Protein tyrosine kinases (PTKs) are a large and diverse multigene family evolved to perform functions that regulate a range of cellular processes, including cell growth, differentiation, death, motility, adhesion, and cell-to-cell communication (Pawson, 1994). They consist of a group of very closely related protein kinases, which are distinguished by common sequence motifs and tyrosine-specific catalytic activity that differs from the broader group of serine/threonine kinases (Robinson, et al., 2000, Manning, 2002 ). While members of the protein kinase superfamily are found throughout all kingdoms of life, tyrosine kinases are restricted to metazoan cells. Tyrosine phosphorylation is a hallmark of metazoans and is associated with a variety of cellular processes directly or indirectly linked to environmental clues and related to the multicellular status of the organism. Dysfunction of cellular phosphorylation is associated with a variety of human diseases, from cancer to diabetes (Hunter, 2002). Each PTK possesses a functional kinase domain capable of catalyzing the transfer of the gamma-phosphate group of ATP to the hydroxyl groups of specific tyrosine residues in peptides. Although phosphotransfer reactions catalyzed by various PTKs are similar with regard to their basic mechanisms, the recognition of substrates by PTKs and, therefore, subsets of proteins phosphorylated by them show a considerable degree of specificity.
Aberrant kinase activity is implicated in a variety of human diseases, in particular those involving inflammatory or proliferative responses, such as cancer. Directly or indirectly, more than 400 human diseases have been connected to protein kinases. The ability to modulate kinase activity therefore represents an attractive therapeutic strategy for the treatment of human illnesses. However, despite a wealth of potential targets, only a handful of kinases are targeted by drug compounds currently on the market.
The PTK superfamily can be divided into two groups according to the presence of transmembrane and extracellular domains, which enable PTKs possessing them to recognize extracellular ligands, in particular, various peptide growth factors. Specific ligands and intracellular signaling pathways induced by them have been identified for many, albeit not for all, membrane-spanning PTKs (Schlessinger, 2000). PTKs lacking the transmembrane and extracellular sequences are referred to as non-receptor or non-transmembrane PTKs. Thirty-two genes encoding for non-receptor PTKs clustered into 10 families are present in the human genome (Robinson, et al., 2000, Manning, 2002 ).
Brk protein tyrosine kinases belong to a novel family of intracellular soluble tyrosine kinases distinct from Src family kinases. This family branched off the Src tyrosine kinase family tree early in evolution. Although developmental expression patterns and functional overexpression in vitro have associated these kinases with growth suppression and differentiation, their physiological functions remain largely unknown.
The Brk family of non-receptor PTKs has four members: Brk, Frk, Srms, and Src42A (Serfas and Tyner, 2003). They are defined by a highly conserved exon structure that is distinct from other major intracellular tyrosine kinase families including c-Src (Lee, et al., 1998). Brk and Frk have been cloned independently from human, mouse, and rat cells by several laboratories, and there are multiple names for these PTKs; Brk is also known as PTK6 and Sik (Lee, et al., 1993;Mitchell, et al., 1994;Vasioukhin, et al., 1995), whereas Frk is also known as Rak, Bsk, Iyk, and Gtk (Cance, et al., 1994,;Lee, et al., 1994;Oberg-Welsh and Welsh, 1995;Sunitha and Avigan, 1996;Thuveson, et al., 1995). Srms was cloned and studied only in mice, but its ortholog is present in the human genome, as well (Kohmura, et al., 1994). Src42A, also known as Dsrc41, was cloned and studied only in Drosophila, and it shares 61% amino acid identity with its putative mammalian ortholog Frk (Serfas and Tyner, 2003;Shishido, et al., 1991). Brk and Frk are expressed specifically in epithelial cells, primarily those of the intestinal tract, and their expression is upregulated in some epithelial tumors. In contrast, Srms expression is ubiquitous, although found most abundantly in lung, liver, spleen, kidney and testis (Kohmura, et al., 1994). Src42A is expressed in a wide range of tissues during embryonic development.
Brk-family PTKs are highly homologous to Src-family PTKs, even more so than are Csk-family PTKs (Robinson, et al., 2000). Their domain structure is very similar to that of Src-family PTKs, consisting of a highly divergent N-terminal sequence followed by an SH3 domain, an SH2 domain, and a tyrosine kinase domain. SH2 and SH3 domains bind to phosphorylated tyrosine residues and proline rich sequences of target proteins respectively (Songyang, et al., 1993, Cohen, 1995 #3503). As found with Src family PTKs, these domains are involved in both intermolecular associations that regulate signaling cascades, and intramolecular associations that autoregulate protein kinase activity (Sicheri and Kuriyan, 1997;Thomas and Brugge, 1997;Xu, et al., 1997). However, unlike Src-family PTKs, most Brk-family PTKs lack the N-myristoylation site and are thus not specifically targeted to the membrane. The only exception from this rule is rodent Frk, which retains the glycine residue in position 2 and is consequently myristoylated and localized to the membrane (Sunitha and Avigan, 1996). Nuclear localization of Brk and Frk has been reported (Cance, et al., 1994, Derry, 2000 #2416;Haegebarth, et al., 2004). Frk, Brk and Src42A, although not Srms, possess tyrosine residues near their C-termini, which might mediate negative regulation of these PTKs in an Src-like fashion. Frk and Src42A have been shown to be phosphorylated by Csk and dCsk respectively (Cance, et al., 1994;Read, et al., 2004).
Overexpression of the epithelial specific tyrosine kinase Frk in a number of cell lines of epithelial and mesenchymal origin resulted in potent growth arrest that may function, in part, through its interaction with pRb (Craven, 1995 #3330; Oberg-Welsh, 1998 #3506; Anneren, 2000 #3537}. Frk has been shown to promote neurite outgrowth in PC12 cells (Anneren, et al., 2000), and is also able to associate with and phosphorylate SHB, the Src homology 2 (SH2) domain adaptor protein, thus regulating versatile signal transduction pathways involved in cell survival, differentiation, and proliferation (Anneren, 2002;Anneren, et al., 2003). Recent studies showed that expression of Frk is able to block breast ductal carcinoma cell proliferation at the G1 phase of the cell cycle (Meyer, et al., 2003), consistent with findings that its expression is progressively lost from human breast tumors (Berclaz, et al., 2000). Furthermore, Frk is involved in the apoptotic response of B-cells to inflammatory cytokines (Anneren and Welsh, 2001) (Welsh, et al., 1999). These findings hinted that Frk might be involved in the regulation of cell differentiation. However, Frk-deficient mice demonstrated no morphological abnormalities in epithelial tissues, no related metabolic or developmental changes, and no increase in the incidence of spontaneous tumors (Chandrasekharan, et al., 2002). The only phenotypic change observed in these mice was a slight decrease in the level of circulating thyroid T3 hormone.
Similarly to Frk, homozygous deletions of Srms in mice have been produced, with no detectable phenotypic effect (Kohmura, et al., 1994). Furthermore, mice deficient for Brk are viable and fertile, suggesting redundant functions for the mammalian Brk family members (Wenjun Bie, 2005). In contrast, loss of function alleles of Src42A cause homozygous lethality in Drosophila (Lu and Li, 1999). Src42A has been identified as a negative regulator of Ras pathway receptor tyrosine kinase signaling (Lu and Li, 1999). Furthermore, Src42A has been show to activate the Bsk signaling pathway in epidermal closure, the Drosophila homolog to Jnk pathway signaling (Tateno, et al., 2000).
The intracellular tyrosine kinase Brk was identified in a screen for protein tyrosine kinases involved in breast cancer (Mitchell, et al., 1994), from the mouse small intestine in a screen for factors that regulate epithelial cell differentiation (Siyanova, et al., 1994), and from cultured human melanocytes (Lee, et al., 1993). Brk expression is restricted to epithelial cells of the skin, gastrointestinal tract, and prostate, with highest levels being expressed in the gastrointestinal tract (Derry, et al., 2003;Llor, et al., 1999;Vasioukhin, et al., 1995). Furthermore, Brk expression is developmentally regulated. It is detected late in gestation in the mouse, at mouse embryonic day 15.5 in the differentiating granular layer of the skin and at embryonic day 18.5 in the differentiating intestine (Vasioukhin, et al., 1995). Brk expression is initiated as cells migrate away from the proliferative zone and begin the process of terminal differentiation. Overexpression of Brk in mouse keratinocytes resulted in increased expression of the differentiation marker filaggrin during calcium-induced differentiation (Vasioukhin and Tyner, 1997). Brk is expressed in many breast carcinoma cell lines and primary breast tumors, but has not been detected in normal human breast tissue (Barker, et al., 1997;Mitchell, et al., 1994), or at any stage of mammary gland differentiation in the mouse (Llor, et al., 1999). Modest increases in Brk levels have been detected in colon tumors and Brk expression increases during differentiation of Caco-2 colon adenocarcinoma cells (Llor, et al., 1999).
While Brk resembles Src structurally with SH3 and SH2 protein-protein binding domains, the tyrosine kinase domain, and the regulatory C-terminus, it lacks the amino-terminal myristoylation signal that localizes Src to the cell membrane, and therefore is not specifically targeted to the membrane (Fig. 1) (Vasioukhin, et al., 1995). Its intracellular localization is flexible and it can be found to be present in the nucleus as well as the cytoplasm or at the membrane (Haegebarth, et al., 2004). The Src homology 3 (SH3) domain is involved in intramolecular interactions that regulate kinase activity, interactions with substrates, cellular localization, and association with other protein targets (Pawson, 1995). SH3 domains bind proline-rich sequences of the consensus PXXP in substrate proteins. The SH2 domain on the other hand is essential in controlling interactions. It recognizes and binds to phosphorylated tyrosine residues, with the specificity being determined by the 3-5 amino acids following the tyrosine residue (Songyang, et al., 1993).
Like Src family members, the SH3 and SH2 domains of Brk engage in intramolecular interactions with the kinase domain to form an autoinhibited conformation (Qiu and Miller, 2002). Brk activity is negatively regulated by tyrosine phosphorylation of its C-terminal tyrosine residue, Tyr-447 in mouse, similar to that of Src-family PTKs (Fig. 1). However, it remains to be determined how this tyrosine becomes phosphorylated in Brk, since it is phosphorylated neither by Brk itself nor by Csk (Qiu and Miller, 2002). Csk is playing this role for Src-family PTKs (Liu, et al., 1993). Phosphorylation on Tyr-447 in Brk causes the intramolecular interaction of this tyrosine with the SH2 domain, which induces the binding of the SH3 domain to the linker region connecting the SH2 domain and the kinase domain, accompanied by the binding of the linker region to the kinase domain. These intramolecular interactions prevent the binding of ATP to the critical catalytic residues rendering Brk inactive. Mutation of the carboxy-terminal tyrosine of Brk to phenylalanine (Y447F) results in increased enzyme activity when overexpressed in epithelial cells, supporting a role for this residue in autoinhibition (Derry, et al., 2000;Qiu and Miller, 2002). However in contrast to increasing its transforming potential, mutation of the Brk regulatory tyrosine resulted in a decrease in the ability of Brk to induce anchorage–independent growth of fibroblasts (Kamalati, et al., 1996).
Analysis of Brk using mutagenesis, mass-spectrometry and enzyme kinetics indicated that Brk is capable of autophosphorylation, which significantly upregulates its kinase activity (Qiu and Miller, 2002). This study mapped the autophosphorylation site of Brk to Tyr-341, a conserved tyrosine residue inside the activation loop (Fig. 1). Autophosphorylation of Tyr-341 in an intermolecular process causes displacement of this tyrosine from a hydrophobic pocket of the PTK catalytic domain, resulting in the correct positioning of all key catalytic residues and the formation of the substrate binding surfaces, thus leading to the full activation of the enzyme.
Thus, physiological regulation of Brk is mediated by modulation of the interactions described above, achieved by (a) phosphorylation/dephosphorylation of the C-terminal regulatory site, and (b) binding of the SH2 and SH3 domains of Brk to various phosphotyrosine- or polyproline-containing proteins.
|Fig. 1: Structure of Src and Brk tyrosine kinases. Src and Brk tyrosine kinases share 44% amino acid identity. Both Src and Brk proteins contain SH3 and SH2 domains that regulate protein-protein interactions as well as a conserved catalytic SH1 domain. The tyrosine at 527 in Src and at 447 in Brk regulates kinase activity. Phosphorylation on these tyrosine residues results in the intramolecular formation of an inactive conformation involving both SH2 and SH3 domains. The lysine at 295 in Src and at 219 in Brk correlates with the ATP binding site and its mutation results in a dominant-negative protein. Tyrosines 416 in Src and 341 in Brk reside in the activation loop and are autophosphorylated resulting in increased kinase activity. In contrast to Src, Brk lacks an aminoterminal consensus myristoylation sequence.|
Like Src, localization of the Brk tyrosine kinase has been correlated with its activities. Brk was localized in the nucleus of normal luminal prostate epithelial cells as well as of well-differentiated but less tumorigenic prostate cancer cells, but it was a predominantly cytoplasmic protein in poorly differentiated tumors and more aggressive tumor cell lines (Derry, et al., 2003). Thus, relocalization of the Brk kinase during development of prostate cancer may indicate the disruption of a signaling pathway important for maintaining the normal phenotype of prostate epithelial cells.
In addition, a correlation between tumorigenicity and the subcellular localization of Brk has also been found in oral squamous cell carcinomas (OSCC) (Petro, et al., 2004). Brk was present in the nucleus and cytoplasm of normal oral epithelium (NOE) and moderately differentiated OSCC cells. However, in poorly differentiated OSCC cells, Brk was localized in perinuclear regions, supporting the notion that subcellular localization of this tyrosine kinase plays a role in determining its growth regulating functions.
Although the exact biological function of the Brk tyrosine kinase is still largely unknown, progress has been made in identifying substrates of this PTK. Brk has been found in complexes with EGF-R (Kamalati, et al., 1996), GTPase activating protein-associated p65 (Vasioukhin and Tyner, 1997), the putative adaptor protein Bks (Mitchell, et al., 2000), the EGF-R family member erbB3/HER3 (Kamalati, et al., 2000), the RNA-binding proteins Sam68, SLM-1 and SLM-2 (Derry, et al., 2000;Haegebarth, et al., 2004), the serine-threonine kinase PKB/Akt (Zhang, et al., 2004), and the focal adhesion protein paxillin (Chen, et al., 2004).
Like c-Src, overexpression of Brk has been shown to sensitize mammary epithelial cells to EGF and to induce transformation of fibroblasts (Kamalati, et al., 1996). The first is likely mediated by the functional interactions of Brk with the epidermal growth factor receptor-related receptor ErbB3, which enhance EGF signaling via the phosphoinositide 3-kinase/Akt pathway (Kamalati, et al., 2000). The use of RNA interference to efficiently and specifically downregulate Brk protein levels in breast carcinoma cells resulted in a significant suppression of their proliferation. Additionally, through the expression of a kinase-inactive mutant, it was shown that Brk can mediate promotion of proliferation via a kinase-independent mechanism, potentially functioning as an adaptor (Harvey and Crompton, 2003). Evidence for a direct interaction between Brk and Akt/Protein kinase B (PKB) has recently been reported (Zhang, et al., 2004). In unstimulated cells, Brk has been shown to associate with and tyrosine-phosphorylate Akt, and herein to inhibit Akt kinase activity and downstream signaling. It has been proposed that Brk kinase activity normally limits basal or “steady state” activity of Akt in resting cells (Zhang, et al., 2004). However, upon stimulation with EGF and subsequent PI3-K activation, the Brk-Akt complex dissociates resulting in activation of Akt signaling. Taken together with the inhibition of receptor tyrosine kinase signaling by Brk family members (Serfas and Tyner, 2003;Zhang, et al., 1999), the biological function of this PTK in normal epithelial cells may be to limit the magnitude of growth factor receptor inputs to multiple intracellular signaling pathways.
Brk has been shown to phosphorylate the RNA-binding proteins Sam68 and the two Sam68-like mammalian proteins SLM-1 and SLM-2, and negatively regulate their RNA-binding activities (Derry, et al., 2000;Haegebarth, et al., 2004). Sam68, SLM-1 and SLM-2 are members of the STAR family of KH (heteronuclear ribonucleoprotein K homology) domain containing RNA binding proteins that regulate different aspects of RNA metabolism, including transport, stability, translation and processing (Lasko, 2003;Lukong and Richard, 2003;Vernet and Artzt, 1997). Although Sam68 can be phosphorylated by other intracellular tyrosine kinases, only Brk has been shown to colocalize with Sam68 in the nucleus where it phosphorylates Sam68, resulting in inhibition of its ability to bind RNA and to function as a cellular Rev homologue (Derry, et al., 2000). Brk further regulates the ability of Sam68 to regulate utilization of specific RNAs in the cytoplasm (Coyle, et al., 2003). Sam68 has also been implicated in a number of cellular processes including transcription, RNA splicing and export, translation, signal transduction, cell cycle progression, and replication of the human immunodeficiency virus and poliovirus (McLaren, et al., 2004;Reddy, et al., 1999;Soros, et al., 2001).
SLM-1 shares many similarities with Sam68; it interacts with many of the same proteins and is also tyrosine phosphorylated by Src during mitosis (Di Fruscio, et al., 1999). SLM-2 was also identified by its ability to interact with RNA-binding motif (RBM) in spermatogenesis and named T-STAR or ETOILE (Venables, et al., 1999). It can regulate the selection of alternative splice sites (Stoss, et al., 2001). Both proteins are tyrosine phosphorylated by Brk, which in turn inhibits their RNA binding abilities (Haegebarth, et al., 2004). The RNA-binding functions of these STAR proteins have been implicated in the posttranscriptional regulation of gene expression. Tyrosine phosphorylation and subsequent inhibition by Brk may regulate specific STAR family signaling pathways.
Inhibition of Sam68’s RNA binding ability has been shown to result in reduced levels of cyclin D1 and inhibition of cell proliferation (Barlat, et al., 1997). It was proposed that in normal epithelial tissues, Brk is positioned to inhibit RNA-binding activities of its nuclear STAR protein family substrates during differentiation (Haegebarth, et al., 2004). Signal transduction pathways have recently been shown to regulate gene expression not only by modifying the activity of transcriptional regulators, but also at the RNA level by posttranscriptional mechanisms (Lasko, 2003;Matter, et al., 2002). Thus, Brk appears to integrate external signals and gene expression regulation by its specific regulation of STAR family signaling pathways.
Recently, paxillin has been identified as a binding partner and substrate of Brk (Chen, et al., 2004). Upon EGF stimulation Brk is catalytically activated resulting in tyrosine phosphorylation of paxillin. This in turn promotes the activation of small GTPase Rac1 via the function of CrkII. Through this pathway, Brk is capable of promoting cell motility and invasion, and functions as a mediator of EGF-induced migration and invasion. This provides the first potential link between Brk and metastatic malignancy (Chen, et al., 2004).
The non-receptor Brk tyrosine kinase shows a highly specific expression pattern in regenerating epithelial linings with highest levels being expressed in the gastrointestinal tract. Its expression is restricted to non-proliferating cells of the villus, suggesting a role for this tyrosine kinase in the regulation of proliferation and differentiation in the intestinal epithelium (Llor, et al., 1999;Vasioukhin, et al., 1995).
The gastrointestinal tract constitutes a tube consisting of three tissue layers – the smooth muscle layer on the outside, the stromal layer in the middle, and the columnar epithelial cell layer on the inside. It can be morphologically and functionally divided into the small and large intestine. The small intestine is further divided into duodenum, jejunum and ileum (Fig. 2A). It is characterized by its complex organization into villi – finger-like projections into the lumen of the intestine, that dramatically increase the absorptive surface area, – and the crypts of Lieberkühn – bottle-shaped invaginations into the submucosa (Fig. 2B). The large intestine on the other hand has only crypts, and instead of villi there is a flat surface epithelium (Fig. 2C). The epithelium of the intestine is a constantly regenerating tissue with well-defined zones of proliferation and differentiation. Gradients in gene expression are established within the epithelium in two dimensions: the vertical, crypt to villus dimension, and the horizontal, duodenum to colon dimension. Furthermore, changes in gene expression are also observed in a temporal dimension – from the developing stage to adulthood.
A small number of intestinal stem cells reside near the bottom of each crypt. These cells slowly divide giving rise to a transient population of progenitor cells that rapidly divide and migrate towards the lumen of the intestine. These clonal populations of proliferating progenitor cells give rise to four different cell types – absorptive enterocytes, mucus-secreting goblet cells, and hormone secreting enteroendocrine cells, all of which are populating the villi and are found in both the small and large intestine. The fourth cell type are the Paneth cells, which produce antimicrobial defensins and reside at the crypt bottom of the small intestine but are absent in the colon (Hocker and Wiedenmann, 1998;Porter, et al., 2002).
The crypt progenitor cells are dividing every 12-16 hours, generating about 200 cells per crypt every day (Potten and Loeffler, 1990). Epithelial homeostasis is ensured through 3 mechanisms. First, cells are being continuously shed at the tip of the villi (small intestine) or the surface epithelium (colon) to counterbalance the crypt cell production (Potten, 1998). Second, the cells in the intestinal epithelium are continuously moving upwards with a transit time of approximately 5 days, with the only exception being made by Paneth cells and the immortal stem cell. Paneth cells differentiate as they migrate downwards to the base of the crypt, where they reside for about 20 days before being phagocytized by their neighbors. And third, proliferation is restricted to the crypt niche, thus resulting in the maintenance of distinct proliferative and differentiated compartments (Hermiston, et al., 1996). Deregulation of the crypt homeostasis is a feature of neoplastic transformation, and is evident in the earliest stage of colon cancer.
|Fig. 2: Schematic presentation of the mammalian digestive tract. (A) Mammalian digestive tract. Digestion starts in the mouth, where food is being macerated and partially digested. The esophagus serves as an alimentary organ, transporting food into the stomach, where it is digested by various enzymes. Most of the nutrient absorption occurs in the small intestine whereas water is absorbed mainly in the large intestine. The cecum serves for the storage of food. (B) Structure of the small intestine. Putative stem cells reside immediately above the Paneth cells. Progenitors stop proliferating at the crypt-villus junction and express differentiation markers. Enteroendocrine, absorptive, and mucus-producing cells migrate upward, whereas Paneth cells migrate downward and localize at the bottom of the crypt. (C) Structure of the large intestine. Stem cells reside at the crypt bottom. Progenitors are amplified by constant division along the bottom two thirds of the crypts, whereas cell cycle arrest and differentiation occur when progenitors reach the top third of the crypts. Paneth cells are absent in the colon.|
Colorectal cancer (CRC) is the second most common type of cancer with one million new cases diagnosed per year worldwide. Approximately 5% of the Western population will develop colorectal malignancies during their lifetime (Jemal, et al., 2002). Patients with a familial risk make up approximately 20% of all patients with CRC, whereas approximately 5-10% are inherited in an autosomal-dominant fashion (Lynch and de la Chapelle, 2003). Genomic instability is a characteristic of all intestinal malignancies.
Hereditary cancer can be divided into two categories based on the presence of polyposis, as exemplified by familial adenomatous polyposis (FAP) and hereditary nonpolyposis colorectal cancer (HNPCC). Patients with FAP develop large numbers of benign adenomatous polyps of the colorectal epithelium early in adulthood (Haggitt and Reid, 1986). Almost invariably, some of these will progress into invasiveness and, ultimately, metastasize. These CRC tumors characteristically display chromosomal instability (CIN) and harbor mutations in various tumor suppressor genes and oncogenes such as APC, K-ras, and p53. They are caused by initial mutations in the tumor suppressor gene adenomatous polyposis coli (APC), whose inactivation also occurs in a large percentage of sporadic CRC (Kinzler, et al., 1991;Nakamura, et al., 1992).
Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal-dominant cancer syndrome that predisposes to multiple primary cancers without intestinal polyposis. HNPCC tumors are predominantly located in the proximal colon (Jass, 1998), and their hallmark is microsatellite instability (MIN) (Thibodeau, et al., 1993). Mutations in mismatch repair (MMR) genes have been identified in HNPCC families. As a consequence of MIN mutations in the -catenin gene, transforming growth factor receptor beta II (TGFBR2) gene, the pro-apoptotic gene Bax, as well as the APC gene arise (Huang, et al., 1996;Rampino, et al., 1997).
The probability that a colorectal cell will acquire the genetic changes leading to a benign tumor is low, but approximately 50% of the Western populations still develop such a tumor by the age of 70. Sporadic CRC is caused by a series of genetic alterations, which begin with benign lesions and eventually lead to fully metastatic tumors (Fig. 3) (Fearon and Vogelstein, 1990). Colorectal tumors occur as a result of the mutational activation of oncogenes coupled with the inactivation of tumor-suppressor genes, followed by mutations in several other genes that are required to produce malignant tumors. These genetic alterations occur in a preferred sequence even though the total accumulation of changes determines the tumor’s biologic properties. In the adenoma-carcinoma sequence, the smallest identifiable lesion is an aberrant crypt focus (ACF). ACFs are very heterogenic which has led to controversial issues regarding their origin and involvement in CRC (Cheng and Lai, 2003). The majority of malignant dysplastic ACFs bears APC mutations, whereas non-malignant hyperplastic ACFs are proposed to arise from activating mutations in K-RAS (Nucci, et al., 1997). Expansion of dysplastic ACFs gives rise to adenomas, which acquire additional mutations and eventually progress to carcinoma in situ (Fig. 3). Colorectal tumors evolve through a series of restriction points, with only those cells acquiring the correct mutational event expanding. However, several signaling pathways are involved in the progression to CRC.
|Fig. 3: CRC development. Correlation between CRC progression and the accumulation of genetic alterations according to Fearon & Vogelstein (1990) (Fearon and Vogelstein, 1990).|
The Wnt signaling pathway has been shown to be of major importance in intestinal cell proliferation and stem cell maintenance, and its deregulation leads to malignant development in the mammalian gut epithelium. There are about 20 different secreted Wnt proteins, which bind to about 10 different Frizzled receptors. Downstream targets of Wnt include β-catenin, and the Tcf/Lef transcription factors. Transduction of canonical Wnt signals results in activation of Dishevelled (Dvl) and inhibition of glycogen synthase kinase 3β (GSK-3β), which in turn causes the stabilization and nuclear translocation of β-catenin. Nuclear β-catenin interacts with members of the Lef/Tcf transcription factor family resulting in activation of target genes (Sancho, et al., 2004). This β-catenin-TCF activity mediates a proliferation/ differentiation switch along the crypt-villus axis (Pinto, et al., 2003;van de Wetering, et al., 2002). Genes whose expression is driven by β-catenin-TCF are present in the proliferating cells of intestinal crypts, while those induced upon cessation of β-catenin-TCF activity are expressed by differentiated cells in the intestinal epithelium. c-Myc has been identified as one of the main mediators of this switch (He, et al., 1998). Furthermore, this gradient of β-catenin-TCF activity imposes restrictions on the migratory behavior of the intestinal epithelial cells by establishing the expression of EphB receptors and their ephrin-B ligands in complementary domains (Batlle, et al., 2002).
In the absence of Wnt signals, β-catenin is found in a multi-protein complex including APC, casein kinase-1 and GSK-3β, which results in phosphorylation and subsequent degradation of β-catenin (Huelsken and Birchmeier, 2001). Consequently, loss of functional APC or stabilizing mutations in β-catenin result in increased levels of nuclear relocalization of β-catenin and constitutive β-catenin signaling (Sansom, et al., 2004). This in turn has been shown to provide the molecular basis for colorectal tumorigenesis (Bienz and Clevers, 2000;Giles, et al., 2003;Polakis, 2000).
The continuous renewal of the intestinal epithelium is driven by the rapid proliferation and constant differentiation of intestinal crypt cells. Tyrosine kinases have been shown to regulate both proliferation and differentiation, and activity of tyrosine kinases has been reported in the intestinal epithelium (Pawson and Bernstein, 1990;Pawson and Hunter, 1994). During development, the proliferating fetal intestinal epithelium contains high levels of proteins phosphorylated on tyrosine residues, but these levels decrease as maturation proceeds (Maher and Pasquale, 1988). Furthermore, the crypt compartment of the intestinal epithelium has been shown to contain fifteen-fold higher levels of tyrosine phosphorylated proteins than the villus epithelium, and the majority of the tyrosine kinases appear to be associated with the cytoskeleton (Burgess, et al., 1989).
Higher levels of cytoskeletal associated non-receptor tyrosine kinase Src protein and activity are found in the crypt epithelial cells (Cartwright, et al., 1993), and the specific activity of Src decreases as primary intestinal crypt cells differentiate (Davidson, et al., 1995). Increased levels of Src family kinase activity have been detected in undifferentiated human colon carcinoma cells, and both the activity and abundance of Src decreases as these cells are induced to differentiate in vitro (Foss, et al., 1989). Overexpression of Src in immortalized colon cells results in a partially transformed phenotype (Pories, et al., 1998). The specific activity of Src and the related kinase Yes is elevated 5-20 fold in most colon carcinoma cell lines, primary carcinomas, and dysplastic and malignant adenomas (Summy and Gallick, 2003). These results suggest that downregulation of Src activity is important for differentiation and that up-regulation is required for growth and transformation of intestinal epithelial cells.
Activation of Src family kinases in colon cancer may occur through a variety of mechanisms and is frequently a critical event in tumor progression. At least four mechanisms are known to up-regulate Src PTK activity in cells (Brown and Cooper, 1996): (1) mutations within the coding region of the src gene, (2) decreased Tyr-527 phosphorylation on Src, (3) subcellular localization of Src and its substrates, and (4) association of Src with other cellular proteins. Mutations of PTKs within their coding regions often result in activated forms of the protein that continually transmit their signal along the pathway in which they participate. The viral form of Src (v-src) has a deletion in the C-terminus that removes the regulatory tyrosine, which leads to a constitutively activated form of the protein. In addition to mutations, overexpression as a result of amplification of PTKs can result in transformation. Compelling evidence indicates that association of Src with other cellular proteins can regulate its activity (Brown and Cooper, 1996). Exactly how Src family kinases contribute to individual tumors remains to be defined, however they appear to be important for multiple aspects of tumor progression, including proliferation, disruption of cell-cell contacts, migration, invasiveness, resistance to apoptosis, and angiogenesis.
The intestinal epithelium is a rapidly renewing tissue in which cell homeostasis is regulated by a balance among proliferation, growth arrest, differentiation and apoptosis. Apoptosis, or programmed cell death, is a cellular response that regulates important processes such as tissue homoeostasis, defense against certain pathogens, and elimination of unwanted cells. In multicellular organisms, mutations in somatic cells affecting critical genes that regulate cell proliferation and survival cause fatal cancers.
The continuous renewal of the intestinal epithelium consists essentially in the production of epithelial cells in the crypts, which differentiate and then migrate toward the apex of the villus or the surface epithelium where they are shed. It was proposed that apoptosis is the main mechanism responsible for the maintenance of appropriate cell numbers by counterbalancing cell division (Potten, 1992). However, it is still unknown if apoptosis or necrosis is responsible for the shedding of cells, and if enhanced rates of apoptosis compensate for high epithelial cell turnover (Merritt, et al., 1995). It has been shown that the anti-apoptotic protein Bcl-2 is expressed in the proliferating crypt cells whereas the pro-apoptotic Bax protein is expressed near the lumen of the large intestine, supporting the idea that apoptosis plays a role in intestinal tissue homeostasis (Vachon, et al., 2001;Vachon, et al., 2000).
Differential involvement of the MEK/Erk and PI3-K/Akt signaling pathways in the regulation of epithelial cell survival has been reported (Gauthier, et al., 2001;Gauthier, et al., 2001). Apoptosis involves the balanced transcription of anti-apoptotic and pro-apoptotic genes, which is regulated by intracellular signal transduction systems. It has been reported that among members of the mitogen-activated protein kinase (MAPK) family, activation of the extracellular signal-regulated kinases (Erk1/2) promotes cell survival, whereas activation of the stress-activated c-Jun N-terminal kinases (p46/p54JNK) and p38MAPK induces apoptosis (Wang, et al., 1998;Wang, et al., 1998;Xia, et al., 1995). However, publications also have provided evidence for a protective role of JNK and p38 against apoptosis (Nishina, et al., 1997;Whitmarsh and Davis, 1996;Zechner, et al., 1998). All MAPKs are regulated as part of core signaling modules downstream from the ras proto-oncogene. These modules contain three tiers, wherein MAPKs are activated by tyrosine and threonine phosphorylation by MAPK kinases (MAP2Ks), which in turn are activated by serine and threonine phosphorylation that is catalyzed by MAPK kinases kinases (MAP3Ks) (Chang and Karin, 2001). Erks respond to mitogenic stimuli, whereas JNKs and p38 kinase respond predominantly to cellular stresses or inflammatory cytokines (Seger and Krebs, 1995).
The serine/threonine kinase Akt/Protein kinase B (PKB) is a major downstream effector of growth factor-mediated cell survival. Growth factor signaling through the phosphoinositide 3-kinase (PI3-K)/Akt pathway has emerged as the major mechanism by which growth factors promote cell survival (Marte and Downward, 1997). Phosphorylation of Akt by PI3-K results in full activation of Akt kinase activity and the subsequent regulation of multiple cellular processes, including the transmission of growth factor-dependent survival signals (Coffer, et al., 1998;Marte and Downward, 1997). Akt inhibits mitochondrial release of cytochrome c (Kennedy, et al., 1997) as well as activation of the death receptor pathway (Gibson, et al., 1999). Signaling through the PI3-K/Akt pathway is ordinarily inhibited by the PTEN phosphatase tumor suppressor (Maehama and Dixon, 1998;Stambolic, et al., 1998;Wu, et al., 1998). Deregulation of this pathway is implicated in a wide range of human cancers. Enhanced signaling from receptor tyrosine kinases, inactivating mutations of the PTEN tumor suppressor (Haas-Kogan, et al., 1998;Li, et al., 1998), and amplification of the PI3-K and/or Akt genes have been reported in various human cancers (Kaufmann and Gores, 2000). All of these changes result in elevated levels of 3-phosphoinositides and enhanced signaling through Akt.
Interestingly, the small intestinal stem cells with their cell cycle time of 12-16 hours and large division potential of about a thousand times during the life span of a laboratory mouse never decline in their proliferative potential and rarely develop carcinogenic mutations (Potten, et al., 2002). In contrast, a relatively high incidence of tumor development is observed in the colon, suggesting a more effective eradication of malignant precursor cells in the small intestine. This is mainly due to the presence of protective mechanisms that ensure the integrity of the small intestinal stem cell genome. Stem cells in the small intestinal crypt are intolerant to genotoxic damage; they do not undergo cell cycle arrest and repair but commit an altruistic p53-dependent cell suicide – apoptosis (Potten and Grant, 1998). This is supported by expression of the pro-apoptotic Bax protein in the small intestinal crypts (Merritt, et al., 1995). Furthermore, small intestinal stem cells have evolved a selective segregation process to ensure that they retain only the template strands of DNA at division and hence reduce the risk of replication-induced genetic errors (Potten, 2004;Potten, et al., 2002). These two processes provide an extremely effective mechanism to ensure the integrity of the genome, thus providing an explanation as to why cancer occurs so rarely in the small intestine. In contrast, stem cells in the colon express the anti-apoptotic Bcl2 protein, favoring cell survival despite genetic damage. These observations provide insight into the mechanisms contributing to the high rates of colon cancer.
Radiation has proven to be a valuable tool to induce cell death, reproductive sterilization, and regenerative proliferation in the highly polarized epithelium of the small intestine (Potten, 2004). Whole-body γirradiation of mice with doses of about 8 Gγ causes p53-dependent apoptosis in the small intestinal crypts with peaks at 3-6 hours (early apoptosis) and 24 hours (late apoptosis) post treatment (Potten, 2004). About 6 apoptosis-susceptible cells are located in each crypt, which lack repair capacity and undergo cell suicide in response to radiation, thus providing a protective mechanism that removes cells with DNA damage.
Opposing effects of inflammation on cancer have been described. It is widely accepted that acute inflammation usually counteracts cancer development, while chronic inflammation promotes cancer development. Acute inflammation results in immediate production of pro-inflammatory cytokines and chemokines, but this process is self-limiting in that the immediate response usually gives way to production of anti-inflammatory cytokines as healing progresses.
Tumors of the lower bowel are the fourth leading cause of human cancer mortality, together with stomach and liver cancer accounting for more than two million deaths annually. Although only a small percentage of human colorectal carcinomas (CRC) are attributable to inflammatory bowel disease (IBD), patients with Crohn’s disease or ulcerative colitis have significantly increased risk of developing CRC and show higher mortality than patients with sporadic CRC (Baisse, et al., 2004;Itzkowitz and Yio, 2004;Munkholm, 2003). While current concepts implicate chronic inflammation as a key element in promoting cancer risk in IBD, a considerable number of IBD patients have quiescent inflammation (Itzkowitz and Yio, 2004;Seril, et al., 2003), suggesting that several factors and pathways activated in normal epithelial cells during IBD contribute to the increased cancer risk. Although no specific infectious agent has yet been causally linked to IBD, it is generally acknowledged that intestinal bacteria initiate the cascade of events leading to chronic enterocolitis in susceptible individuals.
It is presumed that chronic inflammation promotes rather than initiates carcinogenesis in a non-cell-autonomous fashion. Disruption of epithelial barrier function would lead to direct contact between the intestinal microflora and the immune system, resulting in the induction of mitogenic and anti-apoptotic signals essential for mucosal repair and regeneration. Hyper-proliferative and anti-apoptotic responses, together with chronic oxidative stress and inflammation, then create a tumor-promoting microenvironment in which transformed epithelial cells thrive more optimally. This indirect mechanism – inflammation of the submucosa, induced by direct contact with the intestinal microflora – promotes tumor outgrowth in the overlying epithelium. Thus, cell-cell and cell-extracellular matrix adhesions are important cellular features involved in intestinal tissue homeostasis, and are essential for the intestinal epithelium to function as a physiological and structural barrier. Disruption of cell adhesion to neighboring cells and extracellular matrix leads to villus atrophy, epithelial hyperplasia, loss of normal absorptive function, and an increased risk of tumorigenesis (Philip, et al., 2004).
Interestingly, recent evidence supported an additional complementary mechanism. Inflammatory signaling in epithelial cells has been shown to directly result in their inappropriate survival and transformation (Greten, et al., 2004;Rakoff-Nahoum, et al., 2004). This process is supported by the intestinal microflora, which affects epithelial cell properties. The existence of a symbiotic relationship between the intestinal flora and the intestinal epithelial cells involves a protective mechanism, in which commensal bacteria constitutively activate the NF-κB survival pathway through Toll-like receptors (TLR) expressed on epithelial cells, supporting cell survival and protecting the epithelium from injury (Greten, et al., 2004;Rakoff-Nahoum, et al., 2004). This cell-autonomous cell survival response is actually necessary for intestinal homeostasis. However, when stimulated in transformed epithelial cells, this pathway accelerates tumor development in a cell-autonomous fashion and complements the non-cell-autonomous inflammation mechanism (Clevers, 2004).
Cancer is a multi-step process that involves the serial mutation of key genes involved in regulating proliferation, differentiation, survival, and invasive properties of cells (Fig. 3) (Vogelstein and Kinzler, 1993). Mouse models are employed to explore basic mechanisms in the progression from infection to malignancy in the gut.
Azoxymethane (AOM) is a potent carcinogen used to induce colon cancer in rats and mice. It is used to identify other candidate mutant genes in the development of CRC as well as to evaluate the efficacy of preventative treatment for azoxymethane-induced carcinogenesis. AOM is a DNA alkylating agent, which induces tumor formation in the distal colon of susceptible rodents. In this model, mice are injected intraperitonally with 10 mg/kg AOM once a week for the duration of 6 weeks and sacrificed 24 weeks after the last injection. At that time, mice exhibit a high incidence of tumors within the distal colon, with the tumor number greatly dependant on the genetic background. This rodent model aids in the identification of possible preventative approaches to human colon cancer (Corpet and Pierre, 2003).
For IBD and IBD-related CRC, several animal models have been reported. The one most widely used is a mouse model with dextran sodium sulfate (DSS) (Okayasu, et al., 1990) -induced injury of the epithelium accompanied by mild inflammation. To this extend, mice are subjected to 3% DSS in the drinking water for 5 days followed by water only for 3 days (recovery). However, this colitis model needs a long period or repeated administration of DSS to induce colitis and colitis-related CRC, and the incidence and/or multiplicity of induced tumors are relatively low (Okayasu, et al., 2002).
Recently, a novel inflammation-related mouse colon carcinogenesis model has been developed (Tanaka, et al., 2003). Mice were given an initial low dose of AOM (10 mg/kg) followed by 1-week exposure to 2% DSS in drinking water. These mice developed large bowel tumors within 20 weeks, which histologically and biologically strongly resemble the human colitis-related disease. Again, different responses of various strains to IBD-like tumorigenesis have been reported, suggesting the influence of host genetic determinants on the inflammation severity and tumor risk. In general, genetically engineered mice on a C57BL background are more resistant compared with equivalent mutant mice on other genetic backgrounds such as 129/Sv.
A large amount of data showing diverse functions for the Brk tyrosine kinase in the regulation of cell proliferation and differentiation has been accumulated. Despite that, the biological role of this PTK in regulating signaling pathways involved in cell proliferation and differentiation crucial in maintaining a healthy organism is still poorly understood. Brk expression has been detected in breast, colon and prostate tumors as well as in oral squamous cell carcinomas. On the other hand, Brk expression has been correlated with differentiation in keratinocytes and Brk family kinases have been shown to have an inhibitory effect on receptor pathway signaling. To delineate the seemingly controversial role of this novel intracellular tyrosine kinase in proliferation and differentiation, mice carrying a disruption in the brk gene were generated. Since Brk is expressed at highest levels in the gastrointestinal tract, the continuously regenerating intestinal epithelium has been selected as a model system to analyze Brk signaling. In contrast to Src tyrosine kinases, Brk expression is restricted to differentiating cells of the rapidly renewing intestinal epithelium suggesting a role in the regulation of differentiation. The present study is focused on gaining a better understanding of the biological function of Brk in the gastrointestinal tract and its role in the development of cancer.
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