- Email: [email protected]
Colon cancer cells: Pro-invasive signalling
The International Journal of Biochemistry & Cell Biology 38 (2006) 1231–1236
Cells in focus
Colon cancer cells: Pro-invasive signalling Delphine Debruyne a , Maria Jos´e Oliveira a,b , Marc Bracke a,∗ , Marc Mareel a , Ancy Leroy a a
Laboratory of Experimental Cancerology, Department of Radiotherapy and Nuclear Medicine, Ghent University Hospital, De Pintelaan 185, 1P7, B-9000 Ghent, Belgium b Instituto de Patologia e Immunologia Molecular da Universidade do Porto (IPATIMUP), 4200 Porto, Portugal Received 4 November 2005; received in revised form 9 January 2006; accepted 10 January 2006 Available online 13 February 2006
Abstract Colon cancer results from erroneous renewal of the enteric epithelium. Mutations in stem cells, or their proliferative progenitors, cause accumulation of cells that invade into the stroma and continue to divide rather than migrating on top of the basement membrane prior to entering into apoptosis. Many of these changes in invasive activity appear to be related to the invasion-suppressor role of E-cadherin. We have also investigated Listeria monocytogenes and other enteric bacteria, since these bacteria stimulate invasion through the production of a -casein-derived 13-amino acid peptide which is produced by enzymes present in the colon cancer ecosystem. The pro-invasive 13-amino acid peptide signals via small guanosine triphosphatases, which modulate the actin cytoskeleton, and via phosphorylation of the ␦ opioid receptor. The pro-invasive activity of this peptide is neutralized by the ␦ opioid receptor antagonist, naloxone. Since the ␦ opioid receptor belongs to the family of G protein-coupled receptors, implicated in colon cancer cell invasion signalling pathways, it is tempting to speculate that opioids could play a role in mediating this trait of malignant tumours. © 2006 Elsevier Ltd. All rights reserved. Keywords: Colon cancer invasion; Micro-environment; E-cadherin; -Casein peptide; Opioid receptor
Cell facts • Colon epithelial cells are renewed continuously from stem cells, present at the base of the crypts. • Colon cancer cells result from mutations in stem cells or in their proliferating progenitors. • Invasion by colon cancer cells is restricted through cell–cell adhesion and the signalling of the E-cadherin/catenin complex. • Colon cancer cells are sensitive to pro-invasive signalling from enteric bacteria, implicating G protein-coupled opioid receptors in this process.
1. Introduction
∗
Corresponding author. Tel.: +32 9 2403063; fax: +32 9 2404991. E-mail address: [email protected] (M. Bracke).
1357-2725/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2006.01.003
Normal adult colon enterocytes are renewed continuously from stem cells, producing proliferative progenitors that differentiate into absorptive or into secretory
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Fig. 1. Schematic illustration of normal and cancerous colon. The normal sequence (right) comprises proliferation and migration from stem cells in the bottom of the crypt to presence of polarized epithelial cells at the luminal surface. Development of colon cancer (left) starts from mutations in stem cells or their proliferative progenitors (indicated by mitotic figures) transforming into hyperplasia and invasive cancer. The insets illustrate molecular signalling (arrows) between enterocytes (right inset) or cancer cells (left inset) and host cells in their micro-environment. B, bacteria; BM, basement membrane; E-cad, epithelial cadherin; EMT, epithelial–mesenchymal transition; HGF/SF, hepatocyte growth factor/scatter factor; TGF-, transforming growth factor-; Tn-C, tenascin C.
cells. During this process colon enterocytes migrate from the stem cell compartment at the base of the crypt towards the luminal surface, with the basement membrane strictly separating them from the stromal compartment (Radtke & Clevers, 2005). In contrast, invasive colon cancer results from mutations in stem cells or in their proliferative progenitors which breach the basement membrane barrier (Fig. 1). Colon cancer provides a prototype of tumour development for several reasons: its high frequency with a 50% risk of polyps above age 70 and a 5% life time risk of invasive cancer; the occurrence of familial forms such as hereditary non-polyposis colorectal cancer (HNPCC) and familial adenomatous polyposis (FAP); the possibility of observing different precursor lesions, such as polyps and carcinoma in situ, in the same patient. 2. Colon epithelium plasticity: epithelial–mesenchymal transition (EMT) and mesenchymal–epithelial transition (MET) The colon epithelium forms a barrier by means of cell–cell interactions, involving tight junctions, cadherin-based adherens junctions, gap junctions and desmosomes. When, during cancer development, the invasive program is switched on, epithelial cells transform into cells with a mesenchymal phenotype (EMT)
(Trusolino & Comoglio, 2002). These cells lose their normal established position due to alterations in cell–cell contacts, cell–matrix interactions and the cytoskeletal structure. They migrate in the wrong direction, through the basement membrane and into the stroma. Here, dramatic alterations take place, constituting a novel microenvironment for the cancer cell. These include transition from fibroblasts into myofibroblasts, infiltration by leukocytes and the formation of new blood and lymphatic vessels (De Wever & Mareel, 2003). Cancer cells at the invasive front disseminate via the circulation to form a secondary tumour in a distant organ. At this metastatic site, cancer cells may regain the phenotype of the primary tumour, undergoing a mesenchymal–epithelial transition (MET) (Brabletz et al., 2005). The invasion and metastasis programs underlying this EMT/MET appear to result from the involvement of the same cellular activities and molecular signalling pathways that also participate in the maintenance of the normal colon epithelium. Comparison between normal colon and invasive colon cancer, nevertheless, reveals up- and downregulation of multiple tumour-promoter and -suppressor genes, respectively. This becomes clear not only from cDNA microarray analysis, but also from detailed dissection of the invasion-related proteomes and their signalling pathways (Mareel & Leroy, 2003). The E-cadherin/catenin complex serves as our paradigm of
D. Debruyne et al. / The International Journal of Biochemistry & Cell Biology 38 (2006) 1231–1236
involved molecules because of its crucial role in maintenance of the epithelium and its invasion-suppressor function (Behrens, Mareel, Van Roy, & Birchmeier, 1989; Takeichi, 1991). 3. Functional loss: colon cancer cells lose cell–cell contacts 3.1. The key-role of the E-cadherin/catenin complex Epithelial(E)-cadherin is a transmembrane glycoprotein of the type I cadherin superfamily, mediating homotypic (same cell type) homophilic (same molecules) cell–cell adhesion and, through its interaction with the catenins, signal transduction events as well. Circumstantial evidence in favour of E-cadherin functioning as an invasion-suppressor comes from the analysis of human cancers in various stages of progression and from the manipulation of cell lines. More direct evidence was gained from observations on transgenic animals. Each criterion on its own does not suffice to assign a given molecule an invasion-suppressor or an invasionpromoter function but, taken together, they give an estimation of the positive or negative weight of the molecule in the invasion process. In normal colon, immunohistochemical staining of E-cadherin produces a honeycomb like pattern at the lateral borders of the enterocytes. In colorectal cancer, disturbance of this pattern and lower levels or absence of E-cadherin expression is associated with increasing histological grading (higher grades correspond to lower degree of differentiation) and with a worse prognosis (Van Aken et al., 1993). Most colon cancer cell lines are E-cadherin positive and are poor invaders into extracellular matrices unless assisted by stromal cells or by pro-invasive ligands. In a series of seven cell lines studied by Kinsella, Lepts, Hill, and Jones (1994), two had decreased levels of expression of E-cadherin and these two invaded into collagen and into Matrigel in vitro. The causal relationship between E-cadherin expression and invasion in vivo was demonstrated convincingly in transgenic mice bearing pancreatic -cell tumours (Perl, Wilgenbus, Dahl, Semb, & Christofori, 1998), though similar experiments have not yet been done with colon tumours. What is the mechanism of E-cadherin downregulation in colon cancer? Inactivating mutations, such as found in gastric and in lobular breast cancer (Berx & Van Roy, 2001), are absent. By contrast, epigenetic modulation of E-cadherin expression by promoter hypermethylation, a frequent event in several types of cancer, does occur. Methyl-CpG-binding protein (MeCP2) and 5 CpG island hypermethylation, as demonstrated
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by in situ hybridization and by methylation-specific PCR, respectively, did correlate with reduction of Ecadherin expression (Darwanto, Kitazawa, Maeda, & Kitazawa, 2003). Downmodulation of E-cadherin by promoter methylation is reversible and fits with METassociated re-expression in metastases. The hypothesis is that in order to grow at the metastatic site, disseminated mesenchymal cancer cells must regain at least some of their epithelial functions (Brabletz et al., 2005). Transcriptional downregulation is another mechanism of E-cadherin repression that may act in concord with methylation (Fig. 2A). Snail, a member of a multizinc finger protein family of transcription factors, is a strong repressor of the E-cadherin CDH1 gene, interacting specifically with E-boxes in the promoter and causing loss of epithelial function and gain of invasion. In the normal colon Snail is not expressed whereas cancers score positive by immunohistochemistry with a trend towards higher expression in tumours with metastatic ability (Larriba & Mu˜noz, 2005). We have mentioned before the multiplicity of changes related to the invasion program. This is well illustrated by Snail, consistently repressing 167 genes and inducing 23 others (De Craene et al., 2005). One other element of the E-cadherin/catenin invasionsuppressor complex, -catenin, deserves mentioning because it too plays a dominant role in colon cancer as an invasion-promoter (Radtke & Clevers, 2005). Mutations in the -catenin serine/threonine phosphorylation sites preclude its degradation and direct it to the nucleus where it transactivates invasion-promoter genes (Fig. 2B). Upregulation of N-cadherin, described in some E-cadherin-negative cancers, has not been described in colon cancer (Derycke & Bracke, 2004). 3.2. Participation of enteric bacteria: from E-cadherin to opioid receptors E-cadherin led us to the opioid receptors. Since Ecadherin mediates binding of the Listeria (L.) monocytogenes virulence factor Internalin A to enterocytes (Lecuit, 2005), we wondered whether binding of L. monocytogenes would neutralize the invasionsuppressor role of E-cadherin (Oliveira et al., 2003). L. monocytogenes did stimulate invasion of human colon cancer cells into extracellular matrices in vitro, but E-cadherin was not mechanistically implicated. Conditioned medium (CM) prepared from L. monocytogenes incubated on top of collagen type I also stimulated invasion. In this pro-invasive conditioned medium, a 13mer peptide (HKEMPFPKYPVEP) was identified by reverse-phase high-performance liquid chro-
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Fig. 3. Generation of the pro-invasive 13mer peptide (HKEMPFPKYPVEP). Arrows and arcs indicate activation and inactivation, respectively, in the invasion pathway. Dashed arrows indicate links that are not yet confirmed experimentally. Acronyms in alphabetic order: Cdc42, cell division cycle 42; DOR, ␦ opioid receptor; FAK, p125 focal adhesion kinase; G␣␥, G protein; Mpl, Listeria thermolysin-like metalloprotease; PAK1, p21-activated kinase; PI3K, phosphoinositide 3 kinase; RhoA, Ras homologue A.
matography (RP-HPLC) followed by mass spectrometry (MS–MS) and it was shown that the pro-invasive activity could be reproduced by a synthetic 13mer peptide. The sequence of this peptide corresponds to a bovine casein sequence and the source is the tryptic soy broth (TSB) culture medium, adsorbed onto the bacterial surface. Both, collagen and L. monocytogenes, are required to generate the 13mer peptide (Fig. 3). Bacteria isolated from human colon cancer biopsies, were grown in TSB on collagen type I. With CM from such cultures we were able to stimulate invasion in vitro. Moreover, the enzymes needed for cleavage of a -casein source into the pro-invasive 13mer peptide also were present in human colon: when we incubated a synthetic -casein source on top of colon cancer biopsies, we also found the 13mer peptide in the supernatant using
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HPLC/MS–MS. The 13mer peptide-mediated invasion pathway in colon cancer cells was, in part, revealed by transfecting the cells transiently with cDNA constructs encoding dominant-negative and constitutively active proteins and by treating the cells with pharmacological modulators (Oliveira et al., 2003). Signalling was through PI3K activation of Cdc42, which in turn activates PAK1. This serine/threonine kinase connects Cdc42 with RhoA and inactivates it, causing dephosphorylation of FAK and downregulation of focal adhesion complexes. Cdc42 activation explains the increase in filopodia formation, as was observed by F-actin staining of a human colon cancer cell line. All these effects are associated with stimulation of invasion, the 13mer peptide acting as a motility factor. There is evidence that the 13mer peptide acts via the ␦ opioid receptor (DOR). Since in silico analysis revealed sequence homology between the C-terminus of the 13mer peptide and human -casomorphin (a DOR agonist), DOR was suggested as the candidate receptor. Moreover naloxone, a DOR antagonist, inhibited 13mer peptide-stimulated invasion, as well as the increase in serine phosphorylation of DOR after treatment with the 13mer peptide. Opioid receptors belong to the superfamily of G protein-coupled receptors (GPCRs). Although opioid receptors are associated predominantly with the nervous system, they also are present in the immune system and in different cancer cell lines (Fichna & Janecka, 2004). They are activated both by endogenously produced opioid peptides and by exogenously administered opiate compounds. Opioid agonist treatment switches on signal transduction pathways that lead to activation of invasion-associated activities like reorganization of the actin cytoskeleton, and increased cell survival. These are arguments to investigate further the role of opioids in cancer invasion.
Fig. 2. Transcriptional (A) and post-transcriptional (B) modulation of E-cadherin. (A) E-cadherin promoter with positive (in green) and negative (in red) regulators and with co-activators (light green) and co-repressors (light red). Acronyms in alphabetic order: AP2, adaptor protein 2; CtBP, C-terminal binding protein; C/EBP, CCAAT/enhancer-binding protein; DNMT, DNA methyltransferase; ␦ EF1, delta-crystallin enhancer binding factor 1; E47, basic helix–loop–helix transcription factor; HDAC, histone deacetylase; SIP1, Smad interacting protein 1; RB, retinoblastoma tumour-suppressor; SIN3A, histone deacetylase complex subunit; Snail and Slug, zinc finger containing proteins; SP1, specific protein 1; WT1, Wilms’ Tumour 1. Modified after Peinado, Portillo, and Cano (2004). (B) Post-transcriptional regulation of E-cadherin (E-cad) and its cytoplasmic binding protein beta-catenin (-CTN). Ectodomain shedding occurs when matrix metalloproteases (MMPs) cleave a stable 80 kDa soluble fragment (sE-cad). Tyrosine phosphorylation by receptor tyrosine kinases (RTKs), such as the epidermal growth factor receptor and the HGF/SF receptor c-Met, or the cellular homologue of the Rous sarcoma virus oncogenic protein (c-Src), counteracted by protein tyrosine phosphatase (PTP) may lead to endocytosis, association with the E3 ubiquitin ligase Hakai (Japanese for destruction) and degradation (Fujita et al., 2002). By contrast, serine/threonine phosphorylation by glycogen synthase kinase-3 (GSK-3), casein kinase II (CKII) or protein kinase D1 (PKD1) stabilizes the E-cadherin/catenin complex. When accumulating as a result of lack of GSK-3-mediated phosphorylation and degradation through activation of the Wnt signalling pathway (not shown), -CTN in complex with lymphoid enhancer/T-cell factor (TCF/LEF-1) transactivates genes, the products of which (e.g. MMPs) are pro-invasive. Axin and adenomatous polyposis coli (APC) are cytoplasmic partners of -CTN. P in yellow or brown represent protein phosphorylation on serine/threonine or on tyrosine, respectively.
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4. Therapeutic implications One of the aims of analyzing mechanisms of cancer invasion in its various aspects is the development of new strategies for cancer therapy. The three aspects of colon cancer invasion discussed here, namely molecular cancer cell signalling, cross talk of cancer cells with host cells and interaction with enteric micro-organisms, have served successfully as targets in the treatment of human cancer. Cancer cell signal transduction through epidermal growth factor receptor (EGFR) is inactivated by cetuximab, an antibody against the extracellular domain of the receptor that interferes with ligand binding. Cetuximab is used successfully, alone or in combination with chemotherapy, in the treatment of metastatic colon cancers that are resistant to other forms of therapy (Wong, 2005). Formation of new vessels, a prominent part of the host reaction, is evoked mainly by vascular endothelial growth factor (VEGF). Antibodies against VEGF, such as bevacizumab, have been used successfully in colon cancer treatment (Nygren, Sorbye, Osterlund, & Pfeiffer, 2005). The role of bacteria in cancer development has been a matter of debate for some decades. That bacteria may be useful targets for anticancer therapy is illustrated by the decrease of gastric cancer incidence through eradication of Helicobacter pylori (Figueiredo et al., 2002). It is an open question as to whether or not the selective eradication of enteric bacteria could be equally effective in colon cancer. Acknowledgements D. Debruyne and A. Leroy are fellows of the FWO (Fonds voor Wetenschappelijk Onderzoek)-Vlaanderen. M.J. Oliveira is a fellow of the Portuguese Foundation for Science and Technology. The authors thank G. De Bruyne for artwork and I. Hart for reviewing the manuscript. References Behrens, J., Mareel, M. M., Van Roy, F. M., & Birchmeier, W. (1989). Dissecting tumor cell invasion: Epithelial cells acquire invasive properties following the loss of uvomorulin-mediated cell–cell adhesion. J. Cell Biol., 108, 2435–2447. Berx, G., & Van Roy, F. (2001). The E-cadherin/catenin complex: An important gatekeeper in breast cancer tumorigenesis and malignant progression. Breast Cancer Res., 3, 289–293. Brabletz, T., Hlubek, F., Spaderna, S., Schmalhofer, O., Hiendlmeyer, E., Jung, A., et al. (2005). Invasion and metastasis in colorectal cancer: Epithelial–mesenchymal transition, mesenchymal–epithelial transition, stem cells and -catenin. Cells Tissues Organs, 179, 56–65.
Darwanto, A., Kitazawa, R., Maeda, S., & Kitazawa, S. (2003). MeCP2 and promoter methylation cooperatively regulate E. cadherin gene expression in colorectal carcinoma. Cancer Sci., 94, 442–447. De Craene, B., Gilbert, B., Stove, C., Bruyneel, E., van Roy, F., & Berx, G. (2005). The transcription factor Snail induces tumor cell invasion through modulation of the epithelial cell differentiation program. Cancer Res., 65, 6237–6244. De Wever, O., & Mareel, M. (2003). Role of tissue stroma in cancer cell invasion. J. Pathol., 200, 429–447. Derycke, L. D. M., & Bracke, M. E. (2004). N-Cadherin in the spotlight of cell–cell adhesion, differentiation, embryogenesis, invasion and signalling. Int. J. Dev. Biol., 48, 463–476. Fichna, J., & Janecka, A. (2004). Opioid peptides in cancer. Cancer Metastasis Rev., 23, 351–366. Figueiredo, C., Machado, J. C., Pharoah, P., Seruca, R., Sousa, S., Carvalho, R., et al. (2002). Helicobacter pylori and interleukin 1 genotyping: An opportunity to identify high-risk individuals for gastric carcinoma. J. Nat. Cancer Inst., 94, 1680–1687. Fujita, Y., Krause, G., Scheffner, M., Zechner, D., Leddy, H. E. M., Behrens, J., et al. (2002). Hakai, a c-Cbl-like protein, ubiquitinates and induces endocytosis of the E-cadherin complex. Nat. Cell Biol., 4, 222–231. Kinsella, A. R., Lepts, G. C., Hill, C. L., & Jones, M. (1994). Reduced E-cadherin expression correlates with increased invasiveness in colorectal carcinoma cell lines. Clin. Exp. Metastasis, 12, 335– 342. Larriba, M. J., & Mu˜noz, A. (2005). SNAIL vs. Vitamin D receptor expression in colon cancer: Therapeutics implications. Br. J. Cancer, 92, 985–989. Lecuit, M. (2005). Understanding how Listeria monocytogenes targets and crosses host barriers. Clin. Microbiol. Infect., 11, 430– 436. Mareel, M., & Leroy, A. (2003). Clinical, cellular, and molecular aspects of cancer invasion. Physiol. Rev., 83, 337–376. Nygren, P., Sorbye, H., Osterlund, P., & Pfeiffer, P. (2005). Targeted drugs in metastatic colorectal cancer with special emphasis on guidelines for the use of bevacizumab and cetuximab: An Acta Oncologica expert report. Acta Oncol., 44, 203–217. Oliveira, M. J., Van Damme, J., Lauwaet, T., De Corte, V., De Bruyne, G., Verschraegen, G., et al. (2003). -Casein-derived peptides, produced by bacteria, stimulate cancer cell invasion and motility. EMBO J., 22, 6161–6173. Peinado, H., Portillo, F., & Cano, A. (2004). Transcriptional regulation of cadherins during development and carcinogenesis. Int. J. Dev. Biol., 48, 365–375. Perl, A.-K., Wilgenbus, P., Dahl, U., Semb, H., & Christofori, G. (1998). A causal role for E-cadherin in the transition from adenoma to carcinoma. Nature, 392, 190–193. Radtke, F., & Clevers, H. (2005). Self-renewal and cancer of the gut: Two sides of a coin. Science, 307, 1904–1909. Takeichi, M. (1991). Cadherin cell adhesion receptors as a morphogenetic regulator. Science, 251, 1451–1455. Trusolino, L., & Comoglio, P. M. (2002). Scatter-factor and semaphorin receptors: Cell signalling for invasive growth. Nat. Rev. Cancer, 2, 289–300. Van Aken, J., Cuvelier, C. A., De Wever, N., Gao, Y., Mareel, M. M., & Roels, H. J. (1993). Immunohistochemical analysis of E-cadherin expression in human colorectal tumours. Pathol. Res. Pract., 189, 975–978. Wong, S.-F. (2005). Cetuximab: An epidermal growth factor receptor monoclonal antibody for the treatment of colorectal cancer. Clin. Ther., 27, 684–694.
Cells in focus
Colon cancer cells: Pro-invasive signalling Delphine Debruyne a , Maria Jos´e Oliveira a,b , Marc Bracke a,∗ , Marc Mareel a , Ancy Leroy a a
Laboratory of Experimental Cancerology, Department of Radiotherapy and Nuclear Medicine, Ghent University Hospital, De Pintelaan 185, 1P7, B-9000 Ghent, Belgium b Instituto de Patologia e Immunologia Molecular da Universidade do Porto (IPATIMUP), 4200 Porto, Portugal Received 4 November 2005; received in revised form 9 January 2006; accepted 10 January 2006 Available online 13 February 2006
Abstract Colon cancer results from erroneous renewal of the enteric epithelium. Mutations in stem cells, or their proliferative progenitors, cause accumulation of cells that invade into the stroma and continue to divide rather than migrating on top of the basement membrane prior to entering into apoptosis. Many of these changes in invasive activity appear to be related to the invasion-suppressor role of E-cadherin. We have also investigated Listeria monocytogenes and other enteric bacteria, since these bacteria stimulate invasion through the production of a -casein-derived 13-amino acid peptide which is produced by enzymes present in the colon cancer ecosystem. The pro-invasive 13-amino acid peptide signals via small guanosine triphosphatases, which modulate the actin cytoskeleton, and via phosphorylation of the ␦ opioid receptor. The pro-invasive activity of this peptide is neutralized by the ␦ opioid receptor antagonist, naloxone. Since the ␦ opioid receptor belongs to the family of G protein-coupled receptors, implicated in colon cancer cell invasion signalling pathways, it is tempting to speculate that opioids could play a role in mediating this trait of malignant tumours. © 2006 Elsevier Ltd. All rights reserved. Keywords: Colon cancer invasion; Micro-environment; E-cadherin; -Casein peptide; Opioid receptor
Cell facts • Colon epithelial cells are renewed continuously from stem cells, present at the base of the crypts. • Colon cancer cells result from mutations in stem cells or in their proliferating progenitors. • Invasion by colon cancer cells is restricted through cell–cell adhesion and the signalling of the E-cadherin/catenin complex. • Colon cancer cells are sensitive to pro-invasive signalling from enteric bacteria, implicating G protein-coupled opioid receptors in this process.
1. Introduction
∗
Corresponding author. Tel.: +32 9 2403063; fax: +32 9 2404991. E-mail address: [email protected] (M. Bracke).
1357-2725/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2006.01.003
Normal adult colon enterocytes are renewed continuously from stem cells, producing proliferative progenitors that differentiate into absorptive or into secretory
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Fig. 1. Schematic illustration of normal and cancerous colon. The normal sequence (right) comprises proliferation and migration from stem cells in the bottom of the crypt to presence of polarized epithelial cells at the luminal surface. Development of colon cancer (left) starts from mutations in stem cells or their proliferative progenitors (indicated by mitotic figures) transforming into hyperplasia and invasive cancer. The insets illustrate molecular signalling (arrows) between enterocytes (right inset) or cancer cells (left inset) and host cells in their micro-environment. B, bacteria; BM, basement membrane; E-cad, epithelial cadherin; EMT, epithelial–mesenchymal transition; HGF/SF, hepatocyte growth factor/scatter factor; TGF-, transforming growth factor-; Tn-C, tenascin C.
cells. During this process colon enterocytes migrate from the stem cell compartment at the base of the crypt towards the luminal surface, with the basement membrane strictly separating them from the stromal compartment (Radtke & Clevers, 2005). In contrast, invasive colon cancer results from mutations in stem cells or in their proliferative progenitors which breach the basement membrane barrier (Fig. 1). Colon cancer provides a prototype of tumour development for several reasons: its high frequency with a 50% risk of polyps above age 70 and a 5% life time risk of invasive cancer; the occurrence of familial forms such as hereditary non-polyposis colorectal cancer (HNPCC) and familial adenomatous polyposis (FAP); the possibility of observing different precursor lesions, such as polyps and carcinoma in situ, in the same patient. 2. Colon epithelium plasticity: epithelial–mesenchymal transition (EMT) and mesenchymal–epithelial transition (MET) The colon epithelium forms a barrier by means of cell–cell interactions, involving tight junctions, cadherin-based adherens junctions, gap junctions and desmosomes. When, during cancer development, the invasive program is switched on, epithelial cells transform into cells with a mesenchymal phenotype (EMT)
(Trusolino & Comoglio, 2002). These cells lose their normal established position due to alterations in cell–cell contacts, cell–matrix interactions and the cytoskeletal structure. They migrate in the wrong direction, through the basement membrane and into the stroma. Here, dramatic alterations take place, constituting a novel microenvironment for the cancer cell. These include transition from fibroblasts into myofibroblasts, infiltration by leukocytes and the formation of new blood and lymphatic vessels (De Wever & Mareel, 2003). Cancer cells at the invasive front disseminate via the circulation to form a secondary tumour in a distant organ. At this metastatic site, cancer cells may regain the phenotype of the primary tumour, undergoing a mesenchymal–epithelial transition (MET) (Brabletz et al., 2005). The invasion and metastasis programs underlying this EMT/MET appear to result from the involvement of the same cellular activities and molecular signalling pathways that also participate in the maintenance of the normal colon epithelium. Comparison between normal colon and invasive colon cancer, nevertheless, reveals up- and downregulation of multiple tumour-promoter and -suppressor genes, respectively. This becomes clear not only from cDNA microarray analysis, but also from detailed dissection of the invasion-related proteomes and their signalling pathways (Mareel & Leroy, 2003). The E-cadherin/catenin complex serves as our paradigm of
D. Debruyne et al. / The International Journal of Biochemistry & Cell Biology 38 (2006) 1231–1236
involved molecules because of its crucial role in maintenance of the epithelium and its invasion-suppressor function (Behrens, Mareel, Van Roy, & Birchmeier, 1989; Takeichi, 1991). 3. Functional loss: colon cancer cells lose cell–cell contacts 3.1. The key-role of the E-cadherin/catenin complex Epithelial(E)-cadherin is a transmembrane glycoprotein of the type I cadherin superfamily, mediating homotypic (same cell type) homophilic (same molecules) cell–cell adhesion and, through its interaction with the catenins, signal transduction events as well. Circumstantial evidence in favour of E-cadherin functioning as an invasion-suppressor comes from the analysis of human cancers in various stages of progression and from the manipulation of cell lines. More direct evidence was gained from observations on transgenic animals. Each criterion on its own does not suffice to assign a given molecule an invasion-suppressor or an invasionpromoter function but, taken together, they give an estimation of the positive or negative weight of the molecule in the invasion process. In normal colon, immunohistochemical staining of E-cadherin produces a honeycomb like pattern at the lateral borders of the enterocytes. In colorectal cancer, disturbance of this pattern and lower levels or absence of E-cadherin expression is associated with increasing histological grading (higher grades correspond to lower degree of differentiation) and with a worse prognosis (Van Aken et al., 1993). Most colon cancer cell lines are E-cadherin positive and are poor invaders into extracellular matrices unless assisted by stromal cells or by pro-invasive ligands. In a series of seven cell lines studied by Kinsella, Lepts, Hill, and Jones (1994), two had decreased levels of expression of E-cadherin and these two invaded into collagen and into Matrigel in vitro. The causal relationship between E-cadherin expression and invasion in vivo was demonstrated convincingly in transgenic mice bearing pancreatic -cell tumours (Perl, Wilgenbus, Dahl, Semb, & Christofori, 1998), though similar experiments have not yet been done with colon tumours. What is the mechanism of E-cadherin downregulation in colon cancer? Inactivating mutations, such as found in gastric and in lobular breast cancer (Berx & Van Roy, 2001), are absent. By contrast, epigenetic modulation of E-cadherin expression by promoter hypermethylation, a frequent event in several types of cancer, does occur. Methyl-CpG-binding protein (MeCP2) and 5 CpG island hypermethylation, as demonstrated
1233
by in situ hybridization and by methylation-specific PCR, respectively, did correlate with reduction of Ecadherin expression (Darwanto, Kitazawa, Maeda, & Kitazawa, 2003). Downmodulation of E-cadherin by promoter methylation is reversible and fits with METassociated re-expression in metastases. The hypothesis is that in order to grow at the metastatic site, disseminated mesenchymal cancer cells must regain at least some of their epithelial functions (Brabletz et al., 2005). Transcriptional downregulation is another mechanism of E-cadherin repression that may act in concord with methylation (Fig. 2A). Snail, a member of a multizinc finger protein family of transcription factors, is a strong repressor of the E-cadherin CDH1 gene, interacting specifically with E-boxes in the promoter and causing loss of epithelial function and gain of invasion. In the normal colon Snail is not expressed whereas cancers score positive by immunohistochemistry with a trend towards higher expression in tumours with metastatic ability (Larriba & Mu˜noz, 2005). We have mentioned before the multiplicity of changes related to the invasion program. This is well illustrated by Snail, consistently repressing 167 genes and inducing 23 others (De Craene et al., 2005). One other element of the E-cadherin/catenin invasionsuppressor complex, -catenin, deserves mentioning because it too plays a dominant role in colon cancer as an invasion-promoter (Radtke & Clevers, 2005). Mutations in the -catenin serine/threonine phosphorylation sites preclude its degradation and direct it to the nucleus where it transactivates invasion-promoter genes (Fig. 2B). Upregulation of N-cadherin, described in some E-cadherin-negative cancers, has not been described in colon cancer (Derycke & Bracke, 2004). 3.2. Participation of enteric bacteria: from E-cadherin to opioid receptors E-cadherin led us to the opioid receptors. Since Ecadherin mediates binding of the Listeria (L.) monocytogenes virulence factor Internalin A to enterocytes (Lecuit, 2005), we wondered whether binding of L. monocytogenes would neutralize the invasionsuppressor role of E-cadherin (Oliveira et al., 2003). L. monocytogenes did stimulate invasion of human colon cancer cells into extracellular matrices in vitro, but E-cadherin was not mechanistically implicated. Conditioned medium (CM) prepared from L. monocytogenes incubated on top of collagen type I also stimulated invasion. In this pro-invasive conditioned medium, a 13mer peptide (HKEMPFPKYPVEP) was identified by reverse-phase high-performance liquid chro-
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Fig. 3. Generation of the pro-invasive 13mer peptide (HKEMPFPKYPVEP). Arrows and arcs indicate activation and inactivation, respectively, in the invasion pathway. Dashed arrows indicate links that are not yet confirmed experimentally. Acronyms in alphabetic order: Cdc42, cell division cycle 42; DOR, ␦ opioid receptor; FAK, p125 focal adhesion kinase; G␣␥, G protein; Mpl, Listeria thermolysin-like metalloprotease; PAK1, p21-activated kinase; PI3K, phosphoinositide 3 kinase; RhoA, Ras homologue A.
matography (RP-HPLC) followed by mass spectrometry (MS–MS) and it was shown that the pro-invasive activity could be reproduced by a synthetic 13mer peptide. The sequence of this peptide corresponds to a bovine casein sequence and the source is the tryptic soy broth (TSB) culture medium, adsorbed onto the bacterial surface. Both, collagen and L. monocytogenes, are required to generate the 13mer peptide (Fig. 3). Bacteria isolated from human colon cancer biopsies, were grown in TSB on collagen type I. With CM from such cultures we were able to stimulate invasion in vitro. Moreover, the enzymes needed for cleavage of a -casein source into the pro-invasive 13mer peptide also were present in human colon: when we incubated a synthetic -casein source on top of colon cancer biopsies, we also found the 13mer peptide in the supernatant using
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HPLC/MS–MS. The 13mer peptide-mediated invasion pathway in colon cancer cells was, in part, revealed by transfecting the cells transiently with cDNA constructs encoding dominant-negative and constitutively active proteins and by treating the cells with pharmacological modulators (Oliveira et al., 2003). Signalling was through PI3K activation of Cdc42, which in turn activates PAK1. This serine/threonine kinase connects Cdc42 with RhoA and inactivates it, causing dephosphorylation of FAK and downregulation of focal adhesion complexes. Cdc42 activation explains the increase in filopodia formation, as was observed by F-actin staining of a human colon cancer cell line. All these effects are associated with stimulation of invasion, the 13mer peptide acting as a motility factor. There is evidence that the 13mer peptide acts via the ␦ opioid receptor (DOR). Since in silico analysis revealed sequence homology between the C-terminus of the 13mer peptide and human -casomorphin (a DOR agonist), DOR was suggested as the candidate receptor. Moreover naloxone, a DOR antagonist, inhibited 13mer peptide-stimulated invasion, as well as the increase in serine phosphorylation of DOR after treatment with the 13mer peptide. Opioid receptors belong to the superfamily of G protein-coupled receptors (GPCRs). Although opioid receptors are associated predominantly with the nervous system, they also are present in the immune system and in different cancer cell lines (Fichna & Janecka, 2004). They are activated both by endogenously produced opioid peptides and by exogenously administered opiate compounds. Opioid agonist treatment switches on signal transduction pathways that lead to activation of invasion-associated activities like reorganization of the actin cytoskeleton, and increased cell survival. These are arguments to investigate further the role of opioids in cancer invasion.
Fig. 2. Transcriptional (A) and post-transcriptional (B) modulation of E-cadherin. (A) E-cadherin promoter with positive (in green) and negative (in red) regulators and with co-activators (light green) and co-repressors (light red). Acronyms in alphabetic order: AP2, adaptor protein 2; CtBP, C-terminal binding protein; C/EBP, CCAAT/enhancer-binding protein; DNMT, DNA methyltransferase; ␦ EF1, delta-crystallin enhancer binding factor 1; E47, basic helix–loop–helix transcription factor; HDAC, histone deacetylase; SIP1, Smad interacting protein 1; RB, retinoblastoma tumour-suppressor; SIN3A, histone deacetylase complex subunit; Snail and Slug, zinc finger containing proteins; SP1, specific protein 1; WT1, Wilms’ Tumour 1. Modified after Peinado, Portillo, and Cano (2004). (B) Post-transcriptional regulation of E-cadherin (E-cad) and its cytoplasmic binding protein beta-catenin (-CTN). Ectodomain shedding occurs when matrix metalloproteases (MMPs) cleave a stable 80 kDa soluble fragment (sE-cad). Tyrosine phosphorylation by receptor tyrosine kinases (RTKs), such as the epidermal growth factor receptor and the HGF/SF receptor c-Met, or the cellular homologue of the Rous sarcoma virus oncogenic protein (c-Src), counteracted by protein tyrosine phosphatase (PTP) may lead to endocytosis, association with the E3 ubiquitin ligase Hakai (Japanese for destruction) and degradation (Fujita et al., 2002). By contrast, serine/threonine phosphorylation by glycogen synthase kinase-3 (GSK-3), casein kinase II (CKII) or protein kinase D1 (PKD1) stabilizes the E-cadherin/catenin complex. When accumulating as a result of lack of GSK-3-mediated phosphorylation and degradation through activation of the Wnt signalling pathway (not shown), -CTN in complex with lymphoid enhancer/T-cell factor (TCF/LEF-1) transactivates genes, the products of which (e.g. MMPs) are pro-invasive. Axin and adenomatous polyposis coli (APC) are cytoplasmic partners of -CTN. P in yellow or brown represent protein phosphorylation on serine/threonine or on tyrosine, respectively.
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4. Therapeutic implications One of the aims of analyzing mechanisms of cancer invasion in its various aspects is the development of new strategies for cancer therapy. The three aspects of colon cancer invasion discussed here, namely molecular cancer cell signalling, cross talk of cancer cells with host cells and interaction with enteric micro-organisms, have served successfully as targets in the treatment of human cancer. Cancer cell signal transduction through epidermal growth factor receptor (EGFR) is inactivated by cetuximab, an antibody against the extracellular domain of the receptor that interferes with ligand binding. Cetuximab is used successfully, alone or in combination with chemotherapy, in the treatment of metastatic colon cancers that are resistant to other forms of therapy (Wong, 2005). Formation of new vessels, a prominent part of the host reaction, is evoked mainly by vascular endothelial growth factor (VEGF). Antibodies against VEGF, such as bevacizumab, have been used successfully in colon cancer treatment (Nygren, Sorbye, Osterlund, & Pfeiffer, 2005). The role of bacteria in cancer development has been a matter of debate for some decades. That bacteria may be useful targets for anticancer therapy is illustrated by the decrease of gastric cancer incidence through eradication of Helicobacter pylori (Figueiredo et al., 2002). It is an open question as to whether or not the selective eradication of enteric bacteria could be equally effective in colon cancer. Acknowledgements D. Debruyne and A. Leroy are fellows of the FWO (Fonds voor Wetenschappelijk Onderzoek)-Vlaanderen. M.J. Oliveira is a fellow of the Portuguese Foundation for Science and Technology. The authors thank G. De Bruyne for artwork and I. Hart for reviewing the manuscript. References Behrens, J., Mareel, M. M., Van Roy, F. M., & Birchmeier, W. (1989). Dissecting tumor cell invasion: Epithelial cells acquire invasive properties following the loss of uvomorulin-mediated cell–cell adhesion. J. Cell Biol., 108, 2435–2447. Berx, G., & Van Roy, F. (2001). The E-cadherin/catenin complex: An important gatekeeper in breast cancer tumorigenesis and malignant progression. Breast Cancer Res., 3, 289–293. Brabletz, T., Hlubek, F., Spaderna, S., Schmalhofer, O., Hiendlmeyer, E., Jung, A., et al. (2005). Invasion and metastasis in colorectal cancer: Epithelial–mesenchymal transition, mesenchymal–epithelial transition, stem cells and -catenin. Cells Tissues Organs, 179, 56–65.
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