Recent Advances in the Signal Transduction

Current Signal Transduction Therapy, 2009, 4, 6-21

6

Recent Advances in the Signal Transduction Targeting of Colorectal Cancer: The Paradigm of Translational Medicine Haralabos Kalofonosa,* and Petros D. Grivasa,b a

Division of Oncology and Clinical Oncology Laboratory, Medical School, University of Patras, 26504 Patras, Greece; Department of Internal Medicine, Hahnemann University Hospital/Drexel College of Medicine, Philadelphia, PA19102, USA b

Abstract: The dynamic cross-talk between bench–based research and clinical practice is the fundamental characteristic of modern oncology. Colorectal cancer, the second most common cause of cancer–related death in the Western world, is the paradigm of the revolutionary use of targeted sophisticated therapies. The continuous unraveling of the molecular identity of signaling pathways that govern sub-cellular decisions reveals candidate targets. The epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) signaling pathways are considered major tumor-promoting cascades. Three monoclonal antibodies have been approved for the management of metastatic colorectal cancer. Cetuximab and panitumumab target the EGFR-signaling pathway, inhibiting the aberrant transduction of tumor–proliferating signals, as well as promoting tumor cell apoptosis. Bevacizumab is the first antibody inhibiting VEGF–driven formation of new blood vessels, a critical component of tumor metastasis. Randomized clinical trials in patients with colorectal cancer have shown significant clinical benefit and tolerable toxicity with the use of these antibodies. At the era of clinical bioinformatics, better selection of patient subpopulations with tumor-over-expressing specific clinical biomarkers could result in significant enhancement of signal transduction targeting success. This review discusses characteristics of cancer-associated signaling, describes the mechanism of action of the three monoclonal antibodies, focusing on their current role in the management of colorectal cancer.

Key Words: Colorectal cancer, signaling, monoclonal antibodies, cetuximab, panitumumab, bevacizumab. INTRODUCTION Colorectal cancer is the second most common cause of cancer – related death in the Western world with an incident of almost 800.000 new cases yearly. The projected incidence in the United States for 2007 was 153.760 cases, while 52.180 deaths were expected [1]. Advances in the systemic therapy of metastatic colorectal cancer were associated with the introduction of the novel cytotoxics irinotecan and oxaliplatin, which have extended median survival for metastatic disease from 9 months to longer than 20 months [2]. Since the beneficial effect of chemotherapy has reached a plateau, molecular targeted therapy has emerged as a really promising endeavor, especially in the view of modern advances in the understanding of molecular pathomechanics of the disease. The epidermal growth factor receptor (EGFR), PI3K/ AKT/m-TOR, mitogen- activated protein kinase (MAPK) and vascular endothelial growth factor (VEGF) signaling pathways are associated with critical cell functions. Aberrant signaling of these “molecular cascades” could result in upregulation of oncogene transcription and thus tumor formation. Monoclonal antibodies targeting such pathways have been developed and have been incorporated in the management of metastatic colorectal cancer, which is frequently resistant to conventional chemotherapy. This review discusses characteristics of cancer–promoting signaling path*Address correspondence to this author at the Division of Oncology and Clinical Oncology Laboratory, Department of Internal Medicine, Medical School, University of Patras, Patras GR-26504, Greece; Tel: +30-2610999535; Fax: +30-2610-994645; E-mail: [email protected]; [email protected] 1574-3624/09 $55.00+.00

ways, describes briefly the approved monoclonal antibodies and their mechanisms of action, focusing on the elucidation of recent clinical advances on their role in the management of colorectal cancer. It also discusses theories behind targeted therapy, directions and perspectives of future research. EGFR SIGNALING PATHWAY: BIOLOGY AND ROLE The conception and analysis of EGFR signaling pathway has contributed to the disentanglement of molecular mechanisms of tumor biology, development and progression. EGFR is a 170 Kd membrane-spanning glycoprotein comprising of an extracellular ligand-binding domain, a transmembranic domain, and an intracellular cytoplasmic protein domain with tyrosine kinase activity. EGFR belongs to the human epidermal receptor (HER) family of receptor tyrosine kinases, which consists of four receptors, HER1 (EGFR, erb-B1), HER2 (neu, erb-B2), HER3 (erb-B3) and HER4 (erb-B4), that are implicated in cell proliferation, growth and survival [3]. Ligand binding induces receptors to form homo- and hetero-dimers, activates the intracellular kinase domain of each receptor, leading to the phosphorylation of tyrosine residues on each member of the dimer. This causes stereochemical changes in the receptor and increases its affinity with downstream molecules, which bind to the receptor and then form functional complexes in the cytoplasm, in order to transduct the signal in the nucleus, thus (in)activating gene transcription. These genes and the coded proteins are essential for cell proliferation, differentiation, apoptosis, invasion, metastasis and angiogenesis, thus being responsible for tumor progression [4]. The signaling pathways that are activated and the target genes that are expressed vary depending ©2009 Bentham Science Publishers Ltd.

Recent Advances in the Signal Transduction Targeting

on the microconcentrations of adaptor and effector proteins that influence intracellular signal transduction, and on the cellular context of the receptor. Receptor-ligand complexes can also be internalized leading to the termination of the signal [5]. The receptor may be either recycled or degraded, depending on the messages it receives from its microenvironment. Intervention in this last step by altering this process may cause receptor modulation, influencing its turnover and its concentration levels on the membrane [6]. EGFR is stimulated by growth factors, such as epidermal growth factor (EGF), and transforming growth factor (TGFa). These ligands are thought to promote tumor development via EGFR in vitro [7, 8], and they are produced by tumors that also express EGFR, implying that autocrine stimulatory mechanisms may participate in EGFR-driven tumor development [8]. Auto-phosphorylation of tyrosine residues within the COOH-terminal domain of EGFR in the cytoplasm, following EGFR tyrosine kinase activation, initiates a cascade of intracellular signaling pathways [9]. Increased ligand levels, hetero-dimerization, cross-phosphorylation of EGFR and cross-talk between EGFR and other membrane-bound receptors could also contribute to tumor aggressive behavior. The EGFR downstream intracellular signal transduction pathways include MAPK, PI3K/Akt/mTOR, signal transducer

Current Signal Transduction Therapy, 2009, Vol. 4, No. 1

7

and activator of transcription (STAT), downstream protein kinase C and phospholipase D pathways [4, 10]. Aberrant activation of EGFR because of either over-expression or activating mutations can result in the stimulation of tumorpromoting signaling Fig. (1). It has been shown that EGFR signaling appears to play a major role in the development of adenomas and maintenance of carcinomas during colorectal tumorigenesis [11]. Mutations in EGFR gene are found in several tumors [12]. EGFR variant type III (EGFRvIII, de2-7 EGFR, or triangle upEGFR), an EGFR mutant, has a constitutively activated tyrosine kinase domain that is potently transforming. It possesses an in-frame deletion of the extracellular domain and is found in numerous types of human tumors [13]. Two recent studies concluded that EGFRvIII expression in colorectal cancer is rare, while EGFR gene expression is very common [13, 14]. Another study suggested that co-expression of cytoplasmic EGFR and EGFRvIII occurs in approximately one third of Dukes' C colorectal carcinomas and the cytoplasmic expression of EGFR or EGFRvIII is a good indicator of response to radiotherapy [15]. Preclinical data have shown that EGFR expression is associated with tumor motility and invasiveness [16], angiogenesis, and metastatic spread. Approximately 65% to 70%

Fig. (1). EGFR signaling in tumor cells (adapted from commons.wikimedia.org/wiki/Image:EGFR_signaling pathway.png; released into the public domain).

8 Current Signal Transduction Therapy, 2009, Vol. 4, No. 1

of human colon carcinomas have been shown to express EGFR, which has been correlated with more aggressive disease and a poorer prognosis [17]. ANTI-EGFR COMPOUNDS Strategies directed towards the interruption of EGFR signaling pathway have been shown to impair tumor cell proliferation. These include anti-EGFR monoclonal antibodies, immunotoxin conjugates, and tyrosine kinase inhibitors. The latter represent synthetic, mainly quinazoline-derived, low molecular weight molecules that interact with the intracellular tyrosine kinase domain of the receptor, and inhibit ligand-induced receptor phosphorylation by competing for the intracellular Mg–ATP-binding site [18]. Two monoclonal antibodies (MAbs), cetuximab and panitumumab, which block the extracellular domain of EGFR, have been approved for the management of metastatic colorectal cancer. There are direct and indirect antibody-mediated effects, which may be simultaneously present [19, 20]. The interaction between antibody and tumor antigen may trigger tumor cell apoptosis, while inhibition of ligandreceptor interaction can lead to cell death, because target cell is deprived of tumorigenic stimuli. Antagonistic antibodies can ‘remove’ or ‘switch off’ the target antigen and may cause receptor down-modulation, preventing hetero-dimerization and activation of downstream signaling, thus inhibiting proliferating effects. Another mechanism is the formation of an entire anti-idiotype antibody network, which induces the activation of the immune system against cancer cells, simulating the mechanism of vaccination. Additionally, the interaction between antigen and antibody can lead to complement-mediated and antibody-dependent cell-mediated cytotoxicity [21, 22]. Antibodies are directed to specific targets and their efficacy depends on specific antigen characteristics. An ideal antigen should be selectively expressed in tumor cells and should be required for cell survival. There should not be any variation that reduces its significance for cell destiny and should not be secreted to the circulation, because tumor bioavailability may be decreased. Moreover, its concentration and function should not be modulated after binding to the antibody. CETUXIMAB (C225) C225 is a 154 kD murine, chimeric monoclonal antibody that consists of two identical heavy chains (449 aminoacids) and two identical light chains (214 aminoacids). Its predecessor, M225, a murine antibody, was fused to the constant region IgG1 to produce C225 that binds specifically to EGFR. It shows higher affinity compared to natural ligands, thus inhibiting endogenous ligand binding. This interaction inhibits cell cycle progression via up-regulation of p27kip1, a cycle - dependent kinase inhibitor. In addition to inhibiting cell proliferation, it also inhibits angiogenesis, cell motility, invasiveness, metastasis and promotes apoptosis [23]. Specifically, it reduces anti-apoptotic bcl-2, increases pro-apoptotic Bax and caspases 8, 9 via the inhibition of MAPK and AKT pathways [24-26]. Furthermore, it reduces both the level of DNA-damage repair enzyme (DNA protein kinase) and its activity in the nucleus [27]. Anti-angiogenetic effect

Kalofonos and Grivas

is performed through the inhibition of the production of angiogenetic factors, such as VEGF, IL-8, TGF-a, bFGF [2832]. Modulation of EGFR levels and ADCC may also partly explain the anti-tumor activity of cetuximab [33, 34]. CLINICAL DATA Chemotherapy–Resistant Disease (Second or Subsequent Line Setting) Initially, cetuximab demonstrated significant anti-tumor efficacy in the preclinical setting, such as tumor growth inhibition and regression of colorectal tumor xenografts when combined with chemotherapeutic agents [35]. Subsequently, phase I studies analyzing the pharmacokinetic profile of cetuximab have shown a half-life ranging from 4 to 7 hours, which appears suitable for once-weekly dosing [36]. A randomized phase II trial [37] was performed to confirm encouraging results from earlier non-randomized phase II studies, regarding the efficacy of cetuximab in patients with EGFR-expressing metastatic colorectal cancer, refractory to irinotecan-based chemotherapy (progressive disease while on or within 3 months of therapy). In this trial, 329 patients were randomized to receive the combination treatment or cetuximab alone (Table 1). Patients were treated at the dose and schedule of irinotecan on which they had exhibited disease progression. If patients receiving cetuximab alone developed progressive disease (56 patients), they were allowed to receive subsequent therapy with the combination. At study entry, patient and disease characteristics were similar in both arms. Tumor growth control rate was superior in the combination arm (56%) to cetuximab alone arm (32%) (p=0.001) and time to progression also favored the combination arm. No relationship was observed between the intensity of EGFR staining and the response to cetuximab. Furthermore, skin reaction was regarded as a predictive factor for response and survival. These findings supported the interaction of cetuximab with chemotherapy, favoring the investigation of new combinations with other chemotherapeutic agents even in the first-line treatment of colorectal cancer. Despite that almost half of the patients were heavily pretreated with chemotherapy, response rates were higher than those attained with currently used conventional second-line chemotherapeutic regimens. The combination of cetuximab with irinotecan was considered to have an acceptable safety profile without increasing the risks of overlapping side effects. This trial was fairly significant to support the revolutionary approval for the incorporation of cetuximab in the second-line treatment of metastatic colorectal cancer. Similar objective response rates for cetuximab monotherapy were described in two additional studies: a response rate of 9% among 57 patients with irinotecan-refractory metastatic colorectal cancer treated in a multicenter study, and of 11.6% among 346 patients with irinotecan- and oxaliplatin-refractory disease [38, 39]. The benefits of the addition of cetuximab to irinotecanbased chemotherapy were also demonstrated in the MABEL trial of 1147 patients with metastatic colorectal cancer (intent to treat population) [40]. The combination of cetuximab with irinotecan at three different dosing schedules showed that progression-free survival rate at 12 weeks exceeded the pre-

Recent Advances in the Signal Transduction Targeting

Current Signal Transduction Therapy, 2009, Vol. 4, No. 1

dicted rate in all three groups. At 24 months, the progression-free survival rates in the three groups were 29%, 32% and 39%, respectively, with an overall rate of 34%. The median overall survival at the end of the study was 9.2 months. Additionally, in a non-comparative trial, the addition of cetuximab to irinotecan in elderly patients with metastatic colorectal cancer resistant to irinotecan resulted in a median time to tumor progression of 4.5 months [41]. The overall response rate was 23% and disease control rate was 53%, implicating that the efficacy of cetuximab plus irinotecan in an elderly heavily-pretreated patient population is comparable to that in younger patients. The fluoropyrimidine derivative capecitabine is at least as effective as leucovorin plus 5-FU bolus regimens. It displays a favorable toxicity profile and offers the advantages of oral administration. A phase II trial established the safety and anti-tumor efficacy of cetuximab combined with capecitabine and oxaliplatin in the treatment of patients with meTable 1.

9

tastatic colorectal cancer progressing under oxaliplatin-based chemotherapy [42]. The main toxic effects of the combination treatment included neutropenia (12.5%), diarrhea (7.5%), fatigue (2.5%), and neurotoxicity (22.5%), while the probability of 1-year survival was 53.4% (Table 1). New studies have been launched to investigate the safety and efficacy of cetuximab administered every second week in the treatment of metastatic colorectal cancer. A recent phase II trial investigated the irinotecan/cetuximab combination every second week, as second line therapy in 31 patients with metastatic colorectal cancer refractory to fluoropyrimidine/oxaliplatin chemotherapy [43]. A confirmed partial response was observed in one patient and stable disease in seven patients. Severe toxicity included diarrhea, rash, neutropenic fever and neuropathy. The regimen was generally well tolerated, suggesting that cetuximab at the dose of 500 mg/m2, can be safely administered every two weeks.

Significant Phase II/III Clinical Trials Evaluating the use of Cetuximab (cet), Panitumumab and Bevacizumab (bev) in the Management of Metastatic Colorectal Cancer (iri: Irinotecan, BSC: Best Supportive Care, PFS: Median ProgressionFree Survival, OS: Median Overall Survival, RR: Response Rate)

Study (Author)

Regimen

Patients

RR%

PFS

OS

Cunningham et al.

iri+cet

218

22.9 p=0.007

4.1

8.6

Phase II (D-1)

cet

111

10.8

1.5

6.9

Souglakos et al. Phase II

CapOX+cet

40

20

3

10.7

Folprecht et al. Phase II

FOLFIRI+cet

21

67

9.9

33

Tabernero et al. Phase II

FOLFOX+cet

43

72

12.3

40

Min et al Phase II

FOLFIRI+cet

23

39.1

Sobrero et al. Phase III (EPIC)

cet+iri iri

648 650

16.4 p