MITOCHONDRIAL ROS SCAVENGING REDUCE CISPLATIN-INDUCED CELL MIGRATION IN TRIPLE NEGATIVE BREST CANCER

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ITOCHONDRIAL

ROS

SCAVENGING

REDUCES CISPLATIN

-

INDUCED MIGRATION

OF TRIPLE NEGATIVE BREAST CANCER CELLS

Erica Pranzini

- Undergraduate thesis -

Director: Prof. Maria Grazia Tozzi, University of Pisa

Co-Director: Prof. Pierre Sonveaux, University of Louvain (UCL)

Direct Supervisor: Dr. Paolo Ettore Porporato, University of Louvain (UCL)

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ABSTRACT

Metastatic progression is associated with poor prognosis for cancer patients. Tissue invasion and metastasis are among the most well-known hallmarks of cancer, and are characterized by the ability of tumor cells to migrate and invade surrounding tissues. Recently, these two features of aggressive cancers have been found to be promoted by mitochondrial reactive oxygen species (mtROS), justifying the study of mtROS scavengers, such as mitoTEMPO and mitoQ, to prevent tumor metastasis. These agents, however, are expected to be used in combination with chemotherapy, and several chemotherapeutic agents promote ROS production by tumor cells, which could participate in their therapeutic effects. Therefore, the aim of our study was to test the safety of combining mtROS scavengers with chemotherapy. We focused on triple negative breast cancer, a highly metastatic tumor type, and on cisplatin and paclitaxel,

i.e., major chemotherapeutic treatment modalities for this type of cancer. Both drugs

had been previously described to promote ROS production by tumor cells. Using murine 4T1 and human MDA-MB-231 cells as experimental models, we found that cisplatin, but no paclitaxel, increased ATP production, mitochondrial activity and mtROS generation at doses typically achieved in human tissues in the clinics. It also increased tumor cell migration in vitro. Importantly, we report that mitochondria-targeted superoxide scavengers mitoTEMPO and mitoQ repress cisplatin-induced tumor cell migration but do not interfere with the cytostatic and cytotoxic effects of cisplatin and paclitaxel. Our study thus indicates not only that it is possible to combine mitochondrial superoxide scavengers with cisplatin and paclitaxel chemotherapies, but also that these agents can prevent cisplatin-induced tumor cell migration.

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1. INTRODUCTION

Cancer is a generic term for a large group of diseases that involve abnormal cell growth with the potential to invade and spread to other parts of the body (WHO, 2014). Cancer is a leading cause of death worldwide, accounting for 8.2 million deaths in 2012 (World Cancer Report, 2014).

It is well known that carcinogenesis is a multistep progressive process during which successive genetic alterations induce a sequence of cellular and molecular events that promote the transformation of a normal cell into a cancer cell. These events consist in the acquisition of specific features summarized by Hanahan and Weinberg as the “Hallmarks of cancer” (Hanahan and Weinberg, 2000): self-sufficiency in growth signals, insensitivity to growth-inhibitory signals, evasion of programmed cell death (apoptosis), limitless replicative potential, sustained angiogenesis, and tissue invasion and metastasis. Underlying these hallmarks is genome instability, which generates the genetic diversity necessary for the acquisition of such traits. Nevertheless, these alterations are not the sole required for tumor onset and progression, as conceptual progress in the last two decades led to a rediscovery of one of the first hallmarks of cancer, reprogramming of energy metabolism.

Multiple molecular mechanisms in cancer cells converge to alter core cellular metabolism, providing support for the basics needs of dividing cells, i.e., rapid ATP generation to maintain the energetic status, increased biosynthesis of macromolecules and maintenance of appropriate cellular redox status. Increasing amount of evidence indicates that many hallmarks of cancer are under metabolic control, suggesting the importance of metabolic characterization for the development of new therapeutic approaches against cancer (Cairns et al., 2011).

1.1 METABOLIC TRANSFORMATION IN CANCER

The metabolism of tumors has interested cancer biologists since Warburg's experiments in the 1920s, who showed that tumor cells convert large amount of glucose in lactate, even in presence of oxygen (Warburg, 1925). This metabolic phenotype observed in tumor cells, now known as the “Warburg effect” or “aerobic

glycolysis”, consists in a shift from ATP generation through OXPHOS to ATP generation

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Fig. 1.1: Schematic representation of differences between oxidative phosphorylation, anaerobic glycolysis and aerobic glycolysis (“Warburg effect”). In the presence of oxygen, non-proliferating tissues metabolize glucose to pyruvate via glycolysis and oxidize most of that pyruvate in the mitochondria to CO2 during the process of oxidative phosphorylation. Because oxygen is required as the final electron acceptor to completely oxidize the glucose, oxygen is essential for this process. When oxygen is limiting, the cells generate lactate (anaerobic glycolysis). This generation of lactate during anaerobic glycolysis allows glycolysis to continue (by cycling NADH back to NAD+), but results in minimal ATP production when compared with oxidative phosphorylation. Importantly, proliferating cancer cells convert most glucose to lactate even in presence of oxygen (aerobic glycolysis). However, aerobic glycolysis is less efficient than oxidative phosphorylation for generating ATP (from Vander Heiden et al., 2009).

The metabolism of glucose to lactate generates only 2 ATPs per molecule of glucose, whereas OXPHOS generates up to 36 ATP upon complete oxidation of one molecule of glucose (Figure 1.1). This apparent waste of energy in proliferating cancer cells using glycolysis preferentially to OXPHOS even in the presence of oxygen led Warburg to hypothesize that increased aerobic glycolysis in tumor cells was due to defects in mitochondrial respiration function, and that aerobic glycolysis was therefore a necessary adaptation to face a lack of ATP (Warburg, 1956). However, subsequent studies demonstrated that the mitochondrial functionality is not impaired in most

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tumor cells (Weinhouse et al., 1997). This raises the question of why a less efficient metabolism, at least in term of ATP production, would be selected in tumor cells. A first explanation for the glycolytic switch is that tumor cells must respond to important metabolic requirements that go beyond simple ATP demand. Indeed, tumor cells need nucleotides, amino acids and lipids to maintain their high proliferative rate. Thus, metabolism reprogramming can be exploited to support the biosynthesis of these macromolecules. Glucose and glutamine are main metabolic requirements for cancer cells, as they supply most of the carbon, nitrogen, free energy and reducing equivalent necessary to support cell growth and division (DeBerardinis et al., 2007).

Therefore, in proliferating cells, glucose is not fully oxidized to CO2 via OXPHOS in

mitochondria for optimal ATP production. Rather, glucose is mainly driven to the synthesis of macromolecular precursors, such as acetyl-coA for fatty acids, glycolytic intermediates for nonessential amino acids and ribose for nucleotides (Nelson & Cox, 2008). Thus, although ATP hydrolysis provides free energy for some of the biochemical reactions responsible for replication of the biomass, cells cannot use glucose only for ATP production during growth and cell division: they must use it also to generate intermediates for biosynthesis and biomass. In addition, in the case a high rate glycolysis would serve only to produce ATP, the rise in the ATP/ADP ratio would severely impair the flux through glycolytic intermediates, limiting the production of acetyl-coA and NADPH required for macromolecular synthesis (Nelson & Cox, 2008). The excess generation of lactate that accompanies the Warburg effect would appear as an inefficient use of cellular resources and a waste of three carbons that might otherwise be utilized for ATP production or macromolecular biosynthesis. A possible explanation of this phenomenon is that the release of lactate allows faster incorporation of carbon into biomass, which facilitates a rapid turnover of the cells. (Vander Heiden et al., 2009).

In addition to promoting cell proliferation, it is also well known that a glycolytic metabolism arises as an adaptation to hypoxic condition during the early avascular phase of tumor development and in hypoxic tumor areas throughout tumor development, as it allows ATP production when oxygen becomes a scarce resource. Adaptation to the resulting acidic microenvironment that is caused by excess lactate

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production may further drive the evolution of the glycolytic phenotype (Gatenby & Gillies, 2004).

The metabolic phenotype of cancer cells is controlled by intrinsic genetic mutations and external responses to the tumor environment. Oncogenic signaling pathways controlling growth and survival are often activated by the loss of tumor suppressors (e.g. p53 or phosphatase and tensin homolog deleted on chromosome 10 [PTEN]) or the activation of oncoproteins (e.g. phosphaditylnositol-3-kinase [PI3K], K-RAS). These altered signals modify cellular metabolism to reach the requirements for cell division (Cairns et al., 2011). Therefore, it is not surprising that several known oncogenes also alter the metabolic activity of cancer cells (Figure1.2):

-PI3K: The PI3K pathway is one of the most frequently altered signaling pathways in cancer cells. This pathway is activated by mutations of tumor suppressor genes (such as PTEN), and its activation provides not only strong growth and survival signals, but also profoundly affects cell metabolism. The best-studied effector downstream of PI3K is protein kinase B (PKB or AKT1) that stimulates glycolysis by increasing the expression and translocation of glucose transporters to the external membrane, and activates key glycolytic enzymes, such hexokinase and phosphofructokinase 2. Increased and prolonged AKT1 signaling inhibits FOXO transcription factor, resulting in complex transcriptional changes that increase the glycolytic capacity of the cells (Khatri et al., 2005). AKT1 also stimulates signaling through kinase mammalian target of rapamycin (mTOR) by phosphorylating and inhibiting its negative regulator tuberous sclerosis protein 2 (TSC2). mTOR functions as a key metabolic integration point, coupling growth signals to nutrient availability. Activation of mTOR stimulates protein and lipid synthesis and cell growth in response to nutrient and energy availability (Guertin &

Sabatini, 2007). mTOR also indirectly causes other metabolic changes by activating

transcriptional factors, such as hypoxia-inducible factor-1 (HIF-1), even under normoxic conditions.

-HIF-1. HIF-1 plays a key role in reprogramming cancer metabolism by activating the transcription of genes encoding glucose transporters and glycolytic enzymes. HIF-1 is a

heterodimeric transcription factor that consists of an O2-regulated HIF-1α and a

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hydroxylated on two proline residues by prolyl hydroxylase domain protein 2 (PHD2),

which uses O2 and α-ketoglutarate as substrates in a reaction that generates CO2 and

succinate as byproducts. Prolyl-hydroxylated HIF-1α is bound by the von Hippel-Lindau tumor suppressor protein (VHL), which recruits an E3-ubiquitin ligase that targets HIF-1α for proteasomal degradation. Under hypoxic conditions, prolyl hydroxylation

reactions are inhibited by substrate (O2) deprivation and/or by the mitochondrial

generation of reactive oxygen species (ROS) that may oxidize Fe(II) present in the catalytic center of the hydroxylases (Guzy & Schumacker; 2006). Thus, during hypoxia, HIF-1α is rescued from degradation and can migrate into the cell nucleus where it binds to HIF-1β, adaptor protein p300 and RNA polymerase 2 to activate the transcription of its target genes that possess a hypoxia-responsive element (HRE) in their promoter sequence (Semenza et al., 2011). In addition to its stabilization under hypoxic conditions, HIF-1α can also be activated under normoxic conditions by oncogenes, including PI3K, and by mutations that inactivate tumor suppressor genes, such as VHL, succinate dehydrogenase (SDH) and fumarate hydratase (FH) (Kapitsinou & Haase, 2008; Selak, et al., 2005; King et al., 2006). Accumulation of succinate or fumarate directly inhibits PHD2 activity, thus contributing to normoxic HIF-1α stabilization. Once activated, HIF-1 promotes the expression of most glycolytic enzymes and transporters. It is indeed considered to be a main promoter of glycolysis in tumors (Porporato et al, 2011). HIF-1 activates the transcription of genes encoding glucose transporter GLUT1 and GLUT3, as well as genes encoding hexokinase 2 (HK2, which converts glucose to glucose-6-phosphate), lactate dehydrogenase A (LDHA, which converts pyruvate to lactate) and monocarboxylate transporter 4 (MCT4, which transports lactate out of the cell) (Semenza 2010). Moreover, pyruvate dehydrogenase kinase (PDK, which phosphorylates and inactivates the catalytic domain of pyruvate dehydrogenase) is activated by HIF-1. As a result of PDK activation, pyruvate is actively shunted away from mitochondria, thus reducing the flux trough the TCA cycle and the

delivery of NADH and FADH2 to the electron transport chain (ETC). HIF-1 also induces

mitophagy in many human cancer cell lines by activating the transcription of genes encoding the BH3 domain protein BNIP3 (Zhang et al., 2008). The interplay between HIF-1 and metabolism is reciprocal, as it has been shown that lactate itself can directly stabilize HIF-1α in normoxia by inhibiting PHD2 (De Saedeleer et al., 2012). This occurs

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similarly with another isoform, HIF-2α, which is also emerging to regulate metabolic fluxes, particularly glutaminolysis (Pérez-Escuredo et al., 2016).

-c-MYC. Another oncogenic transcription factor that has several important effects on cell metabolism is c-MYC. In addition to its well-described role in controlling cell growth and proliferation, c-MYC has been shown to collaborate with HIF-1 for the activation of several glucose transporters and glycolytic enzymes, including GLUT1, HK2, phosphofructokinase (PFK), enolase, LDHA and PDK1 (Dang et al., 2008). c-MYC also plays a major role in promoting the use of glutamine by cancer cells through the upregulation of glutamine transporters SLC5A1 and SLC7A1 (Gao et al., 2009).

-AMP-activated protein kinase (AMPK). AMPK is a crucial sensor of the energetic status of cells that opposes the effect of AKT1 and functions as a potent inhibitor of mTOR. Cancer cells must overcome this checkpoint in order to proliferate in response to activated growth signaling pathways, even in periods of energetic stress. Several oncogenic mutations and signaling pathways can suppress AMPK signaling, allowing cancer cells to respond to inappropriate and aberrant growth signals. In particular, loss of AMPK signaling promotes the activation of mTOR and HIF-1, and might therefore also support the glycolytic shift (Shackelford et al., 2009).

-p53. Although the transcription factor and tumor suppressor p53 is best known for its role in the response to DNA damage and apoptosis, it is becoming clear that p53 also plays an important role as regulator of cell metabolism. The role of p53 in energy metabolism has been well described by Matoba et al. (2006), who reported that loss of p53 diminishes the synthesis of cytochrome C oxidase. Moreover, even if p53 activates the expression of HK2, it inhibits the glycolytic pathway by upregulating the expression of TP53-induced glycolysis and apoptosis regulator (TIGAR), an enzyme that decreases the levels of glycolytic activator fructose-2,6-bisphoshate. P53 also supports the expression of PTEN by binding to PTEN promoter (Stambolic, 2001), which inhibits the PI3K pathway, thereby decreasing glycolysis. Thus, loss of p53 might also facilitate the acquisition of the glycolytic phenotype (Matoba et al., 2006). In addition, p53 affects energy metabolism by regulating AMPK, mTOR, PTEN and insulin-like growth factor binding protein 3 (IGFBP3) (Feng & Levine, 2010).

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-Ras family. Ras mutations are important in promoting cancer initiation and progression. K-Ras, the most commonly mutated oncogenic Ras in pancreatic cancer, has been shown to affect the shape and function of mitochondria during fibroblast transformation (Chiaradonna et al., 2006). Further studies showed that fibroblasts transformed by K-Ras attenuate OXPHOS by suppressing the activity of respiratory complex I, with a corresponding decrease in the expression level of complex I proteins (Baracca et al., 2010). Similarly, H-Ras-transformed mouse fibroblasts exhibit low mitochondrial respiration and an increased dependency on glycolysis, a sensitivity to glycolytic inhibitors and an insensitivity to OXPHOS inhibitors (Yang et al., 2010). -OCT1. Finally, OCT1 is a transcription factor showing upregulated expression in several human cancer cells. OCT1 might collaborate with p53 in regulating the balance between oxidative and glycolytic metabolism in cancer cells. Indeed, OCT1 regulates a set of genes that increase glucose metabolism and reduce mitochondrial respiration, which includes PDK4, a PDK isoform that has the same function as the PDK enzymes activated by HIF-1 (Shakya A. et al., 2009).

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Fig 1.2: Molecular mechanism driving the Warburg effect in cancer cells. The shift from normal cell metabolism (a) to aerobic glycolysis (b) in cancer cells is driven by multiple oncogenic signaling pathways. PI3K activates AKT, which stimulates glycolysis by directly regulating glycolytic enzymes and by activating mTOR. mTOR has an effect on the glycolytic phenotype by enhancing hypoxia-inducible factor-1 (HIF-1) activity. HIF-1 increases the expression of glucose transporters (GLUT), glycolytic enzymes and pyruvate dehydrogenase kinase 1 (PDK1), which blocks the entry of pyruvate into the tricarboxylic acid (TCA) cycle. MYC cooperates with HIF-1 in activating several genes that encode glycolytic proteins. Tumor suppressor p53 opposes the glycolytic phenotype by suppressing glycolysis through TP53-induced glycolysis and apoptosis regulator (TIGAR), increasing mitochondrial metabolism via SCO2 and promoting the expression of PTEN. Of note, de novo expression of pyruvate kinase M2 (PKM2) in his dimeric form affects glycolysis by slowing the pyruvate kinase reaction and diverting substrates into alternative biosynthetic and reduced NADPH-generating pathways (from Cairns et al., 2011).

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Although the traditional view of cancer metabolism is that cancer cells are dependent on aerobic glycolysis, it has been shown that they rather have a broad spectrum of bioenergetic states, ranging from a predominance of aerobic glycolysis to a predominance of OXPHOS. The Warburg effect plays a vital role in cancer cell proliferation and survival (Vander Heiden et al., 2009). However, to adapt to rapidly changing microenvironments, cancer cells vary in metabolic phenotype as they have a high metabolic flexibility. A good example of this metabolic flexibility lies in the concept of metabolic symbiosis between cancer cells. It has indeed been shown by Sonveaux et al. in 2008 that different populations of cancer cells exist in a given tumor, adapting to the relative abundance of oxygen and nutrients (Figure 1.3). In particular, properly oxygenated cancer cells at the tumor edge mostly rely on OXPHOS for energy production, but they use lactate instead of glucose as a main metabolic fuel, thus sparing glucose for the inner hypoxic tumor core. Lactate produced by the hypoxic population of cells is thus used as an oxidative fuel by surrounding well oxygenated cells. The expression of different lactate transporters define this symbiosis, and it has been reported that glycolytic cancer cells normally express lactic acid transporter MCT4 (optimized for lactate extrusion) (Dimmer et al., 2000), whereas oxidative cancer cells mostly express MCT1, which is optimized for lactate uptake (McCullagh et al., 1996).

Fig. 1.3: Metabolic symbiosis in tumors. Hypoxic cancer cells depend on glucose and glycolysis to produce energy and release lactate (a process facilitated by monocarboxylate transporter 4, MCT4). By contrast, oxygenated cancer cells import preferentially lactate (a process mediated by monocarboxylate transporter 1, MCT1). In the presence of oxygen, lactate is oxidized to pyruvate by lactate dehydrogenase 1 (LDH1), and pyruvate fuels the tricarboxylic acid (TCA) cycle to produce ATP. The metabolic preference of oxidative cancer cells for lactate optimizes glucose delivery to hypoxic/glycolytic cancer cells.

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A complementary model has been that proposed by Lisanti and colleagues in 2009. According to this model, aerobic glycolysis can also occur in cancer-associated fibroblasts (CAFs). It is induced by cancer cells that produce ROS, and results in the production of energy-rich metabolites (such as lactate and ketone bodies) that can be transferred to cancer cells and fuel the TCA cycle, resulting in high ATP production via OXPHOS. Essentially, in this new paradigm termed “The Reverse Warburg Effect”, stromal fibroblasts are forced to feed cancer cells via the transfer of high-energy metabolites (Pavlides et al. 2009). This interplay between CAFs and cancer cells includes a mutual metabolic reprogramming. CAFs acquire a Warburg metabolism and undergo mitochondrial stress as a result of contact with cancer cells. Intracellular contact activates CAFs, triggering increased expression of glucose transporter GLUT1, lactate production and extrusion of lactate by de novo expressed monocarboxylate transporter 4 (MCT4). Conversely, cancer cells, upon contact with CAFs, are reprogrammed toward an aerobic metabolism, with a decrease in GLUT1 expression and an increase in lactate upload via MCT1.

Metabolic reprogramming of both stromal and cancer cells is under the strict control of HIF-1 (Fiaschi et al., 2012; Cirri & Chiarugi, 2011). The theory posits that cancer cells induce oxidative stress in adjacent fibroblasts, which in turn induces the autophagic program via the activation of HIF-1 and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB). During autophagy, both caveolae (a special type of lipid rafts that form invaginations of the plasma membrane marked by Cav-1 and involved in cell signaling [Anderson, 1998]) and mitochondria are destroyed by lysosomal degradation, leading to the production of recycled nutrients to feed cancer cells. Mitophagy further promotes aerobic glycolysis in CAFs (Martinez-Outschoorn et al., 2010a) (Figure 1.4). Oxidative stress in CAFs has also other consequences: the amplification of ROS production feeds back upon cancer cells, inducing DNA damage and aneuploidy, which are characteristic of genomic instability. Thus, ROS production in the stroma could fuel cancer cell evolution via a process of random mutagenesis (Martinez-Outschoorn et al., 2010b).

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Fig 1.4: The Reverse Warburg effect. Cancer cells use oxidative stress to induce autophagy in cancer-associated fibroblasts (CAFs) in order to extract energy-rich recycled nutrients (such as lactate, ketones and glutamine) to fuel their oxidative mitochondrial metabolism (from Martinez-Outschoorn et al., 2010a).

1.2 ROLE OF OXIDATIVE STRESS IN TUMOR PROGRESSION

Frequently, changes in cancer cell metabolism are associated to a redox imbalance and oxidative stress. Oxidative stress is defined as a disturbance in the equilibrium between free radicals (FR), ROS and endogenous antioxidant defense mechanisms (Vishal et al., 2005).

ROS comprise a large group of oxygen-derived small molecules that includes radicals

and non-radical species. Radicals such as superoxide (O2˙⁻), the hydroxyl anion (OH˙)

and peroxyls (RO2˙) are short-lived, highly electrophilic and reactive molecules with an

unpaired electron in their outer shell. Non-radical ROS include hypochlorous acid

(HOCl), ozone (O3), singlet oxygen (1O2) and hydrogen peroxide (H2O2). The initial step

in the formation of these molecules is the transfer of one electron to molecular oxygen

(O2) to form the superoxide anion, which can then be transformed into H2O2

spontaneously or by the activity of superoxide dismutases (SODs). Additional steps in the cascade of ROS production include the reaction of superoxide with nitric oxide

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(NO) to form peroxynitrite (ONOO⁻), the peroxidase-catalyzed formation of HOCl from

H2O2, and the iron-catalyzed Fenton reaction leading to the generation of OH˙

(Murphy, 2009).

Two main sources of ROS are mitochondria and the family of NADPH oxidases (NOXs). The three best-characterized sites in the mitochondria are complexes I, II and III in the mitochondrial ETC. These complexes generate superoxide by the one-electron reduction of molecular oxygen (Brand, 2010).

ROS homeostasis is required for proper cell signaling and cellular fitness in normal cells. Low doses of ROS indeed promote cell survival, growth, proliferation and angiogenesis. However, higher ROS levels can be toxic to the cells, inducing cell proliferation arrest and even cell death (Glasauer & Chandel, 2014). Thus, the availability of ROS at a given site results from the balance between its production from various sources and its disposal by enzymatic and non-enzymatic antioxidants (Hecht et al., 2016). Several types of antioxidants play important roles in ROS homeostasis, including dietary natural antioxidants (e.g. β-carotene, ascorbic acid, tocopherol, selenium and polyphenol metabolites), endogenous antioxidant molecules (e.g. glutathione, α-lipoic acid, coenzyme Q, ferritin, uric acid, bilirubin) and endogenous antioxidant enzymes (e.g. SODs, catalases [CATs], glutathione peroxidases [GPXs], glutathione reductase [Gr], thioredoxin reductase [TRX] and peroxiredoxin reductases [PRXs]) (Valko et al., 2006).

Cancer cells are usually subjected to high levels of ROS and aberrant antioxidant levels. This oxidative stress is a hallmark of many cancers (Glasauer & Chandel, 2014). Reasons for increased ROS production in cancer cells are related to alterations of different signaling pathways. P53 mutations or loss of P53 are associated with increased ROS production and tumor aggressiveness (Attardi & Donehower, 2005). The activation of RAS downstream of the PI3K/AKT/mTOR survival pathway is associated to ROS accumulation due to an increase in mitochondrial metabolism and inhibition of FOXO transcription, which, together, promote antioxidant defense. Oncogenic AKT activation can thereby result in an increased production of ROS from mitochondria through metabolic pathways and impaired ROS scavenging though the inhibition of FOXOs, a family of transcription factors that play important roles in regulating the

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expression of genes involved in cell growth, proliferation, differentiation, and longevity. Furthermore, it has been reported that hypoxia also leads to increased mitochondrial complex III ROS production (Jain et al., 2005). In order to survive under stress conditions, cancer cells adapt and acquire different mechanisms to maintain ROS levels as close as possible to their pro-tumorigenic concentration. Cancer cells upregulate various antioxidant systems at the sites of ROS production, maintaining levels of ROS that allow activating pro-tumorigenic signaling pathways without inducing cell death or senescence.

ROS are produced at specific cellular locations by mitochondria and NOX proteins, and can reversibly oxidize cysteine residues in proteins. The best-characterized class of redox-regulated targets is phosphatases, which can negatively regulate cell signaling thought the dephosphorylation and inactivation of kinases. Kinases and transcription factors are also direct targets of ROS oxidation (Meng et al., 2002).

The role of ROS in cancer is two-sided: moderate levels of ROS can contribute to cancer initiation, progression and spreading through the activation and maintenance of signaling pathways that regulate cellular proliferation, survival, angiogenesis and metastasis (Glasauer & Chandel, 2014) (Figure 1.5). However, excessive levels of ROS can also induce cell cycle arrest, cell death signaling and senescence (Glasauer et al., 2005). Moreover, elevated ROS levels cause mitochondrial DNA damage and mutations and alterations of the mitochondrial genomic functions that seem to be implicated in the process of carcinogenesis (Dayem et al, 2010).

Fig 1.5: Dual effects of ROS in cancer. Low levels of ROS can be mitogenic and initiate cellular adaptations that lead to cell survival, growth, proliferation and differentiation in a controlled manner. When levels are too high, ROS induce cancer cell death (from Glasauer & Chandel, 2014).

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Cancer cells function with higher rates of ROS production and higher levels of antioxidant proteins compared to normal cells (Glasauer & Chandel, 2014). This permits high ROS levels to activate proximal pro-tumorigenic signaling pathways without building up excessively high ROS levels that could induce cell death or senescence. The maintenance of the balance between higher ROS and higher antioxidant defenses allows cancer cells to maintain ROS at optimal levels that promote tumor progression. More specifically, the increased rate of ROS production in cancer cells promote the acquisition of various hallmarks of cancer: sustained proliferation, increased cell survival and disruption of cell death signaling, epithelial to mesenchymal transition (EMT), angiogenesis and metastasis (Porporato et al., 2016). Several biological features of cancer cells are affected by ROS, including:

Cell proliferation. ROS promote cancer cell proliferation by increasing proliferative

signaling pathways like PI3K/AKT/mTOR and Mitogen-activated protein

kinases/extracellular signal–regulated kinases (MAPK/ERK) cascades (McCubrey et al.,

2006). H2O2 oxidizes and inactivates phosphatases, such as protein tyrosine

phosphatase 1B (PTP1B) and phosphatase and PTEN, which both normally inhibit the PI3K/AKT pathway (Salmeen et al., 2003; Kwon et al., 2004). Moreover, ROS also inactivate phosphatases inhibiting MAPK, thus resulting in the activation of mitogen signaling (Seth et al., 2006).

Cell survival. Hyper-activation of the PI3K/AKT pathway by ROS can promote cancer cell survival through inhibition of PTEN (Chen et al., 2005; Nitsche et al., 2012). ROS can also activate and stabilize antioxidant regulator NRF2, protecting cells against oxidative stress, cell death and senescence (Ray et al., 2012). Mechanistically, ROS activate NRF2 though oxidation and inactivation of Kelch-like ECH-associated protein 1 (KEAP1) at redox-sensitive cysteine sites. The oxidative modification of KEAP1 inhibits the proteasomal degradation of NRF2, allowing for its stabilization, nuclear accumulation and pro-survival signaling in cancer cells (Kobayashi et al., 2006), including stimulation of antioxidant defenses (DeNicola et al., 2011).

Angiogenesis. Angiogenesis is important for tumor development insofar it provides nutrients and oxygen for continuous tumor growth. Vascular endothelial growth factor

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(VEGF) is a major angiogenesis inducer and it is regulated at the transcriptional level by HIF-1 in response to hypoxia (Forsythe et al., 1996). ROS induce HIF-1α stabilization through inhibition of PHDs, which leads to increased VEGF synthesis, expression, activation and subsequent angiogenesis and tumor progression (Xia et al., 2007). Metastasis. Cancer metastasis is defined as “the complex process by which certain cancer cells at the primary tumor location acquire the ability to penetrate and infiltrate surrounding normal tissues and/or lymphatic or blood vessels, migrate to a differentiate tissue, take root and proliferate to form a secondary tumor” (Chaffer and Weinberg, 2001). Metastasis needs remodeling and intracellular adaptations, including EMT, reduced cell adhesion, increased migration and degradation of tissue barriers

and extracellular matrix (Chiang & Massague, 2008). These events are considered to

be driven by genetic and/or epigenetic alterations within cancer cells. ROS can play a crucial role in tumor metastasis. In particular, mtROS have been shown to promote metastasis formation by upregulating several redox-sensitive pathways, including Drc and Pyk2, two members of the transforming growth factor β (TGFβ) signaling pathway (Porporato et al 2014). As already mentioned before, ROS further induce HIF-1α stabilization, which in turn is related to various key steps of the metastatic cascade and selects for metastatic variants (Chandel et al., 2000).

Cancer cell detachment is the initial event in metastasis formation, and is characterized by the loss of cell adhesion molecules and the acquisition of a mesenchymal phenotype. This process is termed “epithelial to mesenchymal transition” (EMT), although it is never complete and some epithelial characteristics remain (Christiansen & Rajasekaran 2006). A hallmark of EMT is the loss of E-cadherin, an adhesion molecule frequently lost in human cancers and causal in tumor formation and metastasis. Loss of E-cadherin enhances cancer cell motility and resistance to anoikis (a form of programmed cell death that occurs in anchorage-dependent cells when they detach from the surrounding extracellular matrix) (Chiang & Massague, 2008). EMT is a crucial process during the normal development of multicellular organism, where is controlled by AKT-, Wnt-, Notch-, and Hedgehog-signaling pathways and can be induced by a number of growth factors (such as TGF, epidermal growth factor [EGF], hepatocyte growth factor [HGF] and fibroblast growth factor

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[FGF]). Several transcription factors critical for mesoderm formation, like SNAI1, Zinc finger E-box-binding (ZEB) and certain basic helix-loop-helix (bHLH) factors, are known to induce EMT through the repression of E-cadherin and induction of mesenchymal gene expression (Kalluri & Weinberg, 2009). Recent findings also implicate the HIF pathway in EMT induction, acting through mesodermal fate genes. One of the EMT-regulating factors controlled by HIF is SNAI1, a transcription factor indispensable for mesoderm development. In addition, twist family bHLH transcription factor 1 (TWIST1) was identified as a critical downstream target in hypoxia sensing (Gort et al., 2008). TWIST1 is directly regulated by HIF-2 under hypoxia. While SNAI1-mediated repression of E-cadherin indirectly leads to activation of mesenchymal markers such as Vimentin (Cano et al., 2000), TWIST1 can directly activate N-cadherin expression, a marker of EMT progression (Alexander et al., 2006). Thus, both SNAI1 and TWIST1 appear to be under the control of HIFs and can orchestrate the cadherin switch during EMT.

A second important step for tumor metastasis is the degradation of tissue barriers and extracellular matrix (ECM). Strong evidence for a role of hypoxia and HIFs as regulators of ECM degradation and cancer cell migration comes from the analysis of Erler and colleagues (2006) who identified the ECM crosslinking enzyme lysyl oxidase (LOX) as a direct HIF-1-target gene product. LOX was initially reported as a copper-dependent amine oxidase responsible for the catalysis of collagen and elastin cross-linking within the ECM (Kagan et al., 2003). In this process, LOX catalyzes the exchange of an amine

to an aldehyde group on a peptidyl lysine, producing H2O2 and ammonia as

by-products. However, recent works have shown that LOX may also have intracellular functions, including the regulation of cell differentiation, motility/migration and gene

transcription. LOX regulates cell adhesion through a H2O2-mediated mechanism that

activates Src kinase, leading to downstream changes in cell adhesion and migration.

H2O2 functions by activating various protein tyrosine kinases within cells, including Src

and focal adhesion kinases (FAKs). Although the specific mechanism by which H2O2

activates these proteins is not yet understood, it is known that H2O2 increases the

phosphorylation of various protein tyrosine kinases, thus leading to their activation (Rhee, 1999). Moreover, LOX homologues LOXL2 and LOXL3 interact and cooperate with SNAI1 in the downregulation of E-cadherin in carcinoma progression (Peinado et al., 2005). Of note, HIFs are also implicated in the “invasive switch”, an increase in cell

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motility and tissue invasion that allows cells to colonize oxygen-rich areas. HGF is a pleiotropic cytokine that stimulates proliferation and invasion through its receptor, tyrosine kinase proto-oncogene c-Met, which is frequently overexpressed or mutated in human cancers. HIF directly promotes invasion by inducing c-Met transcription, thus sensitizing cells to HGF stimulation (Pennacchietti et al., 2003). A huge variety of intracellular signaling molecules have been implicated in cell migration, including the MAPK pathway, lipid kinases, phospholipases, Ser/Thr and Tyr kinases and scaffold proteins. However, one particular family of proteins, the Rho GTPases, seems to play a pivotal role in regulating the biochemical pathways most relevant to cell migration (Raftopoulou et al., 2004). Interestingly, fibroblasts lacking HIF-1 fail to upregulate RhoA, a core transducer of integrin signaling critical in stress fiber formation (Greijer et al., 2005). Thus, HIF-1 enhances cell motility by acting at multiple levels to facilitate cancer cell migration towards nutrient- and oxygen-rich areas within oxygen-deprived tissues. Of note, ROS can also promote the activation of matrix metalloproteinases (MMPs) that participate in the degradation of membranes and help detach primary cancer cells from the ECM (Binker et al., 2009)

Anoikis. Even if cancer cells continuously enter the bloodstream, only few of them are able to survive and to produce metastatic tumors. During the invasive process, cancer cells are exposed to different stresses, such as a loss of interaction with their environment, that can trigger anoikis. Cancer cells can escape anoikis by repressing apoptosis. For instance, overexpression of neurotrophin receptor TrkB protects from cell death induced by cell-matrix dissociation (Douma et al., 2004). TrkB expression can be upregulated directly by HIF-1 (Vooijs et al., 2008). Furthermore, TrkB can regulate VEGF expression via HIF1 (Nakamura et al., 2006). In addition, c-Met signaling confers resistance to anoikis and supports anchorage independence (Longati et al., 1996). Furthermore, SNAI1/2 and TWIST1 act on anti-apoptotic and prosurvival pathways by antagonizing p53- and c-Myc-induced apoptosis, respectively (Maestro, et al., 1999; Wu et al., 2005). Thus, HIF signaling increases the metastatic potential of cancer cells by promoting cell survival in the bloodstream and in metastatic colonies (Vooijs et al., 2008).

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Src family kinases are critically involved in the control of cytoskeletal organization and in the generation of integrin-dependent signaling that is triggered by cell attachment to the ECM. It is well known that the activity of the Src family of tyrosine kinases is tightly controlled by the inhibitory phosphorylation of a carboxy-terminal tyrosine residue (Tyr527). This phosphorylation induces an inactive conformation in the kinase. Along with this phosphorylation/dephosphorylation switch, Src undergoes redox regulation during cell adhesion, controlling its activation. In particular, the late phase of Src activation (associated with cytoskeletal rearrangement and cell spreading) is characterized by a strong oxidation of the kinase (Giannoni et al., 2005). This oxidation is mainly mediated by ROS produced by the arachidonate 5-lipoxygenase (5-LOX), the main source of ROS during integrin-signaling. The involvement of Src in the motility and invasiveness of cancer cells is a well-known event (Frame et al., 2002), and mounting evidence confirms the role of Src redox regulation in these events. In invasive breast cancer, treatment with CAT leads to a dose-dependent loss of Src-mediated invasion and motility (Payne et al., 2005). Moreover, Src has been described as playing a key role in anoikis resistance, and its redox sensitivity has an essential role in this function (Giannoni et al., 2008). In metastatic prostate carcinoma cells undergoing a constitutively deregulated production of ROS, Src kinase is constitutively oxidized and activated in the absence of adhesion. This allows a constitutive, Src-dependent and ligand-Src-dependent transphosphorylation of EGFR, activating the ERK- and AKT-mediated pro-survival pathways. Antioxidant treatment of prostatic cancer cells completely abolishes the ligand-independent activation of EGFR and restores the

pro-apoptotic stimuli. Conversely, addition of physiological doses of H2O2 to

untransformed epithelial cells allows them to escape from anoikis, confirming the crucial role of ROS in ensuring anoikis resistance. Together, these evidences highlight the fact that the redox regulation of Src is crucial for tumorigenesis and metastatic spread (Giannoni & Chiarugi, 2014).

Epigenetics. In addition to genetic alteration, epigenetic changes accompany tumor progression and metastasis. Especially, hypermethylation of CpG islands in promoter regions of tumor suppressor genes is frequently seen in cancer cells (Esteller, 2002; Herman et al., 2003). For example, Lim et al. (2008) demonstrated that prolonged ROS

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stress induces a methylation of the E-cadherin promoter via a Snail-dependent pathway. Interestingly, redox stress appears to be associated with Snail upregulation, methylation of the E-cadherin promoter and, consequently, E-cadherin downregulation (Lim et al., 2008). Thus, ROS can trigger DNA methylation of tumor suppressor gene promoters in carcinogenesis and metastasis.

1.4-BREAST CANCER

Breast cancer is the most common type of cancer in women, both in the developed and less developed world. It is estimated that over 500.000 women died from breast cancer in 2011 worldwide (Global Health Estimates, WHO 2013). While breast cancer incidence is still increasing, mortality from breast cancer is decreasing in many Western societies, probably due to the combined effect of early detection and improvements in treatments (Peto et al. 2000; Balmain et al. 2003).

The risk of getting breast cancer increases with age. The cause of breast cancer is complex and not fully understood. Germline genetic alterations of highly penetrant genes, like the tumor suppressor genes BRCA1, BRCA2 and TP53, are suggested to explain only 5-10% of all breast cancer cases (Ford et al. 1995; Martin & Weber, 2000). Thus, the majority of breast cancers are probably caused by lifestyle and environmental factors and/or alterations in a variety of low penetrance breast cancer susceptibility genes (Martin & Weber 2000). The major differences in breast cancer incidence between ethnic groups and geographic areas in general are assumed to be due to life style and environmental factors (diet, smoking, high-alcohol consumption) rather than to differences with respect to ethnic background. Evidence in favor of this conclusion was provided when it was revealed that Japanese women immigrating to America over time adapted a breast cancer risk resembling white Americans (Buell 1973).

Breast cancer survival rates greatly vary worldwide, ranging from 80% or over in North America, Sweden and Japan to around 60% in middle-income countries and below 40% in low-income countries (Coleman et al., 2008). The low survival rates in less developed countries can be explained mainly by the lack of effective screening

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strategies, as well as by the lack of adequate measures of diagnosis and therapy (World Cancer Report 2014).

Breast cancer is a highly heterogeneous disease, which comprises a number of distinct biologically entities with specific pathological features and biological behaviors. Different breast tumor subtypes have different risk factors, clinical presentations, histopathological features, outcome and response to systemic therapies. Thus, stratification of breast cancer by clinically relevant subtypes is very important (Chavez et al., 2010).

For many years, tumors of the breast were characterized by tumor size only. However, this sub-classification proved to be limiting for it was unable to define subgroups sharing similar prognostic and therapeutic characteristics. Subsequently, a histological classification system has been developed, dividing breast cancers into subgroups distinguished by the histological appearance of the tumor. Despite advantages over the system based on the tumor size, this classification also failed to form homogenous breast cancer subgroups. Currently, the most used classification system of breast cancer combines histo-morphological information (such as histological subtype and grading) and TNM staging information, based on tumor size (T), lymph nodes (N) and the occurrence of distance metastasis (M) (Sobin et al., 2009). An even more recent approach to classify breast cancer subgroups is that of gene expression profiling. Breast cancer patients routinely have alterations in the expression of estrogen receptor (ER), progesterone receptor (PR) and the amplification of the receptor tyrosine-protein kinase erbB-2 (HER-2/Neu), a member of the human epidermal growth factor receptor family (Brenton et al., 2005). These markers allow classification of breast cancer tumors as hormone receptor-positive tumors, HER-2/Neu-amplified tumors, and those that do not express ER, PR and do not have HER-2/Neu amplification. The latter group is referred to as triple-negative breast cancer (TNBC).

Generally, hormone receptor-expressing breast cancers present a more favorable prognosis than either those with HER-2/Neu amplification or those that are triple negative. While all breast cancers are treated with chemotherapy, therapeutic

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options are significantly affected by the expression of these three markers. Tumors that express ER and PR are treated with agents that interfere with hormone production or activity. Tumors that have HER-2/Neu are treated with agents that inhibit HER-2/Neu, whereas TNBC tumors are treated predominantly with chemotherapy (Brenton et al., 2005). Importantly, TNBC represents approximately 10-15% of all breast cancers, and patients with TNBC have a poor outcome compared to the other subtypes of breast cancer, not only because hormonal therapy and treatment with targeted therapy (e.g. trastuzumab, effective only to those tumors harboring overexpression of HER-2) are ineffective, but also because these tumors are more aggressive than other breast carcinoma subtypes (Nofech-Mozes et al. 2009). Compared to other breast cancer subtypes, TNBC indeed develops earlier in live and, consequently, more often in pre-menopausal women (Carey et al., 2006). At diagnosis, TNBCs are commonly of high nuclear mitotic grade, of larger tumor size, and they show a more aggressive expression profile with low Bcl-2 but high p53 and KI-67 expression ( (Tian et al., 2008).

In addition to surgery and radiotherapy, chemotherapy is a main option for treating malignant breast cancer and can increase the lifespan of the patient (Decatris, et al., 2004). Cisplatin is an important chemotherapeutic agent widely used for the treatment of a variety of malignancies, including breast cancer. In addition, it is generally accepted that taxanes are among the most active chemotherapeutic agents in the management of metastatic breast cancer (Ghersi et al., 2005). These agents are discussed in chapter 1.5.

1.5-CHEMOTERAPEUTIC DRUGS

Currently, cancer therapy is largely based on chemotherapy, i.e., the administration of cytotoxic or antineoplastic drugs that inhibit the proliferation of cancer cells. This approach is categorized as either neoadjuvant chemotherapy or adjuvant chemotherapy. Neoadjuvant chemotherapy is used before surgery and radiotherapy of the primary tumor in order to decrease the size of the tumor and to destroy any micrometastases (Trimble et al., 1993). Adjuvant chemotherapy is used after surgery to increase the chances of recovery, to destroy neoplastic foci

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residues and to avoid relapses. One can also distinguish mono-chemotherapy, based on the use of only one type of drug (until it loses its effectiveness and is replaced with another) and poly-chemotherapy that uses a combination of drugs having different characteristics, with the aim to delay as much as possible the appearance of chemoresistance. Choosing the right drug to use is closely related to several factors, such as location, size, isotype, tumor stage and patient's clinical characteristics (age, gender…). Among cytotoxic chemotherapies, the most used are alkylating agents, anti-metabolites, mitotic spindle inhibitors and topoisomase II inhibitors. Here, we will briefly review these classes of chemotherapy and then focus on cisplatin and paclitaxel that are at the core of our study.

-Alkylating agents exert their greater cytotoxic action on DNA, causing structural and functional alterations. They are active on proliferating cells in S phase, blocking the cell cycle and leading to cell death. Alkylating agents are used to treat several types of cancers. However, they are also toxic to normal cells, particularly cells that divide frequently, such as those in the gastrointestinal tract, bone marrow, testicles and ovaries. One must distinguish between poly and mono-functional alkylating agents. Poly-functional alkylating agents cause alkylation reactions at the level of DNA bases, promoting the formation of bridges and crosslinks between the two strains, which will thus be unable to separate during the replication and transcription processes, causing the cytotoxic effect. Among poly-functional alkylating agents, one finds platinum coordination complexes such as cisplatin, carboplatin and oxaliplatin. Mono-functional alkylating agents bind to DNA, destabilizing the guanylyl bases and favoring their loss (or depurination) with fragmentation of the double helix.

-Antimetabolites agents have a chemical structure similar to normal metabolites, inhibiting various enzymatic reactions in the synthesis of purines and pyrimidines or their precursors (amino acids). Generally, they act in the S phase of the cell cycle, going to hinder the synthesis of DNA and RNA. Antimetabolites can be further divided into antifolates (drugs that antagonize the action of folic acid such as methotrexate), purine analogues (6-mercaptopurine) and pyrimidine analogs (5-fluorouracil).

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-Mitotic inhibitors are drugs that inhibit mitosis. Mitotic inhibitors are usually derived from natural substances such as alkaloids, and prevent cells from undergoing mitosis by disrupting microtubule polymerization, thus preventing cancer growth. Some examples are taxanes (paclitaxel, docetaxel), Vinca alkaloids and colchicine. The principal mechanism of action of taxanes is the disruption of microtubule function. Microtubules are essential for cell division, and taxanes stabilize GDP-bound tubulin in the microtubule, inhibiting the process of cell division because depolymerization is prevented. In contrast, Vinca alkaloids prevent mitotic spindle formation though inhibition of tubulin polymerization. Both, taxane and Vinca alkaloids are, therefore, named spindle poisons or mitosis poisons, even if they act in different ways.

-Topoisomerase inhibitors are agents designed to interfere with the action of topoisomerases, i.e., enzymes that control the changes in DNA structure by catalyzing breaking and rejoining of the phosphodiester backbone of DNA strands during normal cell cycle. An important class of this type of drugs is anthracyclines (doxorubicin and its derivative epirubicin, used mostly in breast cancer; and daunorubicin, used to treat acute lymphoblastic or myeloblastic leukemias).

Our study focused on the effect of cisplatin and paclitaxel in enhancing ROS production in cancer cells. These two chemotherapeutic drugs are widely used in the clinics for different types of tumors, among which TNBC (Kampan et al., 2015). We focused on cisplatin and paclitaxel according to their well describe effect on oxidative homeostasis and due to the fact these drugs induce a proliferation arrest and a ROS increase thought two very different mechanisms of action (Saad et al., 2004; Alexandre et al., 2006). These differences allow to investigate the pathophysiological consequences of increased ROS production as a consequence to two different mechanisms.

-CISPLATIN: Cisplatin (Figure 1.6) and its analogue carboplatin are nowadays among

the most commonly used antitumor drugs. Cisplatin was first described by Michele Peyrone in 1845, and known for a long time as Peyrone’s salt. Its anticancer proprieties remained unnoticed until the mid-1960s, when Rosenberg and collaborators (1965, 1969) discovered that the electrolysis of platinum electrodes generated a soluble

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platinum complex that inhibited binary fission in E. coli. Although bacterial cell growth continued, cell division was arrested. Amongst the platinum complexes formed, cisplatin was identified as the main antiproliferative agent. In the next years, cisplatin was developed into one of the most widely used drugs in cancer chemotherapy. Cisplatin is highly effective in the treatment of testicular, ovarian, bladder, head and neck, esophageal, small and non-small cell lung, breast, cervical, stomach and prostate cancers. Moreover, interesting new evidence suggests that cisplatin therapy is effective for the treatment of TNBC patients (Silver et al., 2010).

Fig 1.6: Cisplatin structure. Cisplatin is an inorganic and water-soluble platinum complex. After undergoing hydrolysis, it reacts with DNA to produce both intra and interstrand crosslinks. These crosslinks impair replication and transcription of DNA.

Despite its success, cisplatin has several disadvantages that include severe side effects, e.g. nephrotoxicity, neurotoxicity, ototoxicity, nausea and vomiting. These toxic effects limit the dose that can be applied to patients (Pasetto et al., 2006). Despite a consistent rate of initial responses, cisplatin treatment often results in the development of chemoresistance, leading to therapeutic failure (Koberle et al., 2010). Some tumors, such as colorectal and non-small cell lung cancers, have intrinsic resistance to cisplatin, whereas others, such as ovarian or small cell lung cancers, develop acquired resistance after an initial treatment (Galluzzi et al., 2012). Resistance to cisplatin is generally considered as a multifactorial phenomenon mainly due to reduced drug accumulation, inactivation by thiol-containing species, increased repair of platinum-DNA adducts, improved platinum adduct tolerance and failure of cell death pathways (Cepeda et al., 2007). In vitro, these mechanisms are cell line-dependent, thus different cell lines exhibit different levels of tolerance to a same platinum treatment. A way to circumvent cisplatin resistance is the combination of cisplatin with drugs that interfere with specific cisplatin-resistance pathways (Fuertes et al., 2013).

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Drug accumulation: Once cisplatin is intravenously administered to a patient, it rapidly diffuses into tissues and is highly bound to plasma proteins, as a result of the strong reactivity of platinum for sulfur of thiol groups of amino acids, such as cysteine. Therefore, about 90% of platinum in the blood is bound to albumin and other plasma proteins (i.e., ɣ-globulins and transferrin), leading to the sequestration of a great amount of cisplatin molecules (Judson & Kelland, 2000). In the blood where the chloride concentration is relatively high (~100 mM), cisplatin exists mainly in the dichloro neutral form. Loss of the chloride groups from the cisplatin molecule is required for intracellular cisplatin activity (Jamieson & Lippard, 1999; Miller & House, 1991). When the neutral compound enters the cell, the relatively low chloride concentration (2-30 mM) promotes the replacement of one or both chloride leaving groups by water, resulting in positively charged molecule mono and diaquo species

([Pt(H2O)Cl (NH3)2] + and [Pt(H2O)2(NH3)2] 2+ cations) that react with nucleophilic

molecules such as DNA, RNA and proteins (Kartalou & Essigmann, 2001). The biochemical mechanism by which cisplatin crosses the cell membrane still remains unclear. Early studies reported that cisplatin uptake is not inhibited by its structural analogues, and the entry into the cell did not seem to be dependent on an optimum pH (Binks et al., 1990). So, it was suggested that passive diffusion was the main mechanism by which cisplatin enters the cell. However, it was found later that a certain degree of cisplatin uptake is energy-dependent and could be modulated by a variety of pharmacological agents that do not cause a general permeabilization of the membrane and by activation of some intracellular signal transduction pathways. Thus, it has been proposed that part of the initial rate of uptake of cisplatin is due to passive diffusion, but also facilitated diffusion through a gated channel (Gately & Howell, 1993) and a copper transporter (Ishida et al., 2002), leading to propose an active transport for cisplatin in addition to passive diffusion.

Binding to DNA: It is generally accepted that binding of cisplatin to genomic DNA (gDNA) in the cell nucleus is the main event responsible for its antitumor properties. Cisplatin targets gDNA, blocking DNA replication, inducing G2 cell cycle arrest, inhibiting RNA transcription and finally promoting cell death through apoptosis (Zorbas

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& Keppler, 2005; Eastman, 1999). When binding to DNA, cisplatin favors the N7 atom of the imidazole rings of guanosine and adenosine that are the most accessible and nucleophilic reactive sites for platinum coordination to DNA. Cisplatin may form several types of lesions on purine bases of DNA: mono-adducts and bifunctional adducts called intrastrand or interstrand crosslinks (Payet et al., 1993) (Figure 1.7). The binding of cisplatin to DNA appears as a phasing phenomenon that begins with the formation of mono-adducts. However, greater than 90% of mono-adducts then react further to form DNA crosslinks, which blocks replication and/or prevents transcription. Almost 60-65% of these crosslinks are d(GpG) intrastrand, whereas 20-25% are 1,2-d(ApG) intrastrand cross-links. It has been recently shown that the minor 1,3-d(GpTpG) intrastrand cross-link could also be important in the mechanism of action of cisplatin (Fuertes et al., 2003). In fact, the minor 1,3-d(GpTpG) intrastrand cross-link adduct induces an asymmetric arrangement of DNA in nucleosomes in relation to the histone octamer, which affects the translational positioning of the DNA. All crosslinks result in contortion of the DNA. Moreover, DNA-protein crosslinks might also contribute to the cytotoxic activity of cisplatin.

Fig 1.7: Main adducts formed after binding of cisplatin to DNA. (A) 1,2-intrastrand cross-link, (B) interstrand cross-link, (C) monofunctional adduct, and (D) protein-DNA cross-link. The main site of attack of cisplatin to DNA (N7 of guanine) is shown in the central panel (from Cepeda et al., 2007).

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Binding to non-DNA targets: When cisplatin crosses the cell membrane, the cis-Pt(II) center may coordinate to some constituents of the lipid bilayer that contain nitrogen or sulfur atoms, including phospholipids and phosphatidylserine (Jordan & Carmo-Fonseca, 2000). Moreover, in the cytoplasm, many components that present nucleophilic sites may react with cisplatin (i.e., cytoskeletal microfilaments, thiol-containing peptides, proteins and RNA). Interestingly, only 5-10% of covalently bound cell-associated cisplatin is found in the gDNA fraction, whereas 75-85% of the drug binds to proteins and other cellular constituents (Akaboshi et al., 1994). The most important non-DNA target of cisplatin is probably tripeptide glutathione (GSH), which is present in cells at high concentrations (0.5-10 mM). GSH and other thiol-containing biomolecules, such as metallothioneins (MT), bind quickly to platinum. Cisplatin binding to GSH and MT has primarily been associated with negative pharmacological properties, including the development of resistance and toxicity (Lai et al., 1989). As a potent nucleophile, GSH reacts with cisplatin, forming a GSH-platinum complex that is then eliminated from the cell by an ATP-dependent glutathione export pump. On another hand, cisplatin can also alter the activity of enzymes, receptors and other proteins through coordination to sulfur atoms of cysteine and/or methionine residues and to nitrogen atoms of histidine residues (Arnesano & Natile, 2008). In fact, binding of cisplatin to N-terminal methionine (Met1) and histidine (at position 68) of ubiquitin may inhibit the ubiquitin-proteasome pathway, which ends up in cytotoxic events (Peleg-Shulman et al., 2001). In addition, it has been found that cisplatin, besides inhibiting the activity of heat shock protein 90 (Hsp90, an ATP-binding chaperone) in

vitro, efficiently and specifically blocks its C-terminal ATP binding site. The C-terminal

of Hsp90, via its ATP hydrolytic function, is involved in the correct folding of proteins that play a role in signal transduction and cell cycle regulation (Soti et al., 2002).

It has been shown that most cisplatin molecules that enter the cell are bound to proteins rather than DNA, and there is experimental evidence showing that the former type of damage also plays an important role in the initiation of both apoptotic and necrotic pathways (Peleg-Shulman & Gibson 2001). In this scenario, a number of additional properties of cisplatin are now emerging, including activation of signal transduction pathways leading to apoptosis. Such pathways may originate at the level

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of the cell membrane after damage of receptor or lipid molecules by cisplatin, in the cytoplasm by modulation of proteins via interaction of their thiol groups with cisplatin and from DNA damage via activation of the DNA repair pathways (Boulikas & Vougiouka, 2003). In this context, AKT, c-Abl, p53, MAPK/JNK/ERK/p38 and related pathways respond to the presence of DNA lesions (Cepeda et al., 2007). AKT is a most important Ser/Thr protein kinase promoting cell survival, and it protects cells from damage induced by different stimuli as well as by cisplatin (Datta et al., 1999). Cisplatin downregulates X-linked inhibitor of apoptosis protein (XIAP) protein levels and promotes AKT cleavage, resulting in apoptosis in chemosensitive but not in resistant ovarian cancer cells (Fraser et al., 2003). c-Abl is an important protein in the signaling of the DNA damage, which belongs to the SRC family of non-receptor tyrosine kinases (Cepeda et al., 2007; Wang & Lippard, 2005). This molecule acts as transmitter of DNA damage triggered by cisplatin from nucleus to cytoplasm (Shaul, 2000). Accordingly, sensitivity to cisplatin-induced apoptosis is directly related to c-Abl content and could be blocked by c-Abl overexpression (Wang & Lippard, 2005).

Cisplatin further induces oxidative stress and is an activator of stress-signaling pathways, especially of the MAPK cascade. Consequently, treatment with cisplatin induces apoptosis following G2 arrest, but seems not to induce a G1 arrest (Sorenson et al., 1988).

Interestingly, it has further been found that exposure of HeLa human cervix carcinoma cells to a low concentration of cisplatin produces shortening and degradation of telomere, which leads to cell death (Ishibashi & Lippard, 1998). Moreover, recent evidence suggests that cisplatin induces cell death through the activation of other apoptotic pathways. For example, Mandic et al. (2002) demonstrated that cisplatin induces calpain activation, which is an early event preceded by and dependent on an

increase in intracellular Ca2+. Calpain activation induces apoptosis in human melanoma

cells and occurs after c-Jun N-terminal kinase (JNK) activation, but hours before Bak modulation, cytochrome c release and activation of caspase 9, thus suggesting the existence of different apoptotic pathways activated in different phases after the treatment of cells with cisplatin. Specifically, cisplatin activates caspase 12 and upregulates Grp78 in enucleated cells (cytoplasts lacking a cell nucleus) in a

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mechanism associated with a rapid induction of cellular ROS, ER stress and increases in

Ca2+ levels, regardless of the extend of DNA damage (Mandic et al., 2003). It is possible

that it is its ability to activate two rather than a single major pathway of apoptosis that makes of cisplatin a generally efficient anticancer agent.

Cisplatin also induces a clustering of Fas/CD95 at the plasma membrane, long-term growth arrest ("premature cell senescence") and mitotic catastrophe (Havelka et al, 2007). It induces E-cadherin cleavage, resulting in disconnection of the actin cytoskeleton and accumulation of E-cadherin and β-catenin in the cytoplasm (Latifi et al., 2011).

Cisplatin and mitochondria: While most investigations of cisplatin cytotoxicity have been focused on interactions between cisplatin and gDNA, only approximately 5-10% of intracellular platinum is bound to gDNA, with the great majority of the intracellular drug available to interact with other nucleophilic sites on other molecules, including but not limited to phospholipids, cytosolic, cytoskeletal and membrane proteins, RNA, and mtDNA (Fuertes et al., 2003). The electrochemical gradient resulting in a negative charge within mitochondria may play a role in the accumulation of positively charged cisplatin in this organelle (Cullen et al., 2007). Mitochondria contain their own DNA (mtDNA) that is transcribed and translated to synthesize proteins of the mitochondrial ETC (Fernandez-Silva et al., 2003). Cisplatin may crosslink mtDNA that, lacking nucleotide excision repair, is significantly more sensitive than gDNA to the damage (Preston et al., 2001). Indeed, mitochondria are thought to be a major target of cisplatin in cancer cells, and alterations in mitochondrial function (reduced mitochondrial respiration and ATP production) have been investigated in cancer cell chemoresistance (Harper et al., 2002). Recent findings have shown that cells depleted of mtDNA gain significant resistance to cell death induced by cisplatin (Park et al., 2004; Montopoli et al., 2009), strongly suggesting that mtDNA could be a major target of cisplatin. Furthermore, mitochondrial damage by cisplatin has been increasingly studied as a mediator of systemic toxicity, such as gastrointestinal toxicity, ototoxicity (Devarajan et al., 2002) and nephrotoxicity (Hanigan & Devarajan 2003).