The development of sporadic colorectal cancer (CRC) involves genetic and epigenetic changes, including allelic losses in specific chromosomal arms, mutations of oncogenes, tumour suppressor genes and mismatch repair genes, micro-satellite instability, and methylation changes in gene promoters [reviewed [
1]]. Additionally, CRC is frequently associated with altered splicing of tumour suppressor genes, and genes encoding enzymes, growth factors, cytoskeletal and cell adhesion proteins, hormone and growth factor receptors, and transcription factors [see [
2‐
5]]. Importantly, since different splice variants of a given gene can have different or even antagonistic effects on diverse cellular functions, including apoptosis, proliferation, differentiation, angiogenesis and cell motility, a change in the splice variant ratio may actively contribute to tumour progression. Indeed, positive selection for splice variants that encode isoforms with a selective advantage in tumour progression is of potential diagnostic value and could provide therapeutic targets.
Ras proteins are low molecular weight (~21 kD) GTPases which cycle between the GDP-bound (inactive) and the GTP-bound (active) state at the plasma membrane, and thereby regulate cell growth, apoptosis, motility and differentiation. K-
ras activating point mutations occur in about 50% of human sporadic CRC cases and act by stabilising the active GTP-bound configuration, and so promote cellular transformation by constitutive activation of downstream effector pathways, including Raf kinases, phosphatidylinositol 3-kinases (PI3-K), and RalGDS family members [reviewed [
6]]. K-
ras activating mutations play a key role in tumour progression and metastasis in CRC by regulating angiogenesis and protease expression, and cell polarity, adhesion and motility [see [
7‐
9]]. The K-
ras gene encodes two splice variants, K-
ras 4A and 4B, and activating mutations that usually arise at codons 12, 13 or 61, jointly affect both isoforms [reviewed [
6]]. Importantly, since K-Ras oncoproteins differentially promote transformation, cell migration, and anchorage-independent growth, they most probably act in a cooperative manner to drive neoplastic progression [
10]. The ratio of the K-
ras 4A/4B splice variants is reduced in human sporadic CRC in both primary adenocarcinomas and colon cancer cell lines that harbour K-
ras activating mutations, including homozygous mutations [
11,
12]. Since mutationally activated K-Ras 4B has an anti-apoptotic action [
13,
14] and, unlike K-Ras 4A, can promote cell migration [
10], and K-Ras 4B can drive expression of matrix metalloproteinase 2 (MMP-2) which specifically cleaves type IV collagen, and is involved in cell detachment and migration [
15], the altered splicing of the K-
ras oncogene in CRC in favour of K-
ras 4B could contribute to neoplastic progression by enabling the survival of cells with DNA damage and facilitating tumour invasion and, ultimately, metastasis. Indeed, tumour growth and metastasis in human CRC is linked with increased expression of MMP-2 [see [
7]]. However, the finding that the K-
ras 4A/4B ratio is also reduced in CRC cell lines that lack K-
ras activating mutations raises the possibility that a regulated switch in alternative splicing of the K-
ras proto-oncogene may also have a causal role in tumour progression [
12]. The mechanism could involve increased expression of MMP-2 (see above), and/or reduced apoptosis given that the K-Ras 4A proto-oncoprotein exerts a pro-apoptotic action in mouse intestine following etoposide-induced DNA damage, and evidence that the K-Ras proto-oncoproteins have antagonistic effects on apoptosis in embryonic stem (ES) cells: Ras 4A promotes, whereas K-Ras 4B inhibits, apoptosis [
16]. Further, while K-Ras 4B, and probably K-Ras 4A, promote ES cell differentiation following withdrawal of leukaemia inhibitory factor [
16] it is unlikely they do so with similar efficiency since the Raf/MAPK pathway regulates stem cell differentiation [reviewed [
17]] and K-Ras 4A and 4B differ in their ability to activate Raf-1 [
10]. Therefore, in accordance with the 'stem cell model' for cancer formation we proposed that a change in the ratio of K-Ras proto-oncoproteins may further contribute to neoplastic progression by perturbing stem cell differentiation [see [
12]]. Thus, altered splicing of the K-
ras proto-oncogene could drive tumour progression in sporadic CRC by promoting MMP-2 expression, and inappropriate stem cell survival and self-renewal.
To address the hypothesis that alteration in the ratio of the K-Ras proto-oncoproteins in favour of K-Ras 4B can affect tumour formation in the small intestine in the absence of K-
ras activating mutations, K-
ras
tmΔ4A/tmΔ4A mice, which express the K-
ras 4B splice variant only [
18], were crossed with
Apc
Min/+ (
Min) mice. The latter mice harbour a heterozygous germ-line nonsense mutation in the
Apc (adenomatous polyposis coli) tumour suppressor gene, and are predisposed to developing multiple intestinal tumours initiated by loss of the wild-type
Apc allele [reviewed [
19]]. By this means tumorigenesis in the small intestine was compared between
Apc
Min/+, K-
ras
+/+ and
Apc
Min/+, K-
ras
tmΔ4A/tmΔ4A mice that can, and cannot, express K-
ras 4A respectively. This approach was selected since K-
ras
tmΔ4A/tmΔ4A mice are healthy [
16,
18], intestinal tumours in
Min mice do not harbour K-
ras activating mutations [
20], and K-Ras 4A deficiency does not affect K-
ras 4B expression in the small intestine [
16] where, importantly, most (> 95%) intestinal tumours form in
Min mice [reviewed [
19]]. Thus, the effect of K-Ras 4A on tumorigenesis can be examined in the
absence both of its oncogenic allele and of alteration in K-
ras 4B expression as a consequence of K-Ras 4A deficiency.