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Epidermal growth factor receptor signaling in colorectal cancer: preclinical data and therapeutic perspectives
Annals of Oncology 16: 189 – 194, 2005 doi:10.1093/annonc/mdi057
Review
Epidermal growth factor receptor signaling in colorectal cancer: preclinical data and therapeutic perspectives J. P. Spano1*, R. Fagard2, J.-C. Soria3, O. Rixe1, D. Khayat1 & G. Milano4 1
SOMPS, Groupe Hospitalier Pitie´-Salpe´trie`re, Universite´ Pierre et Marie Curie, Paris; 2De´partement de Biochimie et de Biologie Mole´culaire, Hoˆpital Avicenne, Bobigny; 3De´partement de Me´decine, Institut Gustave Roussy, Villejuif; 4Laboratoire d’Oncopharmacologie, CAC Antoine Lacassagne, Nice, France
Received 27 June 2004; revised 11 October 2004; accepted 11 October 2004
Epidermal growth factor receptor (EGFR) belongs to a family of receptors known as the ErbB family (ErbB tyrosine kinase receptors) which comprises four proteins encoded by the c-erbB protooncogene. EGFR is known to activate a cascade of multiple signaling pathways that facilitate tumor growth process. EGFR has been shown to be overexpressed in colorectal cancer patient populations but its prognostic value in colorectal cancer progression remains unclear. The development of a panel of EGFR inhibitors could reduce the proliferation of tumor cells when used alone or in combination with cytotoxic drugs or radiation. This review focuses on the potential role of EGFR signaling in the survival of colorectal tumor cells and the possible modulation of such signaling pathways by EGFR inhibitors so as to increase tumor control or render tumor cells more sensitive to conventional therapy. Key words: colorectal cancer, EGFR, therapy implications
Epidermal growth factor receptor (EGFR) and its signaling pathway The ErbB family of receptor tyrosine kinases comprises ErbB1, 2, 3 and 4. ErbB1, also known as EGFR, is a typical member of the ErbB family having a tyrosine kinase activity, stimulated upon ligand binding [1, 2]. EGFR is a 170-kDa transmembrane glycoprotein composed of an intracellular tyrosine kinase (TK) domain, a transmembrane lipophilic segment and an extracellular ligand-binding domain. Its main autocrine ligands are epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-a), which were described 20 years ago [3, 4] and are known to be activators of DNA synthesis and cell growth in numerous tumor sites [5] including gastrointestinal disease [6]. EGFR catalyzes the transfer of phosphate molecules from ATP to an active site of tyrosine kinase to mediate signals, triggering a cascade of well-identified molecular events that will protect cells from apoptosis, facilitate invasion and promote angiogenesis reaction [7, 8]. After binding its ligand, EGFR is known to homodimerize, then to transphosphorylate several tyrosine kinase domains and thus to initiate intracellular EGFR signaling pathway. This includes the activation of STAT proteins, SRC family kinases, AKT protein and MAP kinases, inducing the transcription of genes involved in cellular processes such as
*Correspondence to: Dr J.-P. Spano, De´partement d’Oncologie Me´dicale, Groupe Hospitalier Pitie´-Salpe´trie`re, 47 Bld de l’Hoˆpital, 75 013 Paris, France. Tel: +33-1-42-16-04-52; Fax: +33-1-42-16-04-65; E-mail: [email protected] q 2005 European Society for Medical Oncology
cell division and survival [9, 10]. Moreover, it has been shown that EGFR can also mediate the activation of the other members of the ErbB family. This explains, in part, its major role in tumor cell growth [2]. AKT is a well-established antiapoptotic kinase, enabling cell survival via activation of IkB kinase, which plays a fundamental positive regulating role in NF-kB, by leading the transcription of anti-apoptotic genes [10] or by antagonizing P21-mediated cell cycle arrest [11]. In addition, AKT, thanks to its several cell effects, can also activate endothelial nitric oxide synthase, which plays a crucial role in angiogenesis [12]. AKT may also permit the activation of telomerase activity by phosphorylation of the human telomerase reverse transcriptase [13]. In addition to all these major roles in tumor growth and promotion, AKT activation may promote the tumor invasion and metastases process by stimulating the matrix metalloproteinase protein [14]. MAPK pathways with PI3K pathway have also been shown to have anti-apoptotic effects in some cell types [2] and mounting evidence suggests that the anti-apoptotic effect of ErbB receptor signaling may be attributed to the activation of PI3K/AKT pathways [2, 15]. Activation of the MAPK kinase pathway signaling also increases the expression of major proteins involved in the cell cycle regulation, such as Mcl-1, in concert with the activation of Mdm2 protein by AKT [10]. Regarding all these findings, the identification of inhibitors of the different EGFR signaling pathways in a new therapeutic strategies setting could contribute to reduced tumor growth by restoring the pro-apoptotic mechanisms and by reducing cell proliferation.
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EGFR expression in colorectal cancer and prognosis Enhanced activity or overexpression of EGFR has been found to be associated with tumor progression and poor survival in various malignancies such as head and neck [16], lung [17], breast [18], gastrointestinal tract [19] and bladder [20] cancers. It has been well documented that overexpression of EGFR in colon cancer may be linked to an advanced stage of the disease [21] or may predict a potential metastatic risk [22]. However, the impact of EGFR expression on survival remains controversial and overexpression of EGFR is not uniformly associated with an unfavorable prognosis. In most cases, immunohistochemical methods were used for the detection of EGFR in colorectal cancer. The variability of IHC is well known and thus EGFR overexpression in colorectal cancer, which ranges from 25% to 82% [7, 22 –26], needs to be more clearly defined. It has already been demonstrated, using an immunohistochemical technique, that the basal level of expression of TGF-a, EGF and their common receptor EGFR, as well as EGFR2 and EGFR3 (EGFR4 not already studied), is higher in colorectal cancer tissue than in surrounding mucosa [7, 22, 27, 28]. In primary cultures of human colorectal carcinomas, higher levels of EGFR expression have also been shown in apical, luminal cell membrane during tumor progression suggesting an increased sensitivity of colon cancer cells to a mitogenic stimulation. There might exist an autocrine growth factor in the colon lumen that possibly plays a critical role during tumor progression [29]. However, despite the experimental and clinical data mentioned above, the overexpression of EGFR in colorectal tumors and the relationship between this expression and the other clinical and histological parameters, including the impact on patients’ prognosis and survival, have not been thoroughly investigated. The heterogeneity of EGFR expression in colorectal cancers may partly be attributed to the different detection techniques, although most are based on immunohistochemistry [23]. Despite these known reproducibility and validation difficulties [30–32], immunohistochemical testing remains one of the most common methods used to assess EGFR expression and has also been well validated for the screening of HER2 receptor [33]. Currently, a quantitative immunohistochemical detection scoring system has been approved to evaluate EGFR expression in colon cancer patients [23].
Correlation between EGFR and other clinicopathological parameters in colorectal cancer patients Possible associations between expression (and overexpression of EGFR) and other clinicohistological parameters in colorectal cancer patients remain unclear. Concerning the tumor site, consistent with the main published studies results [23, 34], there are no differences in EGFR expression between the different tumor sites of the colorectal tract. Only one study has
demonstrated higher EGFR expression in cancers of the distal colon than in rectal cancers [35]. In our own study [36], no correlation was found between EGFR expression and tumor site. Some investigators have even suggested that the higher levels of growth factors and EGFR in the left-side large bowel as opposed to the right could be related to different molecular mechanisms between proximal and distal sites [7]. EGFR expression is not uniform throughout the different regions of colorectal tumors, with a higher intensity or reactivity being found in the deepest regions beyond the muscularis propria [23]. In our own experiment [36], a significant association between high EGFR expression and TNM tumor stage at diagnosis was shown, highlighting a relationship between EGFR overexpression and tumor invasion. This is the largest study showing that EGFR expression is marked in TNM tumor stage T3 [36]. These findings are in agreement with an earlier study that examined fewer adenocarcinomas (14 out of 39 colorectal carcinomas examined had an overexpression of EGF and EGFR associated with serosal invasion) [37]. They also confirm the results of Goldstein et al., who demonstrated that EGFR immunoreactivity was significantly higher in the deepest regions of the tumors compared with the superficial or luminal zones [23]. The expression of EGFR protein has also been compared with tumor differentiation grade. The results appear to be controversial with some studies showing a link [24, 38, 39] and others reporting no relationship between tumor differentiation and EGFR levels [7, 23, 35 –37, 40]. No significant correlation was demonstrated between patient gender and patient age in the above-mentioned published studies, or in our study. EGFR expression was reported to be correlated with more aggressive disease [41], increased risk of metastases [22], advanced tumor stage [42] and higher rates of mesenteric lymph-node involvement [37]. Reference was made to the fact that metastatic lesions present genomic differences from the primary tumor [43, 44]. As for EGFR expression, such variations are also possible, probably with higher expression levels in metastatic lesions compared with the primary neoplasm [45, 46]. In the Goldstein study [23], it is noteworthy that EGFR reactivity was higher in both lymph node and metastases than in primary tumors. Also, EGFR reactivity in the deepest region of the primary tumor had the strongest correlation with EGFR reactivity in lymph node and liver metastases. EGFR expression (i.e. positive or negative) was found to be concordant between metastases and primary tumor whereas, in the study by McKay et al. [24], a discrepancy in EGFR protein expression (also determined by immunohistochemical) was described between primary tumors and lymphnode metastases. These controversial findings emphasize the need for additional investigations on this topic.
Expression and prognostic significance of EGFR in colorectal cancer patients Most of the published series, including our findings [7, 23, 24, 34, 36, 40, 47, 48], have shown no association between EGFR
191 overexpression and disease evolution. However, there are relatively few studies in this area and the different patient populations are not comparable. For instance, regarding Goldstein’s findings [23] using a standardized and validated EGFR scoring system, EGFR immunoreactivity (2+ and 3+) was significantly greater in the deepest region than in the mucosa and superficial submucosa. In this study, EGFR expression was associated with decreased survival (P = 0.0252) when considering EGFR reactivity in this deep region, but not EGFR reactivity at all levels of the tumor wall. Our data are in agreement with these observations and show EGFR overexpressed in stage II disease (T3N0M0) in which the tumor extends through the muscularis propria into pericolonic adipose tissue. This highlights the existence of a tumor area overexpressing EGFR, which could carry a more aggressive growth and a higher metastatic potential. These findings could, therefore, offer an opportunity to test the efficacy of EGFR inhibitors at this stage of the disease [49, 50].
How can EGFR inhibitors impair tumor cell growth and improve tumor control? Blocking EGFR activation would obviously represent an innovative and key strategy in patient care because this therapeutic strategy impairs crucial cellular functions linked to proliferation and survival [30]. EGFR signaling can be targeted by either monoclonal antibodies (C225, etc.) or tyrosine kinase inhibitors [ZD 1839 (gefitinib), OSI774 (erlotinib), CI1033, PKI166, GW572016, etc.], or even by antisense approaches (antisense molecules to EGFR or targeting key regulatory regions of the EGFR) [2, 30, 50 –52]. Several mechanisms of action of the different EGFR inhibitors have been reported in preclinical studies and additional data have been gathered in the clinical setting. Antibodies have the propriety of binding to the extracellular portions of ErbB receptors. For instance, C225 seems to interact with the EGFR domain that is associated with ligand binding [50]. For example, C225 can promote growth arrest by blocking the cell cycle in phase G1 [53]. Ye et al. [54] have confirmed these data and have shown an increase in p27 expression and a decrease in CDK2, 4 and 6 expressions, when applying C225 to an ovarian cancer cell model. In addition, C225 may promote apoptosis, as demonstrated in the study by Huang et al. [53], showing an increase in Bax expression and a decrease in Bcl2 expression. Furthermore, Mandal et al. [55] noted a transfer of Bax from the nucleus to the cytoplasm and the activation of caspase 3, 8 and 9 [56] when the DiFi tumor cells were treated with M225 (the murine equivalent of C225). Regarding DNA repair, C225 can trigger a decrease of expression of nuclear DNA-PK, one of the key proteins involved in DNA repair following radiation and cytotoxic effects [57, 58]. Noteworthy, the truncated forms of ErbB1, i.e. EGFR vIII can also be inhibited by C225 [59]. Finally, C225 combined with chemotherapeutic agents or radiotherapy can also help to
improve tumor growth control in experimental models [60, 61]. For instance, in two refractory human colorectal tumor xenografts (Ht-29 and DLD-1), Prewett et al. [41] demonstrated enhanced tumor growth inhibition when C225 was added to CPT-11 therapy. Noteworthy, C225 was able to reverse CPT-11 resistance in both tumor models [41]. Tyrosine kinase inhibitors are small molecules that interact with the intracellular domain of EGFR. Gefitinib is an oral quinazoline derivative that selectively inhibits the EGFR tyrosine kinase and has already demonstrated inhibition in the colon cancer cell lines GEO that express EGFR and TGF-a, in a dose-dependant manner, with anti-angiogenesis effects [62]. Generally, the growth inhibition of gefitinib was cytotoxic, but an apoptosis effect was observed with higher doses. In fact, by binding with the catalytic kinase domain of EGFR, ZD1839, OSI774, PKI166 [2, 30, 63], and for the pan-HER inhibitor CI1033 [64, 65], have a potential and positive impact on apoptosis. This finding has been documented in vitro with the decreased expression of BCl2 concomitant with the increased expression of Bax, with, curiously, no major effect on caspase 3 activity, in CAL33 cultured cells treated by gefitinib [66]. Gefitinib has also been shown to have a dose-dependant anti-proliferative effect when associated with cytotoxic agents such as oxaliplatin [62]. The sequence of administration of cytotoxic drugs with EGFR inhibitors has also been considered. For instance, recently, a schedule-dependant effect was reported between gefitinib and SN-38. The optimal effect was observed when gefitinib was given after SN-38 [67], in two colon cancer cell lines (HT-39 and LoVo). This suggests a new potential active combination in colon cancer, when considering SN-38 as the active CPT-11 metabolite. Gefitinib has also been demonstrated to be a radiosensitizer, especially when given before and/or during radiation therapy [68]. The mechanism of such radiosensitization may be explained by the fact that anti-EGFR agents may attenuate DNA repair [68, 69] and decrease angiogenesis [70]. Previous studies suggest that the greatest growth inhibition obtained by the combination of gefitinib and radiation therapy was observed in those tumor cell lines with the highest EGFR expression [71, 72]. Moreover, EGFR specific ligands such as TGF-a may play a role in radiosensitivity as demonstrated by Sauter et al. [73]. Low plasma TGF-a levels were associated with radioresistance in patients with head and neck cancers [68, 73]. Finally, as mentioned above, the cellular effects of the combination of EGFR-targeting agents and radiation includes, as first event, the induction of DNA damage by the ionizing radiation that activates several mitogenic signaling cascades, including the Raf-MEK-Erk kinase [74]. Finally, the potential antitumor activity of such EGFR inhibitors provides a rationale for relevant clinical implications. Most EGFR inhibitors are currently in phase III clinical trials either as single agents or in combination with chemotherapeutic agents or with ionizing radiation. Some studies [39, 75] have already been published, leading to cetuximab approval in irinotecan-refractory colorectal cancer patients, in several countries or regions (e.g. USA, Europe and Canada).
192 To date, in the clinical setting, only the monoclonal antibodies have really shown activity, and not the small molecules. One issue requiring particular attention involves the predictive response markers that could help select those patients most likely to respond to EGFR-targeting agents. Some investigators have suggested that cutaneous side-effects might be predictors of efficacy. Extensive skin rashes or acne, for instance, seem to be associated with improved survival following anti-EGFR therapy [76, 77]. Of particular note, the presence of specific mutations in the EGFR gene have recently been shown to be associated with dramatic responses to EGFR inhibitors, as recently published in non-small-cell lung cancer (NSCLC) patients treated by gefitinib or erlotinib [77, 78]. Cappuzzo et al. [79] have also suggested that NSCLC with high levels of expression of phosphor-Akt are the ones most likely to benefit from EGFR-inhibitors. In conclusion, although EGFR is not considered to be a prognostic factor in colorectal cancer patients, it plays a major role in tumor cell proliferation. Convincing preclinical and clinical studies have already demonstrated the efficacy of EGFR inhibitors in advanced colorectal carcinomas and their potential synergistic combinations with chemo- and radiation therapy. Additional efforts and trials are needed to evaluate the role of EGFR inhibitors in the adjuvant setting and to help to establish better predictors of the efficacy of such agents. Since EGFR tumor level itself does not seem to be a valuable predictor of response to anti-EGR therapy, other biomarkers need to be evaluated besides EGFR mutated forms, p-Akt expression or EGFR amplification.
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Review
Epidermal growth factor receptor signaling in colorectal cancer: preclinical data and therapeutic perspectives J. P. Spano1*, R. Fagard2, J.-C. Soria3, O. Rixe1, D. Khayat1 & G. Milano4 1
SOMPS, Groupe Hospitalier Pitie´-Salpe´trie`re, Universite´ Pierre et Marie Curie, Paris; 2De´partement de Biochimie et de Biologie Mole´culaire, Hoˆpital Avicenne, Bobigny; 3De´partement de Me´decine, Institut Gustave Roussy, Villejuif; 4Laboratoire d’Oncopharmacologie, CAC Antoine Lacassagne, Nice, France
Received 27 June 2004; revised 11 October 2004; accepted 11 October 2004
Epidermal growth factor receptor (EGFR) belongs to a family of receptors known as the ErbB family (ErbB tyrosine kinase receptors) which comprises four proteins encoded by the c-erbB protooncogene. EGFR is known to activate a cascade of multiple signaling pathways that facilitate tumor growth process. EGFR has been shown to be overexpressed in colorectal cancer patient populations but its prognostic value in colorectal cancer progression remains unclear. The development of a panel of EGFR inhibitors could reduce the proliferation of tumor cells when used alone or in combination with cytotoxic drugs or radiation. This review focuses on the potential role of EGFR signaling in the survival of colorectal tumor cells and the possible modulation of such signaling pathways by EGFR inhibitors so as to increase tumor control or render tumor cells more sensitive to conventional therapy. Key words: colorectal cancer, EGFR, therapy implications
Epidermal growth factor receptor (EGFR) and its signaling pathway The ErbB family of receptor tyrosine kinases comprises ErbB1, 2, 3 and 4. ErbB1, also known as EGFR, is a typical member of the ErbB family having a tyrosine kinase activity, stimulated upon ligand binding [1, 2]. EGFR is a 170-kDa transmembrane glycoprotein composed of an intracellular tyrosine kinase (TK) domain, a transmembrane lipophilic segment and an extracellular ligand-binding domain. Its main autocrine ligands are epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-a), which were described 20 years ago [3, 4] and are known to be activators of DNA synthesis and cell growth in numerous tumor sites [5] including gastrointestinal disease [6]. EGFR catalyzes the transfer of phosphate molecules from ATP to an active site of tyrosine kinase to mediate signals, triggering a cascade of well-identified molecular events that will protect cells from apoptosis, facilitate invasion and promote angiogenesis reaction [7, 8]. After binding its ligand, EGFR is known to homodimerize, then to transphosphorylate several tyrosine kinase domains and thus to initiate intracellular EGFR signaling pathway. This includes the activation of STAT proteins, SRC family kinases, AKT protein and MAP kinases, inducing the transcription of genes involved in cellular processes such as
*Correspondence to: Dr J.-P. Spano, De´partement d’Oncologie Me´dicale, Groupe Hospitalier Pitie´-Salpe´trie`re, 47 Bld de l’Hoˆpital, 75 013 Paris, France. Tel: +33-1-42-16-04-52; Fax: +33-1-42-16-04-65; E-mail: [email protected] q 2005 European Society for Medical Oncology
cell division and survival [9, 10]. Moreover, it has been shown that EGFR can also mediate the activation of the other members of the ErbB family. This explains, in part, its major role in tumor cell growth [2]. AKT is a well-established antiapoptotic kinase, enabling cell survival via activation of IkB kinase, which plays a fundamental positive regulating role in NF-kB, by leading the transcription of anti-apoptotic genes [10] or by antagonizing P21-mediated cell cycle arrest [11]. In addition, AKT, thanks to its several cell effects, can also activate endothelial nitric oxide synthase, which plays a crucial role in angiogenesis [12]. AKT may also permit the activation of telomerase activity by phosphorylation of the human telomerase reverse transcriptase [13]. In addition to all these major roles in tumor growth and promotion, AKT activation may promote the tumor invasion and metastases process by stimulating the matrix metalloproteinase protein [14]. MAPK pathways with PI3K pathway have also been shown to have anti-apoptotic effects in some cell types [2] and mounting evidence suggests that the anti-apoptotic effect of ErbB receptor signaling may be attributed to the activation of PI3K/AKT pathways [2, 15]. Activation of the MAPK kinase pathway signaling also increases the expression of major proteins involved in the cell cycle regulation, such as Mcl-1, in concert with the activation of Mdm2 protein by AKT [10]. Regarding all these findings, the identification of inhibitors of the different EGFR signaling pathways in a new therapeutic strategies setting could contribute to reduced tumor growth by restoring the pro-apoptotic mechanisms and by reducing cell proliferation.
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EGFR expression in colorectal cancer and prognosis Enhanced activity or overexpression of EGFR has been found to be associated with tumor progression and poor survival in various malignancies such as head and neck [16], lung [17], breast [18], gastrointestinal tract [19] and bladder [20] cancers. It has been well documented that overexpression of EGFR in colon cancer may be linked to an advanced stage of the disease [21] or may predict a potential metastatic risk [22]. However, the impact of EGFR expression on survival remains controversial and overexpression of EGFR is not uniformly associated with an unfavorable prognosis. In most cases, immunohistochemical methods were used for the detection of EGFR in colorectal cancer. The variability of IHC is well known and thus EGFR overexpression in colorectal cancer, which ranges from 25% to 82% [7, 22 –26], needs to be more clearly defined. It has already been demonstrated, using an immunohistochemical technique, that the basal level of expression of TGF-a, EGF and their common receptor EGFR, as well as EGFR2 and EGFR3 (EGFR4 not already studied), is higher in colorectal cancer tissue than in surrounding mucosa [7, 22, 27, 28]. In primary cultures of human colorectal carcinomas, higher levels of EGFR expression have also been shown in apical, luminal cell membrane during tumor progression suggesting an increased sensitivity of colon cancer cells to a mitogenic stimulation. There might exist an autocrine growth factor in the colon lumen that possibly plays a critical role during tumor progression [29]. However, despite the experimental and clinical data mentioned above, the overexpression of EGFR in colorectal tumors and the relationship between this expression and the other clinical and histological parameters, including the impact on patients’ prognosis and survival, have not been thoroughly investigated. The heterogeneity of EGFR expression in colorectal cancers may partly be attributed to the different detection techniques, although most are based on immunohistochemistry [23]. Despite these known reproducibility and validation difficulties [30–32], immunohistochemical testing remains one of the most common methods used to assess EGFR expression and has also been well validated for the screening of HER2 receptor [33]. Currently, a quantitative immunohistochemical detection scoring system has been approved to evaluate EGFR expression in colon cancer patients [23].
Correlation between EGFR and other clinicopathological parameters in colorectal cancer patients Possible associations between expression (and overexpression of EGFR) and other clinicohistological parameters in colorectal cancer patients remain unclear. Concerning the tumor site, consistent with the main published studies results [23, 34], there are no differences in EGFR expression between the different tumor sites of the colorectal tract. Only one study has
demonstrated higher EGFR expression in cancers of the distal colon than in rectal cancers [35]. In our own study [36], no correlation was found between EGFR expression and tumor site. Some investigators have even suggested that the higher levels of growth factors and EGFR in the left-side large bowel as opposed to the right could be related to different molecular mechanisms between proximal and distal sites [7]. EGFR expression is not uniform throughout the different regions of colorectal tumors, with a higher intensity or reactivity being found in the deepest regions beyond the muscularis propria [23]. In our own experiment [36], a significant association between high EGFR expression and TNM tumor stage at diagnosis was shown, highlighting a relationship between EGFR overexpression and tumor invasion. This is the largest study showing that EGFR expression is marked in TNM tumor stage T3 [36]. These findings are in agreement with an earlier study that examined fewer adenocarcinomas (14 out of 39 colorectal carcinomas examined had an overexpression of EGF and EGFR associated with serosal invasion) [37]. They also confirm the results of Goldstein et al., who demonstrated that EGFR immunoreactivity was significantly higher in the deepest regions of the tumors compared with the superficial or luminal zones [23]. The expression of EGFR protein has also been compared with tumor differentiation grade. The results appear to be controversial with some studies showing a link [24, 38, 39] and others reporting no relationship between tumor differentiation and EGFR levels [7, 23, 35 –37, 40]. No significant correlation was demonstrated between patient gender and patient age in the above-mentioned published studies, or in our study. EGFR expression was reported to be correlated with more aggressive disease [41], increased risk of metastases [22], advanced tumor stage [42] and higher rates of mesenteric lymph-node involvement [37]. Reference was made to the fact that metastatic lesions present genomic differences from the primary tumor [43, 44]. As for EGFR expression, such variations are also possible, probably with higher expression levels in metastatic lesions compared with the primary neoplasm [45, 46]. In the Goldstein study [23], it is noteworthy that EGFR reactivity was higher in both lymph node and metastases than in primary tumors. Also, EGFR reactivity in the deepest region of the primary tumor had the strongest correlation with EGFR reactivity in lymph node and liver metastases. EGFR expression (i.e. positive or negative) was found to be concordant between metastases and primary tumor whereas, in the study by McKay et al. [24], a discrepancy in EGFR protein expression (also determined by immunohistochemical) was described between primary tumors and lymphnode metastases. These controversial findings emphasize the need for additional investigations on this topic.
Expression and prognostic significance of EGFR in colorectal cancer patients Most of the published series, including our findings [7, 23, 24, 34, 36, 40, 47, 48], have shown no association between EGFR
191 overexpression and disease evolution. However, there are relatively few studies in this area and the different patient populations are not comparable. For instance, regarding Goldstein’s findings [23] using a standardized and validated EGFR scoring system, EGFR immunoreactivity (2+ and 3+) was significantly greater in the deepest region than in the mucosa and superficial submucosa. In this study, EGFR expression was associated with decreased survival (P = 0.0252) when considering EGFR reactivity in this deep region, but not EGFR reactivity at all levels of the tumor wall. Our data are in agreement with these observations and show EGFR overexpressed in stage II disease (T3N0M0) in which the tumor extends through the muscularis propria into pericolonic adipose tissue. This highlights the existence of a tumor area overexpressing EGFR, which could carry a more aggressive growth and a higher metastatic potential. These findings could, therefore, offer an opportunity to test the efficacy of EGFR inhibitors at this stage of the disease [49, 50].
How can EGFR inhibitors impair tumor cell growth and improve tumor control? Blocking EGFR activation would obviously represent an innovative and key strategy in patient care because this therapeutic strategy impairs crucial cellular functions linked to proliferation and survival [30]. EGFR signaling can be targeted by either monoclonal antibodies (C225, etc.) or tyrosine kinase inhibitors [ZD 1839 (gefitinib), OSI774 (erlotinib), CI1033, PKI166, GW572016, etc.], or even by antisense approaches (antisense molecules to EGFR or targeting key regulatory regions of the EGFR) [2, 30, 50 –52]. Several mechanisms of action of the different EGFR inhibitors have been reported in preclinical studies and additional data have been gathered in the clinical setting. Antibodies have the propriety of binding to the extracellular portions of ErbB receptors. For instance, C225 seems to interact with the EGFR domain that is associated with ligand binding [50]. For example, C225 can promote growth arrest by blocking the cell cycle in phase G1 [53]. Ye et al. [54] have confirmed these data and have shown an increase in p27 expression and a decrease in CDK2, 4 and 6 expressions, when applying C225 to an ovarian cancer cell model. In addition, C225 may promote apoptosis, as demonstrated in the study by Huang et al. [53], showing an increase in Bax expression and a decrease in Bcl2 expression. Furthermore, Mandal et al. [55] noted a transfer of Bax from the nucleus to the cytoplasm and the activation of caspase 3, 8 and 9 [56] when the DiFi tumor cells were treated with M225 (the murine equivalent of C225). Regarding DNA repair, C225 can trigger a decrease of expression of nuclear DNA-PK, one of the key proteins involved in DNA repair following radiation and cytotoxic effects [57, 58]. Noteworthy, the truncated forms of ErbB1, i.e. EGFR vIII can also be inhibited by C225 [59]. Finally, C225 combined with chemotherapeutic agents or radiotherapy can also help to
improve tumor growth control in experimental models [60, 61]. For instance, in two refractory human colorectal tumor xenografts (Ht-29 and DLD-1), Prewett et al. [41] demonstrated enhanced tumor growth inhibition when C225 was added to CPT-11 therapy. Noteworthy, C225 was able to reverse CPT-11 resistance in both tumor models [41]. Tyrosine kinase inhibitors are small molecules that interact with the intracellular domain of EGFR. Gefitinib is an oral quinazoline derivative that selectively inhibits the EGFR tyrosine kinase and has already demonstrated inhibition in the colon cancer cell lines GEO that express EGFR and TGF-a, in a dose-dependant manner, with anti-angiogenesis effects [62]. Generally, the growth inhibition of gefitinib was cytotoxic, but an apoptosis effect was observed with higher doses. In fact, by binding with the catalytic kinase domain of EGFR, ZD1839, OSI774, PKI166 [2, 30, 63], and for the pan-HER inhibitor CI1033 [64, 65], have a potential and positive impact on apoptosis. This finding has been documented in vitro with the decreased expression of BCl2 concomitant with the increased expression of Bax, with, curiously, no major effect on caspase 3 activity, in CAL33 cultured cells treated by gefitinib [66]. Gefitinib has also been shown to have a dose-dependant anti-proliferative effect when associated with cytotoxic agents such as oxaliplatin [62]. The sequence of administration of cytotoxic drugs with EGFR inhibitors has also been considered. For instance, recently, a schedule-dependant effect was reported between gefitinib and SN-38. The optimal effect was observed when gefitinib was given after SN-38 [67], in two colon cancer cell lines (HT-39 and LoVo). This suggests a new potential active combination in colon cancer, when considering SN-38 as the active CPT-11 metabolite. Gefitinib has also been demonstrated to be a radiosensitizer, especially when given before and/or during radiation therapy [68]. The mechanism of such radiosensitization may be explained by the fact that anti-EGFR agents may attenuate DNA repair [68, 69] and decrease angiogenesis [70]. Previous studies suggest that the greatest growth inhibition obtained by the combination of gefitinib and radiation therapy was observed in those tumor cell lines with the highest EGFR expression [71, 72]. Moreover, EGFR specific ligands such as TGF-a may play a role in radiosensitivity as demonstrated by Sauter et al. [73]. Low plasma TGF-a levels were associated with radioresistance in patients with head and neck cancers [68, 73]. Finally, as mentioned above, the cellular effects of the combination of EGFR-targeting agents and radiation includes, as first event, the induction of DNA damage by the ionizing radiation that activates several mitogenic signaling cascades, including the Raf-MEK-Erk kinase [74]. Finally, the potential antitumor activity of such EGFR inhibitors provides a rationale for relevant clinical implications. Most EGFR inhibitors are currently in phase III clinical trials either as single agents or in combination with chemotherapeutic agents or with ionizing radiation. Some studies [39, 75] have already been published, leading to cetuximab approval in irinotecan-refractory colorectal cancer patients, in several countries or regions (e.g. USA, Europe and Canada).
192 To date, in the clinical setting, only the monoclonal antibodies have really shown activity, and not the small molecules. One issue requiring particular attention involves the predictive response markers that could help select those patients most likely to respond to EGFR-targeting agents. Some investigators have suggested that cutaneous side-effects might be predictors of efficacy. Extensive skin rashes or acne, for instance, seem to be associated with improved survival following anti-EGFR therapy [76, 77]. Of particular note, the presence of specific mutations in the EGFR gene have recently been shown to be associated with dramatic responses to EGFR inhibitors, as recently published in non-small-cell lung cancer (NSCLC) patients treated by gefitinib or erlotinib [77, 78]. Cappuzzo et al. [79] have also suggested that NSCLC with high levels of expression of phosphor-Akt are the ones most likely to benefit from EGFR-inhibitors. In conclusion, although EGFR is not considered to be a prognostic factor in colorectal cancer patients, it plays a major role in tumor cell proliferation. Convincing preclinical and clinical studies have already demonstrated the efficacy of EGFR inhibitors in advanced colorectal carcinomas and their potential synergistic combinations with chemo- and radiation therapy. Additional efforts and trials are needed to evaluate the role of EGFR inhibitors in the adjuvant setting and to help to establish better predictors of the efficacy of such agents. Since EGFR tumor level itself does not seem to be a valuable predictor of response to anti-EGR therapy, other biomarkers need to be evaluated besides EGFR mutated forms, p-Akt expression or EGFR amplification.
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