Molecular networks in respiratory epithelium carcinomas

Cancer Letters 295 (2010) 1–6

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Molecular networks in respiratory epithelium carcinomas Athanasios G. Pallis a,b, Michalis V. Karamouzis a, Panagiotis A. Konstantinopoulos a,c, Athanasios G. Papavassiliou a,* a

Department of Biological Chemistry, Medical School, University of Athens, Athens, Greece Department of Medical Oncology, University General Hospital of Heraklion, Crete, Greece c Department of Medicine, Division of Hematology/Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA b

a r t i c l e

i n f o

Article history: Received 6 January 2010 Received in revised form 14 March 2010 Accepted 17 March 2010

Keywords: Respiratory epithelium carcinomas EGFR Ras Raf MAPK PI3K Akt JAK STAT PLC PKC

a b s t r a c t Current anti-cancer research is focused on cell surface receptors targeting, mainly epidermal growth factor receptor and vascular endothelial growth factor receptor, against which a few targeted agents are now available in clinical practice. Recent improvements of our understanding on the intracellular networks that participate in respiratory epithelium carcinogenesis have further elucidated the role of a variety of molecules that represent attractive targets for novel therapeutic strategies. The aim of this review is to explore the potential therapeutic opportunities of the manipulation of these pathways. Ó 2010 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Carcinomas that originate from the respiratory epithelium are the leading causes of cancer-related mortality worldwide [1], with a 5-year survival of approximately 15% for all stages [2]. Their pathogenesis is causally linked with exposure to carcinogens, mainly tobacco, and they are usually diagnosed at advanced stages where available treatments have limited curative potential [3]. Tumorigenesis and progression of lung cancer is dependent on a variety of cross-reacting growth-stimulating pathways which are activated independently of their surrounding normalappearing environment. These pathways consist of extra-

* Corresponding author at: Department of Biological Chemistry, University of Athens Medical School, 75 Mikras Asias Str., 115 27 Athens, Greece. Tel.: +30 210 746 2508/9; fax: +30 210 779 1207. E-mail address: [email protected] (A.G. Papavassiliou). 0304-3835/$ - see front matter Ó 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2010.03.014

cellular growth factors, their membrane receptors and downstream intracellular signal transduction pathways that transfer the signal to key molecules such as cytoskeletal proteins, metabolic regulators, and transcription factors. Membrane receptors can be divided in two categories: G-protein-coupled receptors and receptors with intrinsic kinase activity. The most studied signal transduction pathways are the Ras/Raf/MAPK pathway, the phosphatidylinositol-3-OH kinase pathway, and phospholipase C/protein kinase C pathway. However, it should be noted that these pathways exhibit an extensive intracellular ‘‘cross-talk” and form a complicated network [4]. Apart from these cascades, recently published data have yielded the important role of JAK/STAT [5] and NRF2/KEAP1 [6] pathways. All these advances have enabled the discovery of several potential molecular targets and have driven the development of novel targeted therapies.

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In the present review we will focus on the most important cellular networks and their associated molecules that have been shown to hold a pivotal role in various aspects of malignant transformation and progression in respiratory epithelium. The Epidermal Growth Factor Receptor (EGFR) pathway will not be presented in this review since it has been presented in detail in several other reports [7,8]. Additionally, we will report the various emerging therapeutic strategies against these molecules as well as promising representative compounds, and consider their future perspectives in respiratory epithelium carcinomas therapeutics.

non-small-cell lung carcinomas (NSCLC) [12], and lung cancer cell lines harboring c-Met amplification are dependent on Met for growth and survival [9]. Moreover, c-Met increased gene copy number is an independent negative prognostic factor in surgically resected NSCLC [13]. More importantly, c-Met amplification has been shown to be directly implicated in resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) through ERBB-3 activation [14–16]. Activating mutations of the cMet have also been described in several other human carcinomas [9]. A variety of Met inhibitors are under development and/or early clinical testing [9].

2. Membrane receptors other than EGFR

2.2. Insulin-like growth factor receptor-1

2.1. Met

Insulin-like growth factor receptor-1 (IGFR-1) is a transmembrane protein that holds a significant role in promoting oncogenic transformation, growth and survival of cancer cells [17]. IGFR-1 activation triggers a cascade of reactions involving two main signal transduction pathways [18]: one activates Ras–Raf–MAPK network and the other involves PI3K; therein showing distinct similarities to the family of ERBB receptor protein family (Fig. 1). Deregulation of IGF signaling has been described in several tumor types, including lung cancer. Furthermore, elevated IGF-1 levels have been associated with increased risk of lung cancer, while increased quantity of IGF-Binding Protein 3 has been correlated with reduced risk [17]. Expres-

Met is the receptor for Hepatocyte Growth Factor (HGF) and is frequently overexpressed in respiratory epithelium carcinomas [9]. HGF stimulation of Met can lead to proliferation, increased survival, altered motility, enhanced invasion into extracellular matrix, and more rapid formation of tubules. Thus, Met is considered as an important receptor in respiratory epithelium carcinogenesis [10]. A number of intracellular pathways participate in Met signaling, including, mitogen-activated protein kinase (MAPK), phosphoinositol 3-kinase (PI3K), and phospholipase C-c (PLC-c) [11]. c-Met gene amplification has been reported in 4–7% of

mAb

(e.g. cetuximab)

(e.g. pertuzumab)

mAb (e.g. bevacizumab)

VEGFR TKIs

VEGFR

HER-2

PDGFR

VEGF

EGFR

EGFR

EGFR/HER-2 TKIs (e.g. lapatinib)

Growth Factors

(e.g. gefitinib, erlotinib)

EGFR

EGFR-TKIs

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(e.g. SU5416)

Cell Membrane

Cytoplasm

PI3K IKK

RAS PLC-γ JAK/STAT PDK1

RAF

NF-κB

AKT

IκB

PKC MAPK

NF-κB

Proteasome

Bortezomib

mTOR Translation of proteins

Transcription of target genes

DNA Nucleic acid-directed strategies (e.g. siRNAs, AS-ODN, ribozymes)

Nucleus Tumor growth

Angiogenesis Apoptosis

Differentiation Invasion / Metastasis

Fig. 1. Major molecular networks participating in respiratory epithelium carcinogenesis and potential ways of targeting.

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sion of IGFR-1 has been detected in squamous cell lung carcinomas, although no survival difference has been observed between IGFR-1 positive and negative surgically resected NSCLC patients [19]. Monoclonal antibodies and TKIs against IGFR-1 have been developed and currently being tested in the clinical setting [20]. 3. Intracellular pathways 3.1. Ras/Raf/MAPK pathway This pathway is usually activated via a signal from a tyrosine kinase membrane receptor (Fig. 1). Among the most widely studied receptors leading to the activation of this pathway are EGFR and platelet-derived growth factor receptor (PDGFR). Ras pathway plays an important role in cell cycle regulation, wound healing, tissue repair and cell migration [21]. Additionally, recently published data support the notion that this pathway is involved in angiogenesis through alteration of the expression of genes participating in the formation of new blood vessels [22]. The role of MAPK network in human carcinogenesis is well documented and K-ras mutations have been found in 15–50% of lung cancers. These mutations might contribute to EGFR independent activation of downstream molecules [23], resulting in the emergence of resistance to anti-EGFR agents [24] and are considered mutually exclusive with EGFR mutations in NSCLC [25]. However, despite Ras/Raf/MAPK pathway’s recognized role in lung carcinogenesis, clinical trials with Ras and Raf kinase inhibitors in NSCLC have not shown positive clinical results [26,27]. Similarly, other potential targeting strategies such as Ras protein expression inhibition with nucleic-acid directed techniques (e.g. anti-sense oligonoucleotides) [28], or inhibition of membrane localization through modifications of post-translational modification and trafficking (e.g. farnesyl-transferase inhibitors) [29] have not demonstrated efficacy in the treatment of lung cancer. 3.2. PI3K/AKT pathway The phosphoinositide 3-kinase (PI3K)/Akt pathway has been found to have a prominent role in several crucial cellular processes such as cell-survival, proliferation and differentiation (Fig. 1) [30]. PI3K can be activated by receptor tyrosine kinases (RTKs) or G-protein-coupled receptors [31]. Afterwards, it leads to direct activation of Akt, a serine–threonine kinase, which subsequently upregulates mTOR activity leading to enhancement of translation process, thus resulting in increased cellular growth and survival [32]. The phosphatase and tensin holologue (PTEN) is the most important negative feedback molecule of the PI3K/Akt pathway [33]. The PI3K/Akt signaling is deregulated in many cancers, among them respiratory epithelium carcinomas, mostly through membrane receptor activation and/or somatic mutations of the downstream proteins, leading to constitutive activation of the pathway. The main strategies that are being developed and tested against this signaling network are: (i) dual PI3K-mTOR

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inhibitors, (ii) PI3K inhibitors, (iii) mTOR inhibitors, and (iv) Akt-inhibitors [34]. Dual PI3K-mTOR inhibitors exert their antitumor action by inhibiting the catalytic activity of both PI3K and mTOR [34]. The potential advantages for this class of drugs are straightforward as complete inhibition of PI3K as well as mTOR would be expected to effectively blackout the PI3K-Akt-mTOR signaling [34]. These inhibitors are now tested in the context of phase I/II trials [34]. The PI3K inhibitors can be divided into isoform specific inhibitors or pan-PI3K inhibitors. As reported by Engelman, specific inhibitors may have the advantage that they might be tolerated at doses that result in complete target inhibition without producing adverse side effects, such as immunosuppression and glucose intolerance [34]. PI3K inhibitors are tested in the context of phase I trials in various solid tumors. Two different strategies have been developed for targeting Akt: competing the catalytic site with ATP mimetics and non-catalytic site inhibitors [35]. Although there is a growing bank of preclinical data about this class of drugs there are no clinical data about its efficacy in lung cancer [36]. Temserolimus and everolimus selectively inhibit the kinase mammalian target of rapamycin and consequently block the translation of cell cycle regulatory proteins and prevent overexpression of angiogenic growth factors. Both these agents have been tested in lung cancer [37–39], albeit with moderate results. 3.3. JAK/STAT pathway The Janus kinase (JAK)–signal transducer and activator of transcription (STAT) pathway is another significant intracellular signaling pathway mediating cytokine signals [5]. The pathway is activated by ligand binding to transmembrane receptors (Fig. 1). This leads to receptor dimerization and cross-activation of receptor-associated JAK kinases, which in turn phosphorylate tyrosine residues in the cytoplasmic part of the receptor. The next step is the activation (phosphorylation) of latent cytoplasmic STAT proteins. Phosphorylated STAT proteins dimerize through Src-homology 2 (SH2)-domain–phospho-tyrosine interactions and translocate to the nucleus, where they function as transcriptional activators, regulating several genes encoding cell-survival factors, (e.g. Bcl-2 protein family), genes involved in cell proliferation, (e.g. cyclin D1 and c-myc), and genes implicated in angiogenesis and/or metastasis, (e.g. vascular endothelial growth factor, VEGF) [40]. There are seven STAT proteins (STAT1–4, 5A, 5B and 6) and four JAK kinases [JAK1–3 and tyrosine kinase 2 (TYK2)] in mammals [41]. JAK/STAT pathway is considered as a significant pathway in respiratory epithelium tumorigenesis [42]. STAT1 serves as a potent inhibitor of growth and promoter of apoptosis through signalling by cytokines (interferon-c, interleukin-21) and thus functionally impaired and/or mutated Stat1 could lead to increased risk of cancer [43]. Another potential mechanism by which JAK/STAT pathway increases carcinogenesis is being persistently activated, as it has been described in human tumors and tumor-derived cell lines [5]. Persistently activated STAT proteins enter the nucleus and with other transcriptional co-activators and/or transcription factors lead to increased transcriptional initiation affecting a variety of

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cellular processes such as differentiation, proliferation, cell growth, survival and apoptosis [44]. Furthermore, it has been suggested that STAT proteins, and particularly STAT3, are required to maintain a transformed phenotype [45]. STAT5 is also commonly found to be constitutively activated in certain malignancies [46]. Accumulating evidence unravels STATs molecular interplays, rendering some of them promising targets for lung cancer therapeutics with new developing strategies [5]. 3.4. PLC/PKC pathway Protein kinase C (PKC) consist a family of serine/threonine kinases, which have a crucial role in cell proliferation, differentiation, angiogenesis, and apoptosis [47]. Extracellular signals, such as hormones, growth factors, mitogens and neurotransmitters, interact with their cell surface receptors, and activate phospholipase C (PLC) which in turn generates diacylglycerol (DAG) and inositol triphosphate from inositol phospholipids (Fig. 1) [48]. The latter, in turn, releases calcium from the intracellular store finally resulting in PKC activation [49]. Once activated, PKC phosphorylates proteins and triggers many cellular processes, including cell proliferation, differentiation, membrane transport and gene expression [47]. PKC isozymes are commonly deregulated in respiratory epithelium carcinomas [50]. These isozymes have been implicated as downstream effectors of oncogenic K-ras in lung cancer and it has been shown to be overexpressed in NSCLC [51,52], while a constitutively active catalytic fragment of PKC has been associated with enhanced growth rate in lung cancer cell lines [53]. Additionally, a kinase-deficient PKC isozyme, when expressed, blocks anchorage-independent growth in vitro and tumorigenicity in vivo [49]. Thus, it is clear that PCK pathway is a central player in respiratory epithelium carcinogenesis. However, clinical trials with mRNA antisense oligonucleotides that inhibits PKC expression, in combination with chemotherapy, have been negative [54]. Other blocking strategies are being developed and currently evaluated [50]. 3.5. NRF2/KEAP1 pathway Nuclear factor-erythroid 2 related factor 2 (Nrf2) is a pivotal transcription factor for cell protection from oxidative and electrophilic insults [55]. In the unstressed condition, Kelch-like ECH-associated protein 1 (Keap1) suppresses cellular Nrf2 in cytoplasm and its level is regulated by the Keap1-dependent ubiquitination and proteasomal degradation systems [56]. A number of activating factors such as oxidants, pro-oxidants, antioxidants, and chemopreventive agents inhibit the activity of Keap1dependent ubiquitin ligase and Nrf2 is translocated to the nucleus, where it forms a heterodimer with small Maf proteins and binds to a consensus sequence called the antioxidant response element [6]. Nrf2 induces cellular rescue pathways against oxidative injury, abnormal inflammatory and immune responses, apoptosis, and carcinogenesis [57]. Approximately 15% of patients with lung cancer harbour somatic mutations in KEAP1 that prevent effective NRF2 repression. Two NRF2 ‘hot-spots’ mutations

have been identified in patients with lung cancer, allowing the Nrf2 to escape KEAP1-mediated regulation [58]. Moreover, recent data suggest that Nrf2 transactivates a wide variety of genes, including several ATP-dependent drug efflux pumps [59,60]. Additionally, Nrf2 modulates the proliferation of respiratory epithelial cells through alterations of cellular glutathione levels [61]. Therefore, it seems rational to assume that activation of Nrf2 in cancer cells provides proliferation and survival advantage against the exposure to anti-cancer agents. Recently, it was also reported that functional inhibition of Nrf2 could lead to suppression of both cell proliferation and resistance to the anti-cancer drugs in lung cancer cell lines [62]. No inhibitors of the Nrf2 have been integrated in the clinical trial setting of lung cancer yet. 4. Ubiquitin–proteasome pathway Proteasome is a multicatalytic proteinase complex that is responsible for the degradation of intracellular proteins [63]. Protein degradation is a highly conserved process that involves protein recognition and then degradation by the proteasome (Fig. 1). Several proteasome substrates have been identified and include p21 and p27 (cell cycle inhibitors), p53 (transcription factor), IjB (inhibitor of NF-jB) and Bcl-2 (regulation of apoptosis) [64]. Inhibition of the proteasome activity leads to intracellular accumulation of proteins such as p53, p27, Bad and Bax or activation of stress kinases that lead to activation of the intrinsic apoptosis pathway [65]. Bortezomib is a small-molecule reversible proteasome inhibitor which mediates apoptosis through regulation of the Bcl-2 protein family. This molecule has shown moderate activity against NSCLC and is being further studied [66]. 5. Future perspectives Although chemotherapy has been the backbone of the treatment of respiratory epithelium carcinomas, little substantial benefit has been gained in terms of survival and it is more than clear that this type of treatment has reached an activity plateau [67]. Further improvement in lung cancer therapeutics will presumably require the integration of novel targeted therapies. Molecular networks understanding represent both a challenge and a prerequisite for future lung cancer treatment options. Although some targeted agents have already proved their efficacy in the treatment of respiratory neoplasms [68–71], several issues remain unanswered. The most intriguing assignment is to develop reliable and easily applicable predictive factors that will allow better selection of the patients who are most likely to benefit from a targeted agent. Furthermore, as these compounds will be integrated into clinical practice, potential concerns associated with toxicity and resistance have been arouse. Additionally, the optimal way to effectively integrate these targeted treatment strategies to conventional therapies is still pending. The development of respiratory epithelium tumors is governed by a multistep process linked to several intracel-

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lular pathways and genetic / epigenetic alterations. Thus, using a single targeted agent strategy might not represent the best strategy to substantially improve clinical outcome. Future research is also focused on the development of a combinatorial strategy of agents targeting several different pathways or multi-targeting molecules. It is wide perception that the clarification of the molecular network complexity during respiratory epithelium carcinogenesis will translate to better treatment options. Conflict of interest

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