (Figure 3). Additionally, p53 and p73 can also induce G2/M arrest by repressing the Cdc25C, a phosphatase that

dephosphorylates Cdk1, leading to the cyclin B1-Cdk1 complex activation (Figure 3). It has also been reported that the proteins MCG10 (a RNA-binding protein), Reprimo (a highly glycosylated protein that is thought to play a role in cyclin B1-Cdk1 localization) and B99 (a protein with microtubule localization) are involved in G2 arrest, and their expression is induced by p53 (reviewed in [20, 22]) (Figure 3).

In addition to their multi-faceted roles in cell cycle, p53 family proteins are also engaged in DNA repair mechanisms, contributing to the maintenance of genomic integrity. Eukaryotic cells have several pathways for DNA repair that use unique enzymatic machinery, which is activated depending on the type of DNA damage [35]. In fact, the p53 family proteins can physically interact with or transcriptionally induce different DNA repair proteins. As referred above, these proteins can induce the expression of GADD45, which besides its functions in cell cycle arrest, is also involved in the nucleotide excision repair (NER) mechanism. p53 also interacts with several other factors that play a role in NER, namely transcription factor II human (TFIH, one of several general transcription factors that make up the RNA polymerase II pre-initiation complex) and the helicases XPB and XPD. p53 has been shown to interact with DNA polymerase β to stabilize the interaction between damaged DNA and base excision repair (BER) machinery [20, 22]. The intervention of p53 family proteins in DNA repair is a more complex process. However, herein, it was only intended to provide a brief overview about this issue, since it is not the focus of this thesis.

Apoptosis induction

Apoptosis is the most important form of cell death in multicellular organisms, given its significance in the control of cell proliferation and tissue homeostasis maintenance, as well as in the elimination of harmful or unnecessary cells [36]. This highly complex process is triggered by either internal or external signals, without boosting an immune system response and consequently, occurring without an associated inflammatory process [37]. Apoptotic cells present typical morphological alterations that include cell shrinkage, nuclear condensation and fragmentation, dynamic membrane blebbing, and loss of adhesion to extracellular matrices or to neighboring cells. Biochemical alterations include chromosomal DNA cleavage into internucleosomal fragments, phosphatidylserine externalization, and activation of a family of proteases, called caspases that cleave essential cellular components, leading to proteolytic dismantling of the cell (reviewed in [36, 38]). An aberrant function of any of the regulatory molecules involved in apoptosis, largely contributes to different pathological disorders, such as cancer. In fact, the evasion of apoptosis is a well-

established hallmark of this disease [3]. Therefore, the intensive study of apoptotic mechanisms, as well as of its interveners has enabled the development of effective drugs that, targeting specific apoptotic pathways or proteins, increase the sensitivity of cancer cells to apoptotic stimuli [36]. Because p53 family proteins act as stressor sensors and tumor suppressors, it is not surprising that these proteins have a role in apoptosis induction. In fact, the understanding of the mechanism of apoptosis regulation by p53 family proteins represents an excellent opportunity to discover new anti-cancer therapies. In mammals, there are two pathways by which a cell can initiate apoptosis: the extrinsic (or death receptor-mediated pathway) and the intrinsic (or mitochondria-mediated pathway) (reviewed in [36, 37]) (Figure 4). Mechanistically, the p53 family can induce apoptosis by transcriptional activation of critical regulators of both pathways (reviewed in [20]) (Figure 4). However, in case of p53, it can also induce apoptosis by directly interacting with specific proteins, influencing mitochondrial membrane physiology (reviewed in [22]). In order to understand the involvement of the p53 family in apoptosis, it is required to have a detailed knowledge of both pathways.

Figure 4 - Regulation of intrinsic and extrinsic apoptotic pathways by p53. p53 induces the expression of proteins involved in intrinsic and extrinsic apoptotic pathways. In the extrinsic pathway, the binding of ligands to specific trans-membrane death receptors leads to the recruitment of the adaptor molecule, FADD. Then, the procaspase-8 binds to FADD leading to DISC formation and resulting in caspase-8 activation. Activated caspase-8 directly activates executioner caspases-3, -6, and -7, or cleaves the pro-apoptotic protein Bid. The intrinsic/mitochondrial pathway involves several interactions between different anti- and pro-apoptotic members of the Bcl-2 family in order to induce MOMP and cyt c release. p53 induces the expression of proteins, such as Puma and Noxa, which

bind to and inhibit the activity of the anti-apoptotic proteins Bcl-2/Bcl-xL, allowing the translocation of cytosolic Bax to the mitochondria. This leads to mitochondrial permeabilization and to the release of cyt c from the mitochondria to the cytosol, resulting in the formation of the apoptosome and caspase- 9 activation. The activation of executioner caspases leads to cell death. The green star markers refer to p53 transcriptional targets. Apaf-1: apoptotic protease activating factor 1; Bax: Bcl-2 associated X protein; Bid: BH3 (Bcl-2 homology domain 3) interacting domain death agonist; FADD: Fas- associated protein with death domain; p53AIP: p53-regulated apoptosis inducing protein 1; Puma: p53-upregulated modulator of apoptosis (Adapted from [36]).

The extrinsic pathway begins with the attachment of extracellular ligands, such as tumor necrosis factor (TNF), Fas ligand (Fas-L), and TNF-related apoptosis-inducing ligand (TRAIL), to cell surface death receptors like the type 1 TNF receptor, Fas (also called CD95/Apo-1), and TRAIL receptors DR4 and DR5 (reviewed in [36, 37]) (Figure 4). The activation of these receptors leads to their trimerization and clustering of their intracellular death domain region, which binds to the corresponding protein motif of adaptor proteins such as Fas-associated death domain (FADD) and TNF receptor-associated death domain (TRADD). These adaptor proteins present other interaction domain, called the death effector domain (DED), that interacts with the DED of pro-caspase-8. This binding leads to the formation of the death-inducing signaling complex (DISC), and consequently to pro- caspase-8 activation. In turn, depending on the cell type, active caspase-8 activates the executioner pro-caspases-3, -6 and -7, leading to cell death by damage or destruction of important cellular structures, or promotes the generation of a mitochondria-permeabilizing fragment, called the truncated Bid (tBid) (reviewed in [36, 39]) (Figure 4). Death receptor- mediated apoptosis can be inhibited by a protein called c-FLIP, which will bind to FADD and caspase-8, rendering them ineffective [40, 41]). The intrinsic pathway can be activated by different stimuli that are sensed by several intracellular proteins that transmit signals to mitochondria, leading to mitochondrial outer membrane permeabilization (MOMP), loss of mitochondrial transmembrane potential

(Δψm), and to the release of pro-apoptotic proteins such as cytochrome c (cyt c), apoptosis- inducing factor (AIF), endonuclease G (EndoG), second mitochondria-derived activator of caspase/direct IAP-binding protein with low isoelectric point (Smac/DIABLO), and serine protease high-temperature requirement protein A2 (Omi/HtrA2) (reviewed in [37]) (Figure 4). Once released from mitochondria, cyt c interacts with the apoptosis protease-activating factor (Apaf-1) and pro-caspase-9, forming the apoptosome. The apoptosome formation leads to pro-caspase-9 activation that, in turn, activates pro-caspases-3, -6 and -7, triggering apoptosis (reviewed in [37, 38]) (Figure 4). Smac/DIABLO [42] and Omi/HtrA2 [43] were reported to promote apoptosis by inhibiting the activity of different inhibitors of apoptosis proteins (IAP) (Figure 4). On the contrary, AIF and EndoG can translocate to the nucleus, promoting cell death in a caspase-independent manner, through induction of

chromatin condensation and cleavage of nuclear DNA (reviewed in [36]). The regulation of this apoptotic pathway is mainly controlled by distinct proteins of the Bcl-2 family, whose main function is thought to be the regulation of cyt c release from mitochondria to the cytosol, via alteration of the mitochondrial membrane permeability (reviewed in [44]). This family is composed by pro-apoptotic (e.g., Bax, Bak, Bad, Puma, Noxa, Bid and Bim) and anti-apoptotic (e.g., Bcl-xL and Bcl-2) members (reviewed in [36]). Anti-apoptotic proteins, like Bcl-2 and Bcl-xL, may interact with mitochondrial proteins involved in mitochondrial pores formation, thereby protecting the mitochondrial membrane integrity, and inhibiting the release of cyt c (reviewed in [44]). On the other hand, the activation of the pro-apoptotic protein Bax leads to its translocation from the cytosol to the outer mitochondrial membrane, where it can homodimerize or heterodimerize with Bak or tBid, leading to the disruption of the mitochondrial membrane integrity and potentiating the release of cyt c [45] (Figure 4). Other pro-apoptotic proteins, as Puma and Noxa, exert their pro-apoptotic effects by binding to the anti-apoptotic proteins, blocking their activity (reviewed in [36, 39]). Also, the p53- apoptosis inducing protein 1 (p53AIP1) localizes to mitochondria, where it interacts with Bcl- 2 protein, leading to the release of cyt c and to the consequent apoptosis induction [46]. Therefore, the ratio between pro-apoptotic and anti-apoptotic proteins dictates whether or not cells are committed to die (reviewed in [37, 39]). Regarding to the regulation of the extrinsic pathway by p53 family proteins, p53, TAp73 and TAp63 were shown to up-regulate the expression of the cell death receptor Fas/CD95 [47-49]. TRAIL receptors, DR4 and DR5 [50-52], and caspase-8 [53] have been described as p53 transcriptional targets. TAp63 was shown to induce the expression of DR4, DR5 and TNF-R1 [48] and caspase-8, as well to regulate c-FLIP expression [54]. Concerning to the intrinsic pathway, the p53 family proteins upregulate pro-apoptotic proteins of the Bcl-2 family, namely Bax and Puma [55-57]. Both p53 and p73 induce the expression of Noxa and p53AIP1, with consequent apoptosis induction [46, 58, 59]. p53 was shown to repress the expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL [60], and to up-regulate Apaf-1, caspase-6 and Bid [61-63]. The mitochondrial chloride intracellular channel (mtCLIC/CLIC4), an organellular chloride channel protein that reduces

Δψm, was also shown to be transcriptionally regulated by p53 [64]. Besides its transcriptional function, evidences showed that p53 itself may localize to mitochondria, where it can form a complex with Bcl-xL and Bcl-2, leading to MOMP induction and cyt c release (reviewed in [65]). Also, cytosolic p53 can activate Bax and Bak through direct protein-protein interaction (reviewed in [66]). Similarly to p53, p73 also has transcription-independent pro-apoptotic functions [67]. In fact, it was demonstrated that the transcription-deficient p73 mutant p73R293H (corresponding to the hotspot mutant p53R273H) can still efficiently induce apoptosis in response to TRAIL, by a mechanism that

involves localization of p73 to mitochondria and interaction with mitochondrial p53 [68]. Remarkably, it was shown that p73 is targeted by caspases during apoptosis, and caspase- cleaved p73 fragments localize to mitochondria to up-regulate apoptosis [67].

ROS are generated as products of normal mitochondrial function, and increased ROS levels are caused by oxidative stress. The production of ROS has been associated with p53-induced apoptosis, since several authors showed an increase of the cellular ROS levels upon p53 overexpression (reviewed in [20]). The p53 family proteins commonly regulate a variety of proteins that participate in ROS-mediated apoptosis, such as ferrodoxin reductase and REDD1/HIF-1 (reviewed in [20]). On the other hand, p53 was shown to transactivate p53 inducible genes (PIGs) 3 and 6, which encode redox proteins and are implicated in ROS generation (reviewed in [69]). Other candidates were added to the list of p53-induced pro-oxidant genes, namely BAX, PUMA, and p66Shc. Additionally, p53 was shown to be involved in the suppression of antioxidant proteins, leading to increased cellular ROS production, namely manganese superoxide dismutase (MnSOD), PIG12, and aldehyde dehydrogenase (ALDH) 4 (reviewed in [69]).

1.2. ENDOGENOUS MODULATORS OF P53 FAMILY PROTEINS

The cellular processes in which the p53 family is involved are tightly regulated in order to maintain normal tissue homeostasis. Although the ability of p53, p63 and p73 to respond to cellular stress is necessary to prevent tumor growth, the deregulation of their cellular levels can lead to quite drastic cell death-dependent effects. In particular, excessive cell death can compromise the structure and function of tissues, which is a hallmark of aging, whereas deficient cell death can lead to neoplastic disorders [70]. The p53 family proteins are mainly regulated at post-translational level by several proteins, undergoing modifications (e.g. phosphorylation, acetylation and ubiquitination), which control their turnover and activity [17]. The two major negative modulators of p53 family proteins are the homologs murine double minute 2 (MDM2) and murine double minute X (MDMX, also known as MDM4) proteins (reviewed in [71]). In normal cells, MDM2 and MDMX suppress the activity of p53 family proteins. However, under stress conditions, an inhibition of MDMs allows that p53, p63 and p73 may respond to the damages (reviewed in [17, 71, 72]). As inhibitors of the p53 family activities, MDM2 and MDMX are oncogenic when overexpressed, exhibiting important roles in tumorigenesis and tumor progression [72]. In fact, the amplification or up-regulation of MDM genes is frequently found in many human tumors (reviewed in [73]).

1.2.1. MDM2 AND MDMX: PROTEIN STRUCTURE

MDM2 was discovered as one of the genes amplified on double minute chromosomes in transformed mouse NIH3T3 fibroblasts [74]. Later studies revealed that MDM2 was able to bind to p53, inhibiting its transcriptional activity [75, 76]. MDMX was later identified, through screening of a mouse cDNA expression library, as a novel protein able to interact with p53 [77]. The authors also demonstrated that like MDM2, MDMX co- immunoprecipitated with p53, and its overexpression inhibited the p53 transcriptional activity [77]. Human MDM2 and MDMX are proteins of 491 and 490 amino acids, respectively that share a high degree of structural similarity (Figure 5). Both proteins present a homologous p53-binding domain at the N-terminal that mediates their interaction with the p53 TAD and the inhibition of the p53 transcriptional activity (reviewed in [78]). Another well- conserved region is the RING-finger domain, located at C-terminal end of both proteins. Concerning to MDM2, this domain is important for homo-dimerization and hetero- dimerization with MDMX, via RING-RING interaction. Along with the zinc-finger and acidic domains, the RING-finger domain is essential for the MDM2 function as an E3 ubiquitin ligase (reviewed in [78]). The major difference between MDM proteins is that, contrary to MDMX, MDM2 has both nuclear localization and export sequences, which allow it to shuttle between the cytoplasm and the nucleus. The lack of such sequences indicates that MDMX is primarily localized in the cytoplasm and depends on other proteins, such as MDM2, for nuclear localization (reviewed in [72, 79]) (Figure 5).

Figure 5 – Schematic representation of MDM2 and MDMX domains. Both MDM2 and MDMX proteins comprises a p53-binding domain, an acidic and a zinc-finger domain in their central portion followed by a C-terminal RING-finger domain. Additionally, the MDM2 protein possesses a nuclear localization signal (NLS) and a nuclear export signal (NES) sequence (Adapted from [80]).

1.2.2. P53 FAMILY-RELATED BIOLOGICAL FUNCTIONS OF MDMS

Genetic studies have shown that both MDM2 and MDMX are two essential p53 negative regulators, since the absence of MDMX or MDM2 expression leads to mice embryonic lethality, which was rescued by the concomitant deletion of TP53 [81]. These studies also demonstrated that, despite their high homology, neither MDM2 nor MDMX can compensate for one another in vivo to inhibit p53, showing that both MDMs have non- redundant roles (reviewed in [82]). Regarding the regulation of p53 by MDM2, both proteins form an autoregulatory feedback loop [83, 84]. Once activated, p53 binds to the P2 promoter of MDM2 inducing its transcription. In turn, MDM2 binds to the N-terminal of p53, via its N-terminal hydrophobic region [85], and negatively regulates the p53 by three different mechanisms: i) MDM2 binds to the p53 TAD, thereby preventing its interactions with the basal transcription machinery and transcriptional co-activators, such as p300; ii) MDM2 induces p53 translocation from the nucleus to the cytoplasm, through its nuclear export signal domain, where p53 can no longer act as a transcription factor; iii) MDM2 functions as an E3 ubiquitin ligase leading to p53 ubiquitination and targeting it to degradation (reviewed in [86, 87]) (Figure 6). In fact, high levels of MDM2 favor the p53 poliubiquitination, leading to its proteasomal degradation. Therefore, this process is responsible for the maintenance of low levels of p53 in unstressed cells. On the other hand, the MDM2 RING-finger domain-mediated ubiquitination has also been shown to cause changes in p53 localization (reviewed in [88]). Low MDM2 levels favor monoubiquitination of p53, promoting its nuclear export and preventing its transcriptional activity. In addition, it can also trigger cytoplasmic p53 translocation to mitochondria, where it is deubiquitinated, causing a transcription-independent induction of apoptosis (reviewed in [72, 82, 88]). Similarly to MDM2, MDMX binds to the p53 TAD, inhibiting its transcriptional activity (reviewed in [77, 85]) (Figure 6). MDMX does not possess E3 ligase activity itself, however Lyappan et al (2010) showed that MDMX can be modified within its RING- and zinc-finger domains, giving to this protein a minimal E3 ubiquitin ligase activity [89]. Also, unlike MDM2, MDMX does not induce p53 nuclear export and degradation (reviewed in [72, 73]). More recently, studies showed that MDMX is also involved in the p53 stability and promotes the p53 mitochondrial apoptosis (reviewed in [90, 91]). In fact, under different forms of DNA damage, MDMX can be phosphorylated by protein kinases, and degraded by MDM2, or it can be phosphorylated and exported out of the nucleus, leading in both cases to p53 activation. It was also demonstrated that a fraction of MDMX can be located at mitochondria, where it promotes the mitochondrial localization of phosphorylated p53 and its binding to Bcl-2, with consequent cyt c release and apoptosis (reviewed in [91, 92]).

Still, several evidences point out to an intricate network between MDM2 and MDMX in p53 regulation [82, 93]. Some studies have predicted that the formation of MDMX-MDM2 heterodimers is structurally favored over the formation of homodimers of either protein. It has been shown that MDMX-MDM2 complexes stabilize MDM2 and enhance the ability of MDM2 to ubiquitinate p53 (Figure 6). In the meantime, disruption of the MDMX-MDM2 complex resulted in p53 activation in vivo (reviewed in [82]).

Figure 6 – Regulation of p53 by its endogenous negative modulators MDM2 and MDMX. Both MDM2 and MDMX bind to p53, modulating its transcriptional activity by preventing its interaction with the general transcription machinery. As a homodimer, MDM2 promotes p53 nuclear export to the cytoplasm for ubiquitin-mediated degradation by the proteasome. Additionally, MDM2 can form a heterodimeric complex with MDMX, promoting the degradation of p53 (polyubiquitination). Ub: ubiquitin (Adapted from [94]).

Given the considerable structural homology between the p53 family members, it is plausible that MDMs may also regulate p63 and p73 activities in a similar manner to that described for p53. The ability of MDM2 and MDMX to bind to p73 has been well documented, but the results obtained for the interaction of MDMs with p63 have been controversial (reviewed in [95]). According to Zdzalik et al (2010), the affinities of MDM2

and MDMX for p73 are of the same order of magnitude as those for p53 [96]. The same study also showed that both p63 and p73 interact more strongly with MDMX, suggesting that MDMX may have a stronger impact than MDM2 on the p63 and p73 regulation [96]. This study also supported the interaction between MDMs and p73, as previously suggested by other studies (reviewed in [95]). In fact, regarding to p73, MDMs were shown to bind to the p73 TAD, inhibiting its transcriptional activity. However, unlike what occurs with p53, MDM2 does not promote p73 ubiquitination [97-99]. Instead, MDM2 relocalizes p73 to subnuclear speckles, preventing its interaction with the acetyltransferase p300/CBP and RNA polymerase-associated factor and consequently, repressing the p73 transcriptional activity [98, 100]. Remarkably, MDM2 can also catalyze the addition of the small ubiquitin- like protein NEDD8 to p73, which seems to inhibit their transcriptional activity [101]. Contrariwise, Zdzalik et al (2010) also showed that the interactions between MDMs and p63 are weaker, which may explain the contradictory results reported by several groups [96]. In fact, by co-immunoprecipitation analysis, some groups reported the absence of interaction between p63 and MDM2/MDMX, in human tumor cells, as well as the absence of effect of both MDM proteins on p63 stability and transcriptional activity [99, 102]. Conversely, other studies were able to detect MDM-p63 complexes, although reporting contradictory effects on p63 function [103, 104]. In fact, Kadakia et al (2001), showed that the interaction between p63 and MDMs leads to the downregulation of the p63 transcriptional activity [103]. These authors also showed that MDM2, but not MDMX, induced p63 nuclear export, inhibiting the p63-dependent apoptosis. In turn, Calabró et al (2002) reported that MDM2 binds to p63, leading to an increase of its transcriptional activity [104]. In 2010, it was reported that MDM2 was also able to interact with ΔNp63, which lacks the TAD involved in the interaction with MDM2 [105]. In this work, it was also demonstrated that this interaction promotes the ΔNp63 nuclear export, but not its degradation. More recently, Stindt et al (2014) showed that MDM2 co-immunoprecipitated with both TAp73 and ΔNp73, and to a much lesser extent with TAp63 and ΔNp63. These differences reflected a stronger inhibition of TAp73 and ΔNp73 transcriptional activity by MDM2 when compared to that observed on TAp63 and ΔNp63 [106].

1.3. THE P53 FAMILY IN CANCER TARGETED THERAPY

Currently, around 22 million people with cancer present defects in p53 signaling (reviewed in [107]). Approximately 50% of these cases are tumors with mutations in the TP53 gene, which prevent the proper folding of the p53 protein or directly disrupt its binding to target genes as a functional tetramer. The remaining tumors retain a wt p53 form that

remains inactive due to enhanced degradation or inhibition, frequently caused by overexpression of the negative regulators MDM2 and MDMX, through gene amplification or mutation (reviewed in [82]). Given the critical roles in cancer pathogenesis, particularly in therapy resistance, the reactivation of the p53 function is a promising anti-cancer therapeutic strategy [22]. In tumors retaining the wt p53 inactivated by MDMs, the pharmacological strategies to activate p53 include the: i) repression of MDM2 and MDMX expression; ii) inhibition of the MDM2 E3 ubiquitin ligase activity; and iii) inhibition of the p53-MDM2/MDMX interactions (reviewed in [73, 108]). High levels of MDM2 are commonly found in human cancers, including sarcomas, gliomas, hematologic malignancies, melanomas and carcinomas, while gene amplification and consequent upregulation of MDMX was found in tumors, such as gliomas, soft tissue sarcoma, head and neck squamous carcinoma, retinoblastoma, melanoma, and breast cancer (reviewed in [71]). A possible strategy to reduce the expression of MDM proteins in cancer cells is to specifically target them with interfering RNA (RNAi), despite the delivery and cellular uptake limitations related to this kind of therapy (reviewed in [73]). To date, two small molecules that downregulate MDMX levels, causing a p53-dependent transactivation of pro-apoptotic genes in several cancer cell lines, were identified: the benzofuroxan derivative XI-006 (NSC207895) [109], and the pseudourea derivative XI-011 (NSC146109) [110]. Regarding the development of small molecules that inhibit the MDM2 ubiquitin ligase activity, it was shown that HLI98, a member of the pyrimido-dione-quinoline family, could bind to the MDM2 C-terminal, compromising its function, suppressing p53 degradation, and leading to a p53-dependent apoptosis in tumor cells [111]. Later, a more soluble derivative was identified, HLI373, with higher in vitro potency, although in vivo studies are still required [112]. Other authors identified the small molecule JNJ-26854165 that reactivates p53 by inhibiting MDM2 E3 ubiquitin ligase, and inhibits tumor growth in mouse xenograft models. JNJ-26854165 entered phase I clinical trials (reviewed in [71]). Additionally, two natural products that inhibit the MDM2 E3 ubiquitin ligase activity, the Lissoclinidine B [113] and Sempervirine [114], have been also identified. Among the different strategies to restore the p53 function in tumor cells, the most exhaustive research has been focused on the identification of small molecule inhibitors of the p53-MDM2/MDMX interactions. Targeting protein-protein interactions by small molecules is a challenging task, since it usually involves large and flat surfaces that are difficult to disturb by low molecular weight compounds [86, 107]. However, the knowledge about protein-protein interaction architecture provides a framework to design small molecules that mimic one of the proteins involved in that interaction [86].

In the case of the p53-MDM2 interaction, structural studies revealed that there is a hydrophobic cleft on MDM2 for p53 binding and only just three amino acid residues of p53 (Phe19, Trp23, and Leu26) are crucial for the interaction (reviewed in [107, 108, 115]). Based on this, during the last years, a huge number of small molecule inhibitors of the p53- MDM2 interaction, belonging to different chemical families, such as sulphonamides, quiloninoles, terphenyls, cis-imidazolines, benzodiazepinediones, spiro-oxindoles, pyrrolidine-2-ones and isoindolinones, has been described (reviewed in [115]). The first potent and selective small molecule inhibitors of the p53-MDM2 interaction, named nutlins, were identified by Vassilev et al in 2004. This group showed that this class of cis-imidazoline compounds were able to displace p53 from its complex with MDM2 [116]. Nutlins mimick the three critical amino acid residues of p53 and interact with MDM2 at the p53 binding site (reviewed in [117]). In vitro, nutlin-3a promoted p53 accumulation and activation, triggering the transcription of p53 target genes, cell cycle arrest in G1 and G2 phases, and apoptosis [116]. As expected, nultin-3a exhibited tumor suppression activity in cell lines with functional p53 rather than in cell lines with mutant or deleted p53 [116]. However, the modest in vivo activity of nutlin-3a encouraged its optimization, leading to RG7112, with higher potency and improved pharmacological properties [118]. This compound has already entered phase I clinical trial for the treatment of solid tumors, hematologic neoplasms and leukemia (reviewed in [73]). A structure-based screening of compounds, combined with molecular modeling, led to the identification of spiro-oxindole-based molecules as p53-MDM2 interaction inhibitors [119]. MI-219 is highly specific, with subnanomolar affinity to MDM2, good pharmacokinetic and pharmacodynamic properties. This compound has been shown to increase the levels of p53 and p53-target genes, such as cyclin-dependent kinase inhibitor 1A (CDKN1A, which encodes p21) and MDM2, inducing cell cycle arrest and apoptosis [120]. On the contrary, the furan derivative RITA (reactivation of p53 and induction of tumor cell apoptosis) has been proposed to inhibit the p53-MDM2 interaction, both in vitro and in vivo, by binding to the N-terminal domain of p53, instead of MDM2, inducing p53 accumulation and p53-dependent growth arrest and apoptosis in carcinoma cell lines [121]. However, NMR structural studies performed by Krajewski et al (2005) indicated that this compound did not directly target p53, suggesting that RITA can selectively target cells expressing p53 by other mechanisms, namely by interaction with other binding proteins and co-factors, or by stabilization of the complex against ubiquitin-driven proteolysis [122]. Therefore, more details are still required to better understand the molecular mechanism of action of RITA.

Given the considerable number of compounds described as inhibitors of the p53- MDM2 interaction, Table 1 covers the existing p53-MDM2 interaction inhibitors currently in clinical trials.

Table 1 – Small molecule inhibitors of the p53-MDM2 interaction currently in clinical trials (Adapted from [108]).

ClinicalTrials.gov Compound Study phase identifiers

Phase I trial in advanced solid tumors, NCT00559533 RG7112 (also known as solid tumors, haematological neoplasms NCT01164033 RO5045337) and liposarcomas (all completed) NCT00623870 NCT01143740 RG7112 with cytarabine Phase I in AML (completed) NCT01635296 Phase I in soft tissue sarcoma RG7112 with doxorubicin (completed) NCT01605526 Phase I in advanced malignancies RO5503781 (recruiting) NCT01462175 RO5503781 with cytarabine Phase I in AML (recruiting) NCT01773408 MI-773 (also known as Phase I in malignant neoplasms SAR405838) (recruiting) NCT01636479 Phase I in advanced solid tumor DS-3032b lymphoma (recruiting) NCT01877382 CGM097 Phase I in advanced solid tumors NCT01760525

AML – Acute myeloid leukaemia

Concerning to the p53-MDMX interaction, to date, a very low number of inhibitors have been identified. The p53-binding pockets of MDMX and MDM2 are structurally different, which justifies that MDM2 inhibitors, such as nutlins, have low affinity for the p53- binding pocket of MDMX (reviewed in [108]). Through X-ray crystallographic studies, Popowicz et al (2010) reported the first inhibitor of the p53-MDMX interaction, the small molecule WK298, which binds to MDMX in the p53 binding pocket [123]. However, the biological effects of this compound in tumor cells remain unavailable. Additionally, through a high-throughput screening of chemical libraries, SJ-172550 was identified as a lead compound that disrupts the p53-MDMX interaction [124]. This compound forms a reversible covalent complex with MDMX, and induces p53-dependent cytotoxic effects in MDMX- overexpressing tumor cells [124]. Since both MDMs interact with p53, negatively regulating its activity, the simultaneous inhibition of the p53-MDM2/MDMX interactions, for a full p53 reactivation, has emerged as a promising therapeutic strategy [82]. However, few small molecules with that function were identified so far. The pyrrolopyrimidine-based compound 3a was identified as

a dual inhibitor of the p53-MDM2/MDMX interactions, leading to the increase of p53 and p21 expression levels and to apoptosis in a p53-dependent manner [125]. The indolyl hydantoin RO-5963, an optimization of RO-2443, binds to the p53-binding pocket of MDM2 and MDMX by inducing the formation of dimeric protein complexes [126]. It has a potent inhibitory activity in vitro, leading to a p53-dependent cell cycle arrest and apoptosis in MDMX-overexpressing tumor cells [126]. Although nearly equipotent in vitro, RO-5963 was more effective in disrupting the p53-MDMX interaction than the p53-MDM2 interaction [126]. By fluorescence polarization (FP) and isothermal titration calorimetry (ITC) assays, Qin et al (2014) identified the cis-imidazoline derivative H109 as a dual small molecule inhibitor of MDM2/MDMX [127]. This compound can reactivate the p53 pathway in several cancer cell lines [127]. More recently, the (S)-tryptophanol derivative OXAZ-1 was identified from the screening of a small library of enantiopure tryptophanol-derived oxazolopiperidone lactams as a dual inhibitor of the p53-MDM2/MDMX interactions [128]. It was reported to induce a p53-dependent in vitro tumor growth-inhibitory effect, with p53 stabilization and subsequent up-regulation of p53 transcription targets, such as p21, Puma and Bax, and to trigger a p53- dependent mitochondria-mediated apoptosis [128]. As previously referred, both TP63 and TP73 are rarely mutated in human cancers, thus an appropriate regulation of the p63 and p73 pathways represents an important anticancer therapeutic challenge, particularly in p53-null or mutant p53-expressing tumor cells [17, 129]. In fact, all p53 family proteins function as molecular hubs of a highly interconnected signaling network. Thus, not only the p53 pathway, but the entire p53 family pathway is a primary target for anti-cancer drug development. Particularly, the activation of pro-apoptotic isoforms of p63 and p73 represents an undeniable promising anticancer strategy [129]. Regarding p73, the proper stimulation of TAp73 expression and function in cancer cells represents an appealing anticancer pharmacological approach. Different chemotherapeutic compounds are able to achieve this goal by different ways, including the: i) stimulation of TP73 transcription or inhibition of p73 degradation, leading to increased TAp73 levels; ii) modulation of the expression or function of TAp73 upstream regulators; and iii) displacement of p73 from inhibitory interactions with other cellular proteins, namely MDM2, MDMX, ΔNp73 and mutant p53 (reviewed in [129]). Nevertheless, considering the scope of this thesis, only the therapeutic strategies involving the disruption of the p73- MDM2 interaction will be discussed. Besides blocking the p53-MDM2 interaction in wt p53- expressing cells, some studies showed that nultin-3a was also able to exert a tumor growth inhibitory effect and to induce apoptosis in p53-null [130, 131] or mutant p53-expressing human tumor cells [132]. This p53-independent effect of nultin-3a was attributed to a possible p73 activation, through inhibition of its interaction with MDM2. In fact, since both

p53 and p73 bind to the same MDM2 hydrophobic pocket [97], it was predicted that nutlin- 3a could prevent the formation of p73-MDM2 complexes. Indeed, Lau et al (2008) confirmed that nultin-3a effectively displaces TAp73α from MDM2, in p53-null human colon adenocarcinoma cell line, leading to the increase of the p73 transcriptional activity with up- regulation of p73 targets, such as Noxa, Puma and p21, and to enhanced apoptosis [131]. Moreover, nutlin-3a increases doxorubicin-mediated cell death, in a p53-null neuroblastoma cell line LA155N, by the combined up-regulation of TAp73 and activation of the E2F1 transcription factor [130]. Based on these results, it was postulated that nutlin-3a acts by releasing the E2F1/p73 complex from the inhibitory MDM2 binding, while doxorubicin- mediated DNA damage promotes phosphorylation of E2F1 by Chk1/Chk2 kinases, thus stabilizing E2F1 and promoting E2F1-mediated TAp73 transcription and activation [130]. Concerning to p63, to date, no small molecule inhibitors of its interaction with MDMs have been described (reviewed in [133]).

1.4. YEAST AS A CELL MODEL TO STUDY P53 FAMILY PROTEINS

The yeast Saccharomyces cerevisiae has long been used as a valuable eukaryotic model. Actually, this cell system has greatly contributed to the elucidation of the molecular mechanisms underlying several human diseases, such as cancer [134, 135]. In fact, due to the high degree of conservation of cellular and molecular mechanisms between yeast and higher eukaryotes, S. cerevisiae has been used to study extremely complex signal transduction pathways, in which several cancer-related proteins are involved [136]. As a simpler eukaryotic organism, its genome was the first to be sequenced. It is well-known that at least 31% of the proteins encoded by the yeast genome have a human orthologue, and nearly 50% of human disease-related genes exhibit yeast orthologues (reviewed in [134, 137]). Additionally, it presents some other advantages as a cell model to study human disease-related proteins, such as: easy and low-cost manipulation, short generation time, and high compliance to genetic manipulation, given its well-defined genetic system [134]. Nevertheless, as an unicellular organism, the impossibility to study disease aspects like multicellularity and cell-cell interactions represent the main limitation of this model [136]. Despite this, yeast has proven to greatly contribute to the clarification of complex cellular processes. Additionally, it has been considered an efficient first-line tool for genetic and chemical screenings that has greatly advanced the pharmacotherapy of several human diseases, specially of cancer [134]. The study of cancer-related proteins in the yeast model can involve the adoption of different approaches, which are highly dependent on the degree of conservation of the

protein of interest [136]. If the human gene implicated is conserved in yeast, its function can be directly studied in this organism. However, if the gene has no orthologues in yeast, its study is still possible by heterologous expression. This “humanized yeast” model allows the study of a human protein in a simplified eukaryotic environment, without the interference of other proteins with similar or overlapping functions, or its regulators [136]. This justifies the attractiveness of this cell model for uncovering major aspects of p53 family proteins [136]. In fact, no orthologues of each member of the p53 family, or of any of their endogenous negative modulators, have been identified in yeast so far [138]. Several yeast-based methodologies have been developed for functional, molecular and pharmacological studies of p53 family proteins. In 2003, the technique of functional analysis of separated alleles in yeast (FASAY) was developed to understand the transcriptional status of p53 in cancer cells, as described in [139]. Some modified versions of the FASAY assay allowed many different studies, including the identification of tumor- derived p53 mutations, and the understanding how these mutations could interfere with the p53 function in human tumor cells [140]. This type of assays was also used to compare the transcriptional activity of p53 and p63 on CDKN1A, BAX and MDM2 response elements [141], and to analyze the ability of p53 mutants to inhibit the p73 transcriptional activity [142]. More recently, a FASAY-derived dual-luciferase functional assay, based on the analysis of the p53 transcriptional activity using the Firefly luciferase reporter gene, was developed [143]. This assay has been used in parallel with mammalian cell-based assays, contributing to the investigation of the impact of small molecules and interacting proteins, such as MDM2 and 53BP1, on wt and mutant p53 transactivational activity [143] (Figure 7A). In fact, the effectiveness of this assay was validated with the two aforementioned inhibitors of the p53-MDM2 interaction, nultin-3a [116] and RITA [121]. Later, similar yeast- based transactivation assays were developed for p63 and p73 [144]. Actually, using these assays, Monti et al (2014) demonstrated that both TAp63α and ΔNp63α were transcriptionally active in yeast exhibiting intrinsic differences in transactivation specificities [145]. The yeast two-hybrid (Y2H) assay is the most well-known and used genetic assay to study protein-protein interactions. This assay takes advantage of the properties of the transcriptional factor GAL4, which is composed by two domains, a DNA-binding domain (DBD) and a transactivation domain (AD), that can be separated and individually fused to the interest proteins [146]. This assay was crucial to determine the amino acids of MDM2 that are critical for binding to p53 [147] (Figure 7B). It was also decisive to reveal not only the interaction between the anti-apoptotic translationally controlled tumor protein (TCTP) and p53, but also the critical binding sites between them [148]. The Y2H also allowed the

study of interactions between ΔNp63 and wt/mutant p53 [149] and between TAp63 isoforms with other p53 family members [150]. Additionally, with the Y2H method, the analysis of the affinity between p63/p73 and MDMs became possible [150]. Yeast has been also widely used as a tool to study the role of p53 on cell growth and cell cycle. Nigro et al (1992) showed the induction of growth inhibition by human wt p53 expressed in S. cerevisiae, which was markedly increased by the co-expression of human cell cycle regulated protein kinase CDC2Hs [151]. Latter, it was also demonstrated that the expression of wt p53 in yeast induced a marked growth inhibition associated with S-phase cell cycle arrest [152]. With this toxic effect of p53 in yeast, the development of yeast phenotypic assays, based on simple measurements of cell growth, was possible [152] (Figure 7C). In subsequent studies, the conservation in yeast of the inhibitory effect of MDM2 and MDMX on the p53 activity was demonstrated. Leão et al (2013) showed that the co-expression of p53 with MDM2 and MDMX significantly reduces the p53-dependent growth inhibition and cell cycle arrest in yeast [153, 154]. Additionally, it was proposed that ACT1 is a putative endogenous p53 target gene, based on the fact that the p53-induced yeast growth inhibition and cell cycle arrest were associated with an increase of actin protein levels, which was modulated by natural and chemical regulators of the p53 activity [153, 154] (Figure 7C). These yeast growth inhibitory assays paved the way to the screening of genetic and chemical modulators of the p53 activity.

Figure 7 – Schematic representation of different yeast models frequently used to study p53. (A) Yeast-based p53 dual-luciferase transactivation assay. These assays can use yeast cells

expressing human wt/mutant p53 or co-expressing human wt p53 and a co-factor (e.g., MDM2 or 53BP1). The p53 transcriptional activity is assessed by quantification of the luciferase activity, which is directly proportional to the light units measured in a plate reader. (B) Yeast two-hybrid assay. In this assay, the coding sequence of protein X (Bait) is fused to the GAL4-DBD and the coding sequence of protein Y (Prey) is fused to the GAL4-AD. Upon co-expression, protein-protein interaction is detected because the reconstitution of the transcription factor leads to the expression of a reporter gene. (C) Yeast p53-MDMs phenotypic assay. The expression of wt p53 induces growth inhibition and cell cycle arrest, and increases the actin protein levels, effects that are abolished by both MDM2 and MDMX.

Once developed, these yeast phenotypic assays were used to search for new inhibitors of the p53 interaction with MDM2/MDMX. In fact, using these assays, new small molecule inhibitors of the p53-MDM2 interaction with a xanthone scaffold, such as pyroxanthone 1 [154], α-mangostin and gambogic acid [155], and more recently, with an oxazoloisoindolinone scaffold [156], were identified. In addition, using the same screening approach, the dual inhibitor of the p53 interaction with MDM2 and MDMX, the (S)- tryptophanol derivative OXAZ-1, was identified from a small library of enantiopure tryptophanol-derived oxazolopiperidone lactams [128]. More recently, the effects of human p63 and p73 on cell proliferation and death, as well as the interference of both MDMs on their activities, were also studied in yeast [157]. In this work, it was demonstrated that TAp63, ΔNp63 and TAp73-α isoforms induced a yeast growth inhibition associated with S-phase cell cycle arrest, and increased actin expression levels. Furthermore, MDM2 and MDMX inhibited the activity of all tested p53 family members in yeast, being their effects weaker on TAp63 activity, as observed in mammalian cells [103]. Moreover, nutlin-3a and SJ-172550 were identified as potential inhibitors of the p73 interaction with MDM2 and MDMX, respectively. Collectively, accumulating data have supported the use of the yeast model as an effective tool for biological and pharmacological studies of p53 family proteins.

1.5. AIMS OF RESEARCH

As exposed above, the p53 family proteins are key therapeutic targets in anticancer treatment. This justifies the amazing research performed over the last years around this family of proteins. Despite this, the high complexity of the p53 family pathway, particularly of the molecular mechanisms regulating the interplay between the distinct isoforms of this family, justifies that major issues about the pharmacology and biology of these proteins (especially of p63 and p73) remain unclear.

With the present thesis, we aimed to bring new advances into the pharmacology of p53 family proteins. Most importantly, we envisioned identifying new anticancer drug candidates that may represent new hopes and therapeutic opportunities in the treatment of cancer. To achieve such goals, it was intended to: i) Elucidate the molecular mechanism of action of LEM2, an optimized derivative of the pyroxanthone 1 previously identified as an inhibitor of the p53-MDM2 interaction [154]; ii) Validate, in human tumor cell lines and animal models, the molecular mechanism of action of a potential dual inhibitor of the p53-MDM2/MDMX interactions, previously identified in yeast p53-MDMs screening assays.

II Materials and Methods

2. MATERIALS AND METHODS

2.1. COMPOUNDS Nutlin-3a was obtained from Alexis Biochemicals (Grupo Taper, Sintra, Portugal). Doxorubicin, SJ-172550 were obtained from Sigma-Aldrich (Sintra, Portugal). The xanthone derivative 1-carbaldehyde-3,4-dimethoxy-9H-xanthen-9-one (LEM2) was synthesized, according to described procedures [154], by Prof. Emília Sousa (CIIMAR, FFUP). The enantiopure phenylalaninol-derived oxazolopyrrolidone lactam derivative JL-1 was synthesized, according to the described procedures [156], by Prof. Maria Santos (iMed.ULisboa, FFUL). All tested compounds were dissolved in dimethylsulfoxide (DMSO; Sigma-Aldrich; Sintra, Portugal).

2.2. YEAST CELLS

2.2.1. YEAST TARGETED SCREENING ASSAY Saccharomyces cerevisiae (strain CG379 Matα, ade5, his2, leu2-112, trp1-289, ura3-52; Kil-O) (Yeast Genetic Stock Center, University of California, USA) expressing human p53 or TAp73α alone and combined with human MDM2 were obtained in previous works [157]. For expression of human proteins, cells were diluted to 0.05 optical density at

600 nm (OD600) in induction selective medium with 2% (w/w) galactose (Sigma-Aldrich, Sintra, Portugal), 1% (w/w) raffinose (Acros Organics; JMGS, Odivelas, Portugal), 0.7% (w/w) yeast nitrogen base without amino acids from Difco (Quilaban, Sintra, Portugal), and all the amino acids required for yeast growth (50 mg/mL) except leucine and tryptophan. Yeast cells were incubated at 30 ºC, under continuous orbital shaking (200 r.p.m.). Co- transformed yeast cells were incubated in selective induction medium in the presence of 0.1 - 50 μM nutlin-3a (positive control), 1-100 μM xanthone derivative LEM2, or 0.1% DMSO only, until 0.4 OD600 achieved with control yeast (transformed with the empty vectors incubated with DMSO only). Yeast growth was analyzed by counting the number of colony- forming units (CFU) per mL (CFU/mL) after 2 days incubation at 30 ˚C on Sabouraud Dextrose Agar from Liofilchem (Frilabo, Porto, Portugal).

2.2.2. YEAST CELL CYCLE ANALYSIS Flow cytometric analysis of DNA content was performed using Sytox Green Nucleic Acid, as described in [152]. Briefly, 1 x 107 cells were fixed in 70% (v/v) ethanol, incubated

with 250 μg/mL RNase A (Sigma-Aldrich, Sintra, Portugal) and 1 mg/mL proteinase K (Sigma-Aldrich, Sintra, Portugal), and further incubated with 10 μM Sytox Green Nucleic Acid from Invitrogen (Alfagene, Carcavelos, Portugal).

2.3. HUMAN TUMOR CELL LINES

2.3.1. HUMAN TUMOR CELL LINES AND GROWTH CONDITIONS The human colon adenocarcinoma HCT116 cell line harboring wt p53 (HCT116 p53+/+) and its isogenic derivative, in which p53 has been knocked out (HCT116 p53-/-), the human breast adenocarcinoma MCF-7, and the osteosarcoma SJSA-1 cell lines were kindly provided by Dr. A. Inga (University of Trento, Italy). Cell lines were routinely cultured in RPMI with ultraglutamine medium from Lonza (VWR, Carnaxide, Portugal) supplemented with 10% fetal bovine serum from Gibco (Alfagene, Carcavelos, Portugal), and maintained in a humidified incubator at 37 °C with 5% CO2 in air.

2.3.2. SULFORHODAMINE B (SRB) ASSAY For the analysis of the effect of compounds on the in vitro growth of human tumor cells, 5 x 103 cells/well were plated in 96-well plates and incubated for 24 h. Cells were then exposed to serial dilutions of compounds (from 0.12 to 50 μM). The effect of compounds was analyzed following 48 h incubation, using the SRB assay. Briefly, following fixation with 10% trichloroacetic acid (Sigma-Aldrich, Sintra, Portugal), plates were stained with 0.4% SRB (Sigma-Aldrich, Sintra, Portugal) and washed with 1% acetic acid (Sigma-Aldrich, Sintra, Portugal). The bound dye was then solubilized with 10 mL Tris-Base and the absorbance was measured at 510 nm in a microplate reader (Biotek Instruments Inc., Synergy MX, USA). The solvent of compounds (DMSO) corresponding to the maximum concentration used in these assays (0.25%) was included as control. The concentration of compound that causes a 50% reduction in the net protein increase in cells (GI50, growth inhibition of 50%) was determined for all tested compounds.

2.3.3. ANALYSIS OF CELL CYCLE AND APOPTOSIS Human tumor cells were plated in 6-well plates at a final density of 1.5 x 105 cells/well and incubated for 24 h. Cells were then treated with the GI50 and/or with twice the GI50

(2xGI50) concentration of compounds, or DMSO only for 24 or 48 h. For cell cycle analysis, cells were fixed in ice-cold 70% ethanol and incubated at 37 ºC with RNase A (Sigma- Aldrich, Sintra, Portugal) at a final concentration of 20 μg/mL for 15 min, and further

incubated with 50 μg/mL propidium iodide (PI) from Molecular Probes (Alfagene, Carcavelos, Portugal) for 30 min, followed by flow cytometric analysis. For apoptosis analysis, cells were analyzed by flow cytometry using the Annexin V-FITC Apoptosis Detection Kit I from BD Biosciences (Enzifarma, Porto, Portugal), according to the manufacturer’s instructions. Briefly, human tumor cells were suspended in binding buffer and then incubated with 5 μL of FITC-Annexin V and 5 μL of PI for 15 min at room temperature. Cells were then analyzed by flow cytometry.

2.3.4. WESTERN BLOT ANALYSIS To prepare the whole protein extracts from human tumor cells, cells were lysed with RIPA buffer containing EDTA-free protease inhibitor cocktail from Sigma-Aldrich (Sintra, Portugal). For mitochondrial and cytosolic fractions of human tumor cells, the Mitochondrial Fractionation Kit from Active Motif (Frilabo, Porto, Portugal) was used according to the manufacturer’s instructions. Whole protein extracts were quantified using the Coomassie staining Bradford from Sigma-Aldrich (Sintra, Portugal). Proteins (40 μg) were electrophoresed using a 10% SDS- PAGE and transferred to a Whatman nitrocellulose membrane from Protan (VWR, Carnaxide, Portugal). Membranes were blocked with 5% milk and probed with a mouse monoclonal anti-p53 (DO-1), anti-MDM2 (D-12), anti-Bax (2D2), anti-PUMA (B-6), anti- PARP (C2-10), and anti-cytochrome c (A-8) followed by an anti-mouse horseradish- peroxidase (HRP)-conjugated secondary antibody. For p73, p21 and MDMX detection, membranes were probed with a rabbit polyclonal anti-p73 (AB7824), anti-p21 (C-19) and anti-MDMX (A300-287A), respectively, followed by an anti-rabbit HRP-conjugated secondary antibody. For loading control, membranes were stripped and reprobed with a mouse monoclonal anti-GAPDH (6C5). For analysis of mitochondrial and cytosolic fractions, membranes were reprobed with the loading controls mouse monoclonal anti-GAPDH (6C5) or anti-Cox4 (F-8), used to exclude putative contamination of cytosolic and mitochondrial fractions, respectively. All antibodies were purchased from Santa Cruz Biotechnology (Frilabo, Porto, Portugal), except the anti-MDMX obtained from Bethyl Laboratories (bioNova cientifica, Madrid, Spain), and anti-p73 obtained from Millipore (Grupo Taper, Sintra, Portugal). The signal was detected with the ECL Amersham kit from GE Healthcare (VWR, Carnaxide, Portugal) and with the Kodak GBX developer and fixer from Sigma- Aldrich (Sintra, Portugal).

2.3.5. CO-IMMUNOPRECIPITATION (CO-IP) ASSAY The co-IP assay was performed using the Pierce Classic Magnetic IP and Co-IP Kit from Thermo scientific (Dagma, Carcavelos, Portugal), according to the manufacturer’s instructions. Briefly, cells were plated at the final density of 5 x 105 cells/plate and incubated for 24 h. Cells were then treated with 2.88 and 3.60 μM LEM2, 7 and 14 μM JL-1 or DMSO only for 24 h. After cell lysis and protein lysate separation, 500 μg of total protein was incubated with a rabbit polyclonal anti-p73 from Millipore (Grupo Taper, Sintra, Portugal), with a mouse monoclonal anti-p53 (DO-1), or with a mouse immunoglobulin G (IgG, control) from Santa Cruz Biotechnology (Frilabo, Porto, Portugal), for 12 h at 4 ˚C. Immunocomplexes were precipitated using magnetic beads. Detection of p53, p73, MDM2, MDMX, and GAPDH (loading control) in whole cell lysate (input) and immunoprecipitated fraction was performed by Western blot analysis as described in section 2.3.4.

2.3.6. ANALYSIS OF MITOCHONDRIAL MEMBRANE POTENTIAL (ΔΨM)

Analysis of Δψm was performed by flow cytometry. Tumor cells were plated in 6-well plates at a final density of 1.5 x 105 cells/well. After 24 h incubation, HCT116 p53-/- cells were treated with the GI50 concentration of LEM2 or DMSO only for 8 h. Cells were harvested and incubated with 1 nM 3,3'-Dihexyloxacarbocyanine Iodide, DiOC6(3), from Alfagene (Molecular probes, Carcavelos, Portugal), for 30 min at 37 °C; cells treated with 50 μM carbonilcyanide p-triflouromethoxyphenylhydrazone (FCCP; Sigma-Aldrich, Sintra,

Portugal), for 15 min at 37 ºC, were used as positive control of dissipation of Δψm.

2.3.7. ANALYSIS OF REACTIVE OXYGEN SPECIES (ROS) GENERATION Analysis of intracellular ROS generation was performed by flow cytometry. Briefly, tumor cells were plated in 6-well plates at a final density of 1.5 x 105 cells/well and incubated for 24 h. Cells were treated with the GI50 concentration of LEM2 or DMSO only for 48 h.

Cells were harvested and stained with 10 μM H2DCFDA from Life Technologies (Alfagene, Carcavelos, Portugal), for 30 min at 37 ºC.

2.3.8. GENOTOXICITY STUDIES BY MICRONUCLEUS ASSAY Genotoxicity was analyzed by cytokinesis-blocked micronucleus assay in lymphocytes as described in [158]. Fresh peripheral blood samples were collected from healthy volunteers into heparinized vacutainers. Blood samples, suspended in RPMI medium supplemented with 10% FBS, were treated with the GI50, 2xGI50 and 3xGI50 concentrations of JL-1, LEM2, DMSO only or 1μg/mL cyclophosphamide (known mutagenic

agent; positive control; Sigma-Aldrich, Sintra, Portugal) for 44 h. Cells were thereafter treated with 3 μg/mL cytochalasin B (cytokinesis preventive; Sigma-Aldrich, Sintra, Portugal) for 28 h. Lymphocytes were isolated by density gradient separation (Histopaque- 1077 and -1119; Sigma-Aldrich, Sintra, Portugal), fixed in 3:1 methanol/glacial acetic acid, and stained with Wright stain (Sigma-Aldrich, Sintra, Portugal). For each sample, 1000 binucleated lymphocytes were blindly scored using a Leica light optical microscope (Wetzlar); the number of micronuclei per 1000 binucleated lymphocytes was recorded.

2.3.9. IN VITRO MIGRATION ASSAYS The impact of JL-1 on migration of HCT116 p53+/+ cell lines was studied performing an in vitro migration assay, commonly used as a parameter of in vivo tumor cell invasiveness: the Wound Healing Scratch assay [159]. In this assay, about 5 x 105 cells/well were grown to confluence in 6-well plates for 24 hours. Using a sterile 200 μL tip, a fixed- width wound was created in the cell monolayer and the GI10 concentration of JL-1 (or DMSO only) was added to medium. Cells were thereafter photographed at different time-points of treatment, using the Microscope AE 2000 from Motic, and the Digital Camera Motican 5MP, until complete closure of the wound.

2.3.10. IN VIVO EXPERIMENTS Xenograft tumor assays were performed with human HCT116 p53+/+ and HCT116 p53-/- tumor cell lines in BALB/c Nude mice purchased from Charles-River Laboratories (Barcelona, Spain) and housed under pathogen free conditions in individual ventilated cages. Briefly, 1 x 106 HCT116 cells (in PBS) were inoculated subcutaneously in the dorsal flank of each mice. Tumor dimensions were assessed by caliper measurement and their volumes were calculated (tumor volume = (length  width2) / 2). Treatment was started when tumors reached volumes of approximately 100 mm3 (which occurred 14 days after the grafts). Mice were then treated twice a week with 50 mg/kg JL-1 or vehicle (control) by intraperitoneal injection during two weeks. Tumor volumes and body weights were monitored twice a week until the end of the treatment. Animals were sacrificed by cervical dislocation at the end of the study or when tumors reached 1500 mm3 or the animals present any signs of morbidity. After sacrifice, tumors were removed and photographed. The following number of animals was used: HCT116 p53+/+ and HCT116 p53-/- cell lines (Control- 6, Treatment-6).

2.4. YEAST AND HUMAN TUMOR CELL LINES

2.4.1. FLOW CYTOMETRIC DATA ACQUISITION AND ANALYSIS For the flow cytometric analysis the AccuriTM C6 flow cytometer from BD Biosciences (Enzifarma, Porto, Portugal) and the CellQuest software from BD Biosciences (Enzifarma, Porto, Portugal) were used. Cell cycle phases were identified and quantified using ModFit LT software (Verity Software House Inc., Topsham, USA).

2.4.2. STATISTICAL ANALYSIS Data were analyzed statistically using the GraphPad software. Differences between means were tested for significance using the Student’s t-test (*p < 0.05; **p < 0.01; ***p < 0.001).

III LEM2: a new small molecule inhibitor of the p73-MDM2

interaction

3. LEM2: A NEW SMALL MOLECULE INHIBITOR OF THE P73- MDM2 INTERACTION

Xanthones are phenolic compounds well-known for their multiple functions, including anticancer activities (reviewed in [160]). In the last years, several molecular modifications in the tricyclic xanthone scaffold have led to compounds with increased in vitro growth inhibitory activity against a wide range of tumor cells (reviewed in [160, 161]), particularly with wt p53 and overexpressed MDM2 [162, 163]. Additionally, the anti-tumor activity of two natural xanthones, such as gambogic acid and α-mangostin, has been related to the activation of a p53-dependent pathway in several works [164-166]. More recently, a work performed by our group led to the identification of pyranoxanthone 1 (LEM1; 3,4-dihydro-12-hydroxy-2,2-dimethyl-2H,6H-pyrano(3,2- b)xanthen-6-one) (Figure 8) as the first inhibitor of the p53-MDM2 interaction with a xanthone scaffold [154]. In that work, using a yeast p53-MDM2 interaction screening assay, the activity of a library of xanthone derivatives on the p53-MDM2 interaction was investigated. Among the tested xanthones, LEM1 significantly reduced the MDM2 inhibitory effect on the p53 activity. The effect of this compound was also analyzed in wt p53-expressing human tumor cell lines, as breast adenocarcinoma MCF-7 and colon adenocarcinoma HCT116 cell lines. In accordance with what was observed in yeast, LEM1 mimicked the activity of the known small molecule inhibitor of the p53-MDM2 interaction, nutlin-3a, in human tumor cells, leading to the successful activation of p53 and downstream cell signaling [154]. In the present thesis, the molecular mechanism of action of LEM2, a LEM1 derivative, was investigated in order to obtain an optimized inhibitor of the p53-MDM2 interaction. Despite this initial aim, the results obtained showed that LEM2 was not an inhibitor of the p53-MDM2 interaction, but instead it was an inhibitor of the p73-MDM2 interaction.

Figure 8 – Chemical structure of 3,4-dihydro-12-hydroxy-2,2-dimethyl-2H,6H-pyrano(3,2- b)xanthen-6-one (LEM1).

3.1. RESULTS

3.1.1. LEM2 DOES NOT INTERFERE WITH THE P53-MDM2 INTERACTION

The effect of LEM2 as inhibitor of the p53-MDM2 interaction was analyzed using a previously developed yeast assay to search for inhibitors of this interaction [154]. These yeast assays are based on the fact that the human wt p53 induces a marked growth inhibition, associated with S-phase cell cycle arrest, in S. cerevisiae, which is abolished by human MDM2. As in mammalian cells, inhibitors of the p53-MDM2 interaction reduce the impact of MDM2 on the p53 activity, leading to the restoration of the p53-induced yeast growth inhibition [152]. The efficiency of this yeast assay was validated through the recent identification of LEM1 as an inhibitor of the p53-MDM2 interaction [154]. Using this approach, the effect of 1-100 μM LEM2 was compared to that obtained with the positive control, nutlin-3a. For the tested concentration range, LEM2 did not reduce the negative effect of MDM2 on the p53-induced growth inhibition (data not shown). Despite the results obtained in yeast, the tumor cell growth inhibitory effect of LEM2, and the involvement of the p53 pathway in its activity, was analyzed. For that, HCT116 cell lines with wt p53 (HCT116 p53+/+) and its p53-null isogenic derivative

-/- +/+ -/- (HCT116 p53 ) were used. The GI50 values obtained for LEM2 in p53 and p53 HCT116 cells (Table 2) indicated that this small molecule strongly inhibited the growth of both tumor cell lines. Additionally, contrary to nutlin-3a, no significant differences in the growth inhibitory effect of LEM2 were observed between p53+/+ and p53-/- HCT116 cells (Table 2). This result indicated that the p53 pathway was not involved in the antitumor activity of LEM2.

Table 2 – GI50 values obtained for nultin-3a and LEM2 in human colon adenocarcinoma HCT116 tumor cell lines.

a GI50 (μM) Compounds HCT116 p53+/+ HCT116 p53-/-

Nutlin-3a 3.67 ± 0.34 24.0 ± 0.7*

LEM2 0.64 ± 0.01 0.72 ± 0.02

a The GI50 was determined for all tested compounds after 48 h incubation, using the SRB assay. Cells were exposed to serial dilution of nutlin-3a (positive control; from 0.62 to 50 μM) or LEM2 (from 0.12 to 10 μM). Data are mean ± S.E.M. of three independent experiments. Values significantly different from HCT116 p53+/+ (*p < 0.05).

In order to confirm the results obtained in yeast, the ability of LEM2 to block the p53-MDM2 interaction was assessed using the co-IP assay, in HCT116 p53+/+ tumor cells. It was shown that 2.56 and 3.20 μM LEM2 (corresponding to 4xGI50 and 5xGI50 concentrations, respectively) did not interfere with the amount of MDM2 co- immunoprecipitated with p53 (Figure 9). This result supported the absence of effect of LEM2 on the p53-MDM2 interaction.

Figure 9 – LEM2 did not disrupt the p53-MDM2 interaction in HCT116 p53+/+ cells. HCT116 p53+/+ cells were treated with 2.56 and 3.20 µM LEM2 or DMSO only for 24 h. HCT116 p53+/+ cells lysates were immunoprecipitated (IP) with p53 or mouse immunoglobulin G (IgG) antibodies, followed by immunoblotting with MDM2 and p53 antibodies; whole cell lysate (input); immunoblots are representative of two independent experiments; GAPDH was used as loading control.

3.1.2. IDENTIFICATION OF LEM2 AS A POTENTIAL INHIBITOR OF THE P73-MDM2

INTERACTION USING THE YEAST SCREENING ASSAY

Since HCT116 p53-/- tumor cells are also p63-null cells [131] (also confirmed by Western blot analysis; data not shown), it was hypothesized if LEM2 could interfere with the p73 pathway. To address this issue, a yeast-based assay previously developed to search for inhibitors of the p73-MDM2 interaction [157] was used. Like for p53, in this assay, although the expression of human MDM2 does not interfere with the yeast cell growth and cell cycle progression, its co-expression with p73 significantly reduces the p73-induced yeast growth inhibition (Figure 10A and 10B) and cell cycle arrest (Figure 10C). The efficiency of this yeast assay was validated by testing nutlin-3a (a known inhibitor of the p73-MDM2 interaction) [157]. Using this approach, the effect of 0.1 – 50 μM LEM2 on the reversion of p73-induced yeast growth inhibition by MDM2 was evaluated. From the concentration-response curves obtained, it was shown that LEM2 presented similar effects to nutlin-3a with reversion of the MDM2-inhibitory effect (Figure 10A). Similarly to nultin-3a, when yeast cells co-expressing p73 and MDM2 were treated

with 10 μM LEM2 for 40 h, approximately 98% and 72% of the p73-induced growth inhibition (Figure 10B) and S-phase cell cycle arrest (Figure 10C), respectively, were reestablished without interfering with the activity of p73 or MDM2 when expressed alone. These results showed that LEM2 was able to inhibit the negative effect of MDM2 on p73, behaving as a potential inhibitor of the p73-MDM2 interaction in yeast.

Figure 10 – LEM2 reduces the inhibitory effect of MDM2 on p73 activity in yeast. (A) Effect of 0.1 – 50 μM LEM2 and nutlin-3a on the reversion of p73-induced yeast growth inhibition by MDM2, after 42 h incubation. The growth of yeast cells co-expressing p73 alone and MDM2 was evaluated by CFU counts; results were plotted setting as 100% the growth achieved with yeast cells expressing p73 alone with DMSO only; data are mean ± S.E.M. of five independent experiments; values significantly different from DMSO only are indicated (***p < 0.001). (B) Effects of 10 μM nutlin-3a and 10 μM LEM2 on the growth of control yeast, yeast expressing only p73 or MDM2, and yeast co-expressing p73 and MDM2, after 40 h incubation. Results were estimated considering 100% growth the number of CFU obtained with control yeast (empty

vectors). Data are mean ± S.E.M. of five independent experiments; values obtained with yeast co-expressing p73 and MDM2 treated with the compound significantly different from DMSO only are indicated (*p < 0.05). (C) Effects of 10 μM nutlin-3a and 10 μM LEM2 on the yeast cell cycle progression of control yeast, yeast expressing only p73 or MDM2, and yeast co-expressing p73 and MDM2, after 40 h incubation. Yeast cell cycle phases were quantified by flow cytometry; data are mean ± S.E.M. of three independent experiments; values significantly different from DMSO only are indicated (*p < 0.05).

3.1.3. LEM2 ACTIVATES THE P73 PATHWAY IN HUMAN TUMOR CELLS THROUGH

INHIBITION OF THE P73-MDM2 INTERACTION

The molecular mechanism of action of LEM2, as a potential inhibitor of the p73- MDM2 interaction, was thereafter ascertained in the p53- and p63-null HCT116 cells.

The results obtained showed that the growth inhibitory effect of LEM2, at GI50 concentration, was associated with the induction of G2/M-phase cell cycle arrest (Figure 11A) and apoptosis (Figure 11B), with PARP cleavage (Figure 11C).

Figure 11 – LEM2 induces cell cycle arrest, apoptosis and PARP cleavage in HCT116 p53- /- cells. (A) LEM2-induced cell cycle arrest was determined after 24 h treatment with 0.72 µM LEM2. Cell cycle phases were analyzed by flow cytometry using PI staining; data are mean ± S.E.M. of three independent experiments; values significantly different from DMSO are indicated

(*p < 0.05). (B) LEM2-induced apoptosis was determined after 48 h treatment with 0.72 µM LEM2. Apoptosis was analyzed by flow cytometry using FITC-Annexin V and PI; data are mean ± S.E.M. of three independent experiments; values significantly different from DMSO are indicated (*p < 0.05). (C) LEM2-induced PARP cleavage was analyzed by Western blot after 48 h treatments with 0.72 µM LEM2 or DMSO only; immunoblots are representative of two independent experiments; GAPDH was used as loading control.

Additionally, it was observed that 0.72 μM LEM2 led to ROS generation (Figure

-/- 12A) and ∆ψm dissipation (Figure 12B) in HCT116 p53 cells.

-/- Figure 12 – LEM2 induces ROS generation and ∆ψm dissipation in HCT116 p53 cells. (A) LEM2-induced ROS generation was determined after 48 h treatment with 0.72 µM LEM2. (B) LEM2-induced ∆ψm dissipation was determined after 8 h treatment with 0.72 µM LEM2. In (A) and (B), for the assessment of ROS generation and ∆ψm, cells were stained with H2DCFDA and DIOC6(3), respectively, and analyzed by flow cytometry; FCCP (C+) was used as positive control; histograms are representative of two independent experiments; M2 cursor indicates the subpopulation analyzed; Δ values correspond to the increase in the percentage of H2DCFDA positive cells (A) and of cells with ∆ψm dissipation (B) after treatment with LEM2.

To assess the activation of the p73 pathway by LEM2, the protein expression levels of p73 and of its target genes, such as MDM2, p21 and Bax, were checked by Western blot analysis in HCT116 p53-/- cells. The results obtained showed that 0.72 μM LEM2 increased the expression levels of the p73, MDM2, p21 and Bax (Figure 13A). Together, these results supported the reactivation of the p73 pathway in tumor cells.

Since LEM2 led to ∆ψm dissipation and increased Bax production, it was hypothesized if this compound could induce a p73-dependent mitochondria-mediated apoptotic pathway. For that purpose typical events of a mitochondrial apoptotic pathway, such as Bax translocation to mitochondria and cyt c release from the mitochondria to

cytosol, were checked in p53-null tumor cells treated with 0.72 μM LEM2. However, LEM2 did not trigger any of these events (data not shown). The ability of LEM2 to block the p73 interaction with MDM2 was thereafter demonstrated by co-IP assay, in HCT116 p53-/- cells. In fact, at 3.60 μM (corresponding to 5xGI50 concentration), a visible decrease of the amount of MDM2 co- immunoprecipitated with p73 was observed (Figure 13B). These results confirmed that LEM2 was an inhibitor of the p73-MDM2 interaction.

Figure 13 – LEM2 increases the expression levels of p73 and up-regulates p73 target genes by blocking the p73 interaction with MDM2 in HCT116 p53-/- cells. (A) Western blot analysis was performed after 24 h treatment with 0.72 µM LEM2 or DMSO only. Immunoblots are representative of two independent experiments; GAPDH was used as loading control. (B) HCT116 p53-/- cells were treated with 2.88 and 3.60 µM LEM2 or DMSO only for 24 h. HCT116 p53-/- cells lysates were immunoprecipitated (IP) with p73 or rabbit immunoglobulin G (IgG) antibodies, followed by immunoblotting with MDM2 and p73 antibodies; whole cell lysate (input); immunoblots are representative of two independent experiment; GAPDH was used as loading control.

3.1.4. LEM2 HAS NO IN VITRO GENOTOXICITY

Genotoxicity refers to the capacity of a substance to damage the genetic information within a cell. The assessment of a genotoxic potential of a certain compound is typically determined in an early phase of drug development process, as an important part of hazard identification [167]. The genotoxicity of LEM2 was cytogenetically tested by assessing its ability to induce micronucleus (MN) in cultured peripheral lymphocytes of normal individuals. In fact, when compared to samples treated with DMSO only, 0.72,

1.44 and 2.16 μM LEM2 (corresponding to GI50, 2xGI50 and 3xGI50 concentrations, respectively) did not increase the number of MN in lymphocytes (Figure 14). These results showed that LEM2 has no apparent in vitro genotoxic activity.

Figure 14 – LEM2 has no in vitro genotoxicity. The induction of micronucleus (MN) was tested in cultured peripheral lymphocytes of normal individuals, after 44 h treatment with LEM2, DMSO or cyclophosphamide, using cytokinesis-blocked micronucleus assay. The number of MN per 1000 binucleated lymphocytes. Cells were treated 1 μg/mL cyclophosphamide were used a positive control (C+); data are mean ± S.E.M. of three independent experiments; values significantly different from DMSO are indicated (***p < 0.001).

3.2. DISCUSSION

The tight regulation of p53 by MDM2, via an autoregulatory feedback loop, is a well-known process [83, 84]. As mentioned above, MDM2 also binds to other p53 family proteins, such as p73, repressing its transcriptional activity [97]. p73 plays an important role in chemosensitivity of cancer cells, particularly when p53 is inactivated or absent. In fact, several studies referred that cisplatin and gamma-irradiation treatment lead to p73 stabilization and enhanced apoptosis in cell lines, such as HCT116, Saos-2 and mouse fibroblasts 3T3 cells (reviewed in [168]). In this manner, the activation of the p73 tumor

suppressive activity, through inhibition of MDM2, represents an appealing anticancer therapeutic strategy. In 2008, Lau et al showed that p53-null and mutant p53-expressing cells were also sensitive to the inhibitor of the p53-MDM2 interaction, nutlin-3a, which suggested the activation of a p53-independent pathway by this compound [131]. These authors proved that nutlin-3a was also an inhibitor of the p73-MDM2 interaction, causing p73 accumulation and inducing the expression of p73 transcriptional targets [131]. Actually, these results were in accordance with the fact that p73 binds to the same hydrophobic pocket of MDM2 targeted by nulin-3a [98]. With this work, we intended to elucidate the molecular mechanism of an optimized derivative of LEM1, a compound previously identified by our group as an inhibitor of the p53-MDM2 interaction [154]. For that, a yeast-based screening assay was used to study the effect of LEM2 on p53-MDM2 interaction. However, surprisingly, the compound LEM2 had no impact on the p53-MDM2 interaction in yeast. Accordingly, this compound presented a p53-independent growth inhibitory effect and did not block the p53-MDM2 interaction in p53-expressing tumor cells. Despite this, LEM2 exhibited a potent growth-inhibitory effect in p53- and p63-null tumor cells, suggestive of a possible involvement of the p73 pathway in its activity. Based on this, and on the reported inhibitory ability of nultin-3a to block the p73-MDM2 interaction [131], the effect of LEM2 on the p73-MDM2 interaction was checked using a yeast-based assay previously developed by our group to search for inhibitors of this interaction [157]. As expected, LEM2 behaved as an inhibitor of the p73-MDM2 interaction in yeast. In order to confirm the results obtained in yeast, the activation of a p73 pathway by LEM2 was checked in p53- and p63-null tumor cells. In accordance with yeast, in these tumor cells, LEM2 led to p73 activation and stabilization, which is in agreement with the reduction of the MDM2 inhibitory effect on p73. In fact, LEM2 led to the activation of the p73 pathway through inhibition of the p73 interaction with MDM2. By analysis of cell cycle progression and Annexin-V staining, it was also observed that LEM2 tumor growth-inhibitory effect was associated with the induction of a G2/M- phase cell cycle arrest and apoptosis, which is in accordance with the increase of p21 (a major cell cycle regulator) and Bax (a pro-apoptotic protein of the Bcl-2 family) protein levels, as well as with PARP cleavage (a well-established substrate for caspase-3, during apoptosis) [169, 170]. Additionally, LEM2 induced ROS generation and ∆ψm dissipation, processes closely related to the activation of a mitochondria-mediated apoptotic pathway. Mitochondria-targeted agents have been widely recognized due to their high efficiency against chemotherapy-refractory cancer cells. In fact, drug-induced mitochondrial-apoptotic pathway has been considered an appealing anticancer strategy due to the high susceptibility of cancer cells to mitochondrial perturbations when

compared to their normal counterparts [170]. As reported for p53, p73 was also shown to induce a mitochondria-mediated apoptosis either by the direct transactivation of the Bax promoter, increasing its protein levels, or by an indirect mechanism mediated by the induction of Puma, which in turn promotes Bax mitochondrial translocation (reviewed in [171]). In physiological conditions, Bax exists as an inactive monomer in the cytosol, but, upon stress stimuli, Bax oligomerizes and translocates to the mitochondria, where it triggers ∆ψm dissipation and MOMP, ultimately leading to cyt c release and caspase activation [171]. In conformity with this, LEM2 was explored as an inducer of a p73- dependent mitochondria-mediated apoptotic cell death. However, despite inducing ∆ψm dissipation and increased Bax production, LEM2 did not trigger Bax translocation to mitochondria and the release of cyt c to cytosol, for several different experimental conditions tested (concentrations of LEM2, treatment periods, among others). These results demonstrated that LEM2 does not induce a p73-dependent mitochondria- mediated apoptotic pathway in human tumor cells. Lastly, LEM2 showed to be a non- genotoxic compound. Further work is still underway to support the activation of a p73-dependent pathway by LEM2, and to check the binding site of this compound. The inability of LEM2 to inhibit the p53-MDM2 interaction, in opposition to nutlin-3a that binds to MDM2, lead us to hypothesize that LEM2 may probably bind to p73 instead of MDM2. Despite this, relevant insights about the mode of action of this compound are already provided in this study. In conclusion, in this work, a new inhibitor of the p73-MDM2 interaction with potent tumor growth inhibitory effect was identified. To our knowledge, LEM2 represents the second inhibitor of the p73-MDM2 interaction reported until present, exhibiting a much higher potency than the reported nutlin-3a in p53-null tumor cells. Great therapeutic applications can be therefore envisioned for LEM2, particularly against p53- null of mutant p53-expressing tumor cells. Additionally, LEM2 may open the way to new derivatives with improved pharmacological properties.

IV JL-1: a dual inhibitor of the p53 interaction with MDM2/MDMX

4. JL-1: A DUAL INHIBITOR OF THE P53 INTERACTION WITH MDM2/MDMX

As referred above, MDM2 and MDMX, overexpressed in many human cancers, are two major endogenous inhibitors of wt p53 function (reviewed in [73]). Therefore, the development of small molecules that can abrogate this inhibitory effect of MDMs on p53 represents an exciting strategy in anticancer drug discovery [71, 73]. In fact, several studies demonstrated that the treatment of wt p53-expressing tumor cells with inhibitors of the p53 interaction with MDM2/MDMX leads to a pronounced tumor growth-inhibitory effect, associated with increased p53 protein levels and with the up-regulation of its target genes (reviewed in [126]). To date, most of the research has been focused on the identification of inhibitors of the p53-MDM2 interaction. However, as previously referred, given its independent but also cooperative role with MDM2, MDMX has also shown to be essential for the fine regulation of p53 (reviewed in [172]). Moreover, despite the structural similarities of MDMX and MDM2, MDMX-overexpressing cancer cells showed to be highly resistant to MDM2-only inhibitors, which is a consequence of their inability to abolish the inhibitory effect of MDMX on p53 activity (reviewed in [71, 126]). These observations support that the simultaneous inhibition of MDM2 and MDMX, for a full p53 reactivation in tumor cells, represents an appealing anticancer therapeutic strategy in cancer [73]. To date, only four dual inhibitors of the p53 interaction with MDM2/MDMX have been described. In a previous work performed by our group, from the screening of a small library of phenylalaninol-derived oxazolopyrrolidone lactams using yeast-based assays, compound JL-1 was identified as a potential dual inhibitor of the p53-MDM2/X interactions. In these assays, human p53 induced a marked growth inhibition, which was abolished by human MDM2 and MDMX, and reestablished by inhibitors of these interactions (nutlin-3a for the p53-MDM2 interaction [154]; SJ-172550 for the p53- MDMDX interaction [153]). Contrary to nultin-3a and SJ-172550, JL-1 was able to revert both the MDM2- and MDMX-inhibitory effects. With this work, it was intended to validate, in human tumor cells, the molecular mechanism of action of JL-1 as an inhibitor of the p53-MDM2/MDMX interactions.

4.1. RESULTS

4.1.1. JL-1 ACTIVATES A P53-DEPENDENT PATHWAY IN WT P53-EXPRESSING

HUMAN TUMOR CELLS

To study the molecular mechanism of action of JL-1 as inhibitor of the p53- MDM2/MDMX interactions, the effect of this compound was ascertained in HCT116

+/+ -/- p53 and in HCT116 p53 cell lines. The GI50 values obtained for JL-1, after 48 h treatment, in p53+/+ and p53-/- HCT116 cells (Figure 15A), revealed that JL-1 has a p53- dependent growth-inhibitory effect. In fact, the potency of JL-1 was significantly reduced when the p53 pathway was knocked out in HCT116 p53-/- cells.

Further results showed that the growth-inhibitory effect of JL-1, at the GI50 (7 μM) and 2xGI50 (14 μM) concentrations, was associated with G0/G1-phase cell cycle arrest (Figure 15B), induction of apoptosis (Figure 15C), and PARP cleavage (Figure 15D), in HCT116 p53+/+, but not in HCT116 p53-/- cells.

Figure 15 – JL-1 has in vitro antitumor activity and induces cell cycle arrest, apoptosis and +/+ -/- PARP cleavage in p53 , but not in p53 HCT116 cells. (A) The GI50 concentration of JL-1 was determined in p53+/+ and p53-/- HCT116 cells, after 48 h treatment, using the SRB assay; data are mean ± S.E.M. of four independent experiments; values significantly different from HCT116 p53+/+ cells are indicated (**p < 0.01). (B) JL-1-induced cell cycle arrest was determined after 24 h treatment. Cell cycle phases were analyzed by flow cytometry using PI staining; data are mean ± S.E.M. of three independent experiments; values significantly different from DMSO are indicated (*p < 0.05; **p < 0.01). (C) JL-1-induced apoptosis was determined after 24 h

treatment. Apoptosis was analyzed by flow cytometry using FITC-Annexin V and PI; data are mean ± S.E.M. of three independent experiments; values significantly different from DMSO are indicated (*p < 0.05; ***p < 0.001). (D) JL-1-induced PARP cleavage was analyzed by Western blot analysis after 24 h treatment; immunoblots are representative of three independent experiments; GAPDH was used as loading control.

Additionally, by western blot analysis, it was shown that JL-1 led to p53 stabilization and to the up-regulation of major p53 transcriptional targets, as revealed by the increased protein levels of MDM2, MDMX, Bax, Puma and p21 in p53+/+, but not in p53-/-, HCT 116 cells (Figure 16A). The ability of JL-1 to block the p53 interaction with MDM2 and MDMX was thereafter demonstrated using the co-IP assay, in HCT116 p53+/+ cells. At 7 and 14 μM, a visible decrease of the amount of MDM2 and MDMX co-immunoprecipitated with p53 was observed (Figure 16B). Altogether, these results demonstrated that JL-1 was an activator of a p53 pathway in human tumor cells by blocking the p53-MDM2/X interactions.

Figure 16 – JL-1 increases the expression levels of p53 and up-regulates p53 target genes by blocking the p53 interaction with MDM2/MDMX in p53+/+, but not in p53-/- HCT116 cells. (A) Western blot analysis was performed after 24 h treatment with 7 μM JL-1 or DMSO only; immunoblots are representative of three independent experiments; GAPDH was used as loading

control. (B) HCT116 p53+/+ cells were treated with 7 and 14 μM JL-1 or DMSO only for 24 h, and were immunoprecipitated with p53 or mouse immunoglobulin G (IgG) antibodies, followed by immunoblotting with MDM2, MDMX and p53 antibodies; whole cell lysate (IP); immunoblots are representative of three independent experiments; GAPDH was used as loading control.

The effect of JL-1 was further tested in two other wt p53-expressing human tumor cell lines that overexpress one of the MDM proteins, namely in MDMX-overexpressing breast cancer MCF-7 cells and in MDM2-overexpressing osteosarcoma SJSA-1 cells.

+/+ Although with higher GI50 values than the obtained in HCT116 p53 cells (11.8 ± 0.7 for SJSA-1 and 13.3 ± 0.5 μM for MCF-7; Figure 17A), JL-1 also presented an evident growth-inhibitory effect in SJSA-1 and MCF-7 tumor cells, associated with the induction of cell cycle arrest (at G0/G1-phase in MCF-7 and at G0/G1- and S-phases in SJSA-1 cells; Figure 17B), and apoptosis induction (Figure 17C), with PARP cleavage (Figure 17D). Moreover, also in SJSA-1 and MCF-7 tumor cells, JL-1 led to p53 stabilization (Figure 17E and 17F, respectively), and to the up-regulation of several p53 transcription targets, as demonstrated by the increase of the protein levels MDM2 and Bax (the remaining targets are still under study) in SJSA-1 cells (Figure 17E), and of MDM2, MDMX, Bax, Puma and p21 in MCF-7 cells (Figure 17F). Altogether, this set of results demonstrated that JL-1 was also able to activate the p53 pathway in tumor cells mostly inhibited by MDM2 and MDMX.

Figure 17 – JL-1 induces growth inhibition associated with cell cycle arrest and apoptosis in MDM2- and MDMX-overexpressing tumor cells through activation of the p53 pathway. (A) The GI50 concentration of JL-1 was determined in SJSA-1 and MCF-7 cells, after 48 h

treatment, using the SRB assay; data are mean ± S.E.M. of four independent experiments. (B) JL-1-induced cell cycle arrest was determined after 24 h treatment. Cell cycle phases were analyzed by flow cytometry using PI staining; data are mean ± S.E.M. of three independent experiments; values significantly different from DMSO are indicated (*p < 0.05; **p < 0.01; ***p < 0.001). (C) JL-1-induced apoptosis was determined after 24 h treatment. Apoptosis was analyzed by flow cytometry using FITC-Annexin V and PI; data are mean ± S.E.M. of three independent experiments; values significantly different from DMSO are indicated (**p < 0.01; ***p < 0.001). (D) JL-1-induced PARP cleavage was analyzed by Western blot analysis after 24 h treatment; immunoblots are representative of three independent experiments; GAPDH was used as loading control. (E) Western blot analysis was performed in SJSA-1 cells, after 24 h treatment with 11.8 μM JL-1. (F) Western blot analysis was performed in MCF-7 cells, after 24 h treatment with 13.3 μM JL-1. In (E) and (F), immunoblots are representative of three independent experiments; GAPDH was used as loading control.

4.1.2. JL-1 HAS NO IN VITRO GENOTOXICITY

The genotoxicity of JL-1 was cytogenetically tested by assessing its ability to induce MN in cultured peripheral lymphocytes of normal individuals. When compared to samples treated with DMSO only, 7, 14 and 21 μM JL-1 (corresponding to GI50, 2xGI50 and 3xGI50 concentrations, respectively) did not increase the number of MN in lymphocytes (Figure 18). These results demonstrated that JL-1 has no apparent in vitro genotoxic activity.

Figure 18 – JL-1 has no in vitro genotoxicity. The induction of micronucleus (MN) was tested in cultured peripheral lymphocytes of normal individuals, after 44 h treatment with JL-1, DMSO or cyclophosphamide, using cytokinesis-blocked micronucleus assay. The number of micronucleus per 1000 binucleated lymphocytes. Cells treated 1 μg/mL cyclophosphamide were used as positive control (C+); data are mean ± S.E.M. of three independent experiments; values significantly different from DMSO are indicated (**p < 0.01).

4.1.3. JL-1 PREVENTS THE IN VITRO TUMOR CELL MIGRATION

Cell migration is an essential process at many stages of embryonic development, for tissue repair and immune function. However, the deregulation of the cell motile behavior largely contributes to pathological processes, including tumor metastasis (reviewed in [173]). In fact, the acquired capability of tumor cells to invade and metastasize was established as a hallmark of cancer [2]. As such, the impact of JL-1 on the invasive ability of HCT116 p53+/+ cell lines was investigated by performing migration assays. In the Wound Healing Scratch assay, the images captured at the beginning and upon 20 and 24 h treatment showed that JL-1 inhibited the HCT116 p53+/+ cell migration, and the subsequent wound closure, when compared to cells treated with DMSO only (Figure 18). These results demonstrated that JL-1 is capable to inhibit the migration of human colon adenocarcinoma cells.

Figure 19 – JL-1 prevents the migration of HCT116 p53+/+ cells. HCT116 p53+/+ confluent cells treated with 3 μM JL-1 (or DMSO only) were observed at different time points (0, 20 and 24 h) in the wound healing assay.

4.1.3. JL-1 HAS POTENT IN VIVO ANTITUMOR ACTIVITY

To evaluate the in vivo antitumor potential of JL-1, mice xenograft models carrying HCT116 p53+/+ tumor cells were used. The results revealed that four intraperitoneal administrations (twice a week) of 50 mg/kg JL-1 inhibited the growth of HCT116 p53+/+ tumors (Figure 19). The in vivo effect of JL-1 in HCT116 p53-/- tumor xenografts, to confirm its p53-dependent antitumor activity, is under analysis.

Figure 20 – JL-1 has in vivo antitumor activity. BALB/c nude mice carrying HCT116 p53+/+ xenografts were treated with 50 mg/kg JL-1 or vehicle (control); values significantly different from control mice are indicated (**p < 0.01, ***p < 0.001).

4.2. DISCUSSION

Several anticancer strategies have been devised to rectify a dysfunctional p53 pathway, particularly due to the overexpression of MDM2 or MDMX. The simultaneous inhibition of the p53 interaction with MDM2 and MDMX has gained increasing attention as an anticancer therapeutic strategy, since it will conduct to a full p53 reactivation in wt p53-expressing tumor cells [71]. In this work, we identified a new dual inhibitor of the p53-MDM2/MDMX interactions, the enantiopure phenylalaninol-derived oxazolopyrrolidone lactam JL-1. Given the low number of this type of inhibitors, the identification of compound JL-1 may represent an important contribution to this field. In a previous work, a yeast targeted assay was used for the screening of inhibitors of the p53-MDM2/MDMX interactions from a small library of phenylalaninol-derived oxazolopyrrolidone lactams. From this analysis, the compound JL-1 was identified as a potential dual inhibitor of the p53-MDM2/MDMX interactions. In the present work, the molecular mechanism of action of JL-1 was validated both in vitro tumor cell lines and in vivo xenograft models. In fact, using human-derived isogenic tumor cell lines with and without p53, it was verified that the in vitro tumor growth inhibitory effect of JL-1 was highly dependent on p53, since a significant reduction of its potency was observed in the absence of the p53 pathway. Consistently with this, it was shown that the antiproliferative effect of JL-1 was associated with the induction of cell cycle arrest and apoptosis, in p53-expressing tumor cells, but not in p53-null tumor cells. Similarly, JL-1 led to the stabilization of p53 protein levels, which is indicative of an inhibition of the

MDM2-mediated p53 degradation, and to the up-regulation of several p53 transcriptional targets in a p53-dependent manner. Finally, it was confirmed that JL-1 was able to block the p53-MDM2/MDMX interactions. Several evidences are therefore provided in this work supporting a dual inhibition of the p53-MDMs interactions by JL-1. In this work, it was also shown a potent antitumor activity of JL-1 against wt-p53 expressing human tumor cells with high levels of MDM2 and MDMX. Particularly, JL-1 overcame the frequently reported resistance of MDMX-overexpressing tumor cells to MDM2-only inhibitors [71, 126]. This is of greatest importance since the overexpression of MDMs represent common mechanisms developed by tumor cells for disabling or attenuating specific p53 functions or the entire pathway [117]. Actually, in these cells, JL-1 also presented a growth-inhibitory effect, associated with the induction of cell cycle arrest and apoptosis, p53 stabilization, and up-regulation of p53 transcription targets. The earning ability to migrate and invade allows tumor cells to spread within the tissues and, consequently, to disseminate from the primary tumor to a distinct organ, fact that accompanies the tumor progression to more aggressive phenotypes. In fact, invasion and metastasis are the most frequent cause of death for cancer patients [174]. The loss of p53 function not only sustains tumor initiation and progression, but also allows tumor cells to more quickly acquire a metastatic phenotype [175]. In fact, p53 can regulate the transcription of several genes involved in the mechanisms underlying cell migration and invasion, such as cell motility and adhesion, signal transduction, inhibition of angiogenesis, extracellular matrix and cytoskeleton organization (reviewed in [175, 176]). Based on this, it is expected that the restoration of the p53 function will lead to tumor regression and metastasis prevention. The results obtained in this work revealed that low doses of JL-1 prevented the migration of these cells. Beyond that, JL-1 showed to be a non-genotoxic compound. Finally, assays in xenograft mice models carrying wt p53-expressing tumor cells confirmed the in vivo antitumor activity of JL-1. Additional studies in xenograft mice models carrying p53-null tumor cells are underway in order to confirm the p53-dependent antitumor activity of JL-1. These results further support the promising antitumor properties of JL-1. Although relevant findings about the mode of action of JL-1 were attained in this work, other studies must be performed in order to further elucidate this issue. In particular, it would be important to analyze the binding site of JL-1 (MDMs or p53), using biophysical assays. In conclusion, the results obtained in this work reveal the identification of a new selective activator of the p53 pathway through the dual inhibition of the p53- MDM2/MDMX interactions. Besides this, evidences are provided by both in vitro and in vivo assays for the potential of JL-1 as anticancer agent.

V General discussion and final remarks

5. GENERAL DISCUSSION AND FINAL REMARKS

Diverse oncogenic events result in the activation of p53 family proteins and the induction of p53 family-dependent cell cycle arrest and apoptosis [95]. Therefore, the deregulation of the p53 family pathway plays a critical role in tumorigenesis [177]. As such, this family of proteins has been recognized as an appealing therapeutic target in cancer treatment. In fact, given its functional diversity in halting the propagation of cells carrying oncogenic lesions, as well as in blocking invasion and metastasis, the inactivation of the function of p53 family proteins is a very frequent event during tumor development, mainly through interaction with negative protein partners [108]. Nevertheless, many aspects of the molecular basis of these protein-protein interactions remain not completely clarified. Additionally, despite the amazing efforts that have been developed in order to find new and better molecular strategies to re-establish the p53 function, small molecule modulators of the activity of p53 family proteins, particularly of p63 and p73, with improved pharmacological properties are highly required [177]. In the present work, two new activators of the p53 and p73 pathways were identified. For that, the yeast cell system, having proved its importance earlier as highly effective first-line assay model [178], largely contributed for our findings. In fact, in previous works, it was shown that, as in human cells, also in yeast human MDM2 and MDMX were able to inhibit the p53 and p73 activity [152, 157]. These results revealed that yeast could be used as a model organism for a simplified cell-based screening assay to search for inhibitors of the interaction of p53 and p73 with MDMs [154, 157]. Herein, we provide a proof-of-concept for the effectiveness of this cell model as a screening tool with the identification of a new inhibitor of the p73-MDM2 interaction (the xanthone LEM2), and of a new dual inhibitor of the p53-MDM2/MDMX interactions (the phenylalaninol-derived oxazolopyrrolidone lactam derivative JL-1). For long, the p73 pathway has been considered an attractive target for anticancer drug development. In fact, several experimental and clinical evidences demonstrated that p73 can potentially emulate p53 functions in cancer cells, inducing apoptosis after DNA damage (reviewed in [58]). Several compounds that modulate the levels and activity of p73 by acting, either directly or indirectly, on its regulators were described (reviewed in [129]). However, to date, just the small molecule nutlin-3a was described as inhibitor of the p73-MDM2 interaction [131]. In this work, we identified a new inhibitor of this interaction, LEM2, which demonstrated a much higher in vitro antitumor potency than nutlin-3a in p53-null tumor cells. Although further work is required to confirm the molecular mechanism of LEM2, considerable therapeutic applications may be

anticipated for this compound, particularly against tumor cells with null or mutant p53. Additionally, LEM2 may open the way to a new class of activators of the p73 pathway with improved antitumor pharmacological properties. On the other hand, the dual inhibition of the p53-MDM2/X interactions is emerging as an exciting therapeutic approach, since it ensures the full activation of the p53 functions [72]. Despite this, only a few number of this type of p53 activators were described to date. A great contribution is therefore provided by the present work to this area with the identification of JL-1 as a dual inhibitor of the p53 interaction with MDMs. This compound exhibited a p53-dependent in vitro growth inhibitory activity, and promising in vivo antitumor properties without apparent in vitro genotoxic activity. Additionally, it was capable of inhibiting the invasion of wt p53-harboring tumor cells, which reinforce its potential use as anticancer drug. Besides this, a new scaffold of dual inhibitors of p53-MDMs interaction was identified, which optimization may lead to derivatives with improved pharmacological properties as activators of the p53 pathway. The exploitation of synergic effects between these compounds (LEM2 and JL-1) and conventional chemotherapeutic drugs, to improve induced cell death, would further expand the therapeutic applicability of these compounds as anticancer agents. As a whole, with this project a relevant contribution was provided to the pharmacology of p53 family proteins, with the identification of promising small-molecule activators of the p53 and p73 pathway that can be further explored as potential anticancer agents.

VI References

6. REFERENCES

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