the protective action of dietary fiber against colorectal cancer ( crc ) has been attributed to the fermentation of fiber by anaerobic bacteria in the colon , producing butyrate 1 , an inhibitor of histone deacetylases ( hdaci ) , which produces cell cycle arrest , differentiation , and/or apoptosis of crc cells 2 - 4 . wnt activity derives from the accumulation of beta - catenin , which becomes associated with dna - binding tcf factors ; the resulting beta - catenin - tcf complexes stimulate transcriptional activity from target genes 8 . the constitutive activation of wnt signaling , primarily due to mutations in the apc and beta - catenin genes , promotes tumorigenesis in the colon 8 . wnt signaling also plays a role in rna metabolism that may have consequences for tumorigenesis 9,10 . for example , the wnt factor beta - catenin has been shown to influence various steps of rna metabolism in crc cells , modulating the levels of gene products relevant to colonic tumorigenesis , including cox-2 and cyclin d1 10 . since wnt activity can influence rna metabolism 10 and butyrate can influence wnt signaling 5 - 7 , the findings that butyrate can directly modulate rna metabolism is provocative . for example , butyrate inhibits the splicing of rna from the wnt - target gene c - myc , important for many forms of cancer including crc 11 , and also alters alternative splicing of the survival motor neuron ( smn ) gene , resulting in enhanced smn levels in vitro and in vivo 
 12 . in addition , butyrate influences the relative levels of calcitonin and calcitonin gene - related peptide mrnas 13 , the ratio of which is controlled by alternative polyadenylation . this latter finding suggests that butyrate modulates 3 ' end processing as well as splicing . these findings indicate that wnt signaling , and the activation of this signaling by butyrate , influences rna metabolism , and that altered rna metabolism modifies patterns of gene expression related to tumorigenesis . further , wnt signaling itself can be differentially modulated by the products of alternative rna processing , suggesting the possibility of bidirectional crosstalk between wnt activity and rna metabolism . as part of our study of differential gene expression affecting colonic tumorigenesis , a full human genome microarray analysis ( genus biosystems ) was performed on rna derived from hct-116 crc cells , to determine genes modulated by a physiologically relevant concentration ( 5 mm ) of butyrate . the expression of a number of genes involved with rna metabolism was found to be influenced by butyrate . for example , butyrate significantly ( p < 0.001 ) decreased by 2.4-fold the expression of the splicing factor gene srp20 ; this latter action by butyrate may mediate its therapeutic activity , since srp20 has been linked to crc tumorigenicity 14 . expression of the intronless gene c - jun , which can promote colonic tumorigenesis ( see below ) , was upregulated by butyrate 9.5-fold ( p < 0.001 ) . furthermore , the expression of other genes important in rna metabolism was up- or downregulated by butyrate treatment of hct-116 cells . genes whose expression was upregulated include the nuclear rna export factor nxf3 , while a number of genes exhibited downregulation of expression , including genes whose products influence 3 ' end processing ( cstf3 , several other cpsf and cstf genes , and pabpn1 ) , as well as genes whose products influence rna stability ( hnrnp d ) and splicing ( srsf10 and ptbp1 ) . the modulation , by butyrate , of the expression of cstf3 , srsf10 , ptbp1 , and pabpn1 is also influenced by wnt activity , as the degree of that modulation was decreased in hct-116 cells expressing an inducible form of dominant - negative tcf4 ( dn - tcf4 ) , which is a potent inhibitor of canonical wnt signaling 8 . to evaluate effects of wnt activity on overall intron splicing efficiency in crc cells , we utilized the luciferase - beta galactosidase double reporter system from the eperon laboratory 15 which is based on a tropomyosin tmp3 intron . two plasmids , ptn23 and ptn24 , were used , which differ in the structure of the tmp3 intron sequences . with efficient intron splicing , coding sequences from both the luciferase and beta - galactosidase cassettes are fully included in the final transcript and both reporter activities are detected . however , in the absence of splicing , only beta - galactosidase reporter activity is observed . thus , the ratio of luciferase to beta - galactosidase reporter activity is indicative of splicing efficiency . sw620 crc cells exhibit a relatively high basal level of canonical wnt signaling that is strongly inhibited by dn - tcf4 ( 5 - 7 and refs . we performed a pilot experiment by cotransfecting the ptn23 or ptn24 vectors with a control vector or with the dn - tcf4 expression vector . inhibition of wnt activity in sw620 cells enhanced splicing efficiency by more than three - fold with the ptn23 vector and more than 1.5-fold with the ptn24 vector ( fig . 1 ) . this demonstrates wnt activity - specific effects on intron splicing in a well characterized crc cell line , with the general trend of increased intron splicing with repressed wnt activity . based on these preliminary findings , a review of the literature was conducted to evaluate evidence for physiologically relevant cross - talk between wnt activity and rna processing that is ( a ) of relevance to crc , and ( b ) potentially modifiable by butyrate / hdacis , and thus possibly amenable to preventive and/or therapeutic approaches against crc . as part of our study of differential gene expression affecting colonic tumorigenesis , a full human genome microarray analysis ( genus biosystems ) was performed on rna derived from hct-116 crc cells , to determine genes modulated by a physiologically relevant concentration ( 5 mm ) of butyrate . the expression of a number of genes involved with rna metabolism was found to be influenced by butyrate . for example , butyrate significantly ( p < 0.001 ) decreased by 2.4-fold the expression of the splicing factor gene srp20 ; this latter action by butyrate may mediate its therapeutic activity , since srp20 has been linked to crc tumorigenicity 14 . expression of the intronless gene c - jun , which can promote colonic tumorigenesis ( see below ) , was upregulated by butyrate 9.5-fold ( p < 0.001 ) . furthermore , the expression of other genes important in rna metabolism was up- or downregulated by butyrate treatment of hct-116 cells . genes whose expression was upregulated include the nuclear rna export factor nxf3 , while a number of genes exhibited downregulation of expression , including genes whose products influence 3 ' end processing ( cstf3 , several other cpsf and cstf genes , and pabpn1 ) , as well as genes whose products influence rna stability ( hnrnp d ) and splicing ( srsf10 and ptbp1 ) . the modulation , by butyrate , of the expression of cstf3 , srsf10 , ptbp1 , and pabpn1 is also influenced by wnt activity , as the degree of that modulation was decreased in hct-116 cells expressing an inducible form of dominant - negative tcf4 ( dn - tcf4 ) , which is a potent inhibitor of canonical wnt signaling 8 . to evaluate effects of wnt activity on overall intron splicing efficiency in crc cells , we utilized the luciferase - beta galactosidase double reporter system from the eperon laboratory 15 which is based on a tropomyosin tmp3 intron . two plasmids , ptn23 and ptn24 , were used , which differ in the structure of the tmp3 intron sequences . with efficient intron splicing , coding sequences from both the luciferase and beta - galactosidase cassettes are fully included in the final transcript and both reporter activities are detected . however , in the absence of splicing , only beta - galactosidase reporter activity is observed . thus , the ratio of luciferase to beta - galactosidase reporter activity is indicative of splicing efficiency . sw620 crc cells exhibit a relatively high basal level of canonical wnt signaling that is strongly inhibited by dn - tcf4 ( 5 - 7 and refs . therein ) . we performed a pilot experiment by cotransfecting the ptn23 or ptn24 vectors with a control vector or with the dn - tcf4 expression vector . inhibition of wnt activity in sw620 cells enhanced splicing efficiency by more than three - fold with the ptn23 vector and more than 1.5-fold with the ptn24 vector ( fig . this demonstrates wnt activity - specific effects on intron splicing in a well characterized crc cell line , with the general trend of increased intron splicing with repressed wnt activity . based on these preliminary findings , a review of the literature was conducted to evaluate evidence for physiologically relevant cross - talk between wnt activity and rna processing that is ( a ) of relevance to crc , and ( b ) potentially modifiable by butyrate / hdacis , and thus possibly amenable to preventive and/or therapeutic approaches against crc . at least 60% of human genes exhibit alternative splicing , and microarray methodologies have been developed to analyze the patterns of pre - mrna splicing in different cells and under varied conditions 16 - 18 . tumor - specific splicing variants have been identified and some of these variants influence wnt activity 19 - 22 . for example , the splicing factor srp20 is a wnt target gene ; srp20 upregulates crc cell proliferation and has been linked to tumorigenesis 14 . it is known that variants of tcf / lef factors such as tcf1 and tcf4 , which bind to beta - catenin and drive wnt transcriptional activity , can be generated by alternative splicing 23 . further , lee et al . 10 demonstrated that an rna aptamer that interferes with the interaction between beta - catenin and tcf4 in crc cells not only repressed wnt activity but also regulated the alternative splicing of rna from an adenovirus e1a minigene . conversely , alternative splicing influences wnt activity ; thus , while the full length form of sulfatase 1 ( sulf1a ) stimulates wnt signaling , a shorter sulf isoform ( sulf1b ) produced by alternative splicing inhibits wnt activity 19 . thus , cross - talk between rna splicing and wnt signaling activity is bidirectional , and is associated with colonic neoplasia . importantly , wnt activity is influenced not only by the expression of the splicing factor srp20 14,22 but also by that of asf / sf2 22,24 . thus , changes in the relative levels of srp20 and asf / sf2 results in altered rna splicing and differential expression of rac1b , a rac1 variant overexpressed in certain colon tumors that contributes to cell survival 14,22 , and which can enhance wnt signaling 25 . srp20 enhances skipping of the rac1 alternative exon 3b in crc cells , while asf / sf2 promotes exon 3b inclusion ; thus , asf / sf2 increases and srp20 decreases rac1b expression . increased levels of wnt activity enhance expression of srp20 , which in turn results in repressed expression of the rac1b variant 22 . rac1b is more highly expressed in crc cells with lower levels of wnt activity , promoting survival of these low - wnt activity cells 20 . conversely , the expression of asf / sf2 is blocked by phosphatidylinositol 3-kinase ( pi3-k ) signaling ; therefore , formation of rac1b is ultimately controlled by levels of wnt and pi3-k signaling 22,24 . crc cells characterized by low levels of wnt activity and pi3-k signaling utilize rac1b to promote survival ; however , crc cell types with higher levels of wnt and pi3-k signaling exhibit low or no expression of rac1b and utilize alternative signaling pathways to promote cell survival 20 . these findings are consistent with our observations that very high levels of wnt activity induced by hdacis promote apoptosis of crc cells , since lower levels of wnt signaling , associated with increased expression of rac1b , promote cell survival 5 - 7 . further , rac1b itself upregulates wnt activity when overexpressed in hct-116 cells , a cell line that normally expresses relatively low levels of rac1b 25 . this upregulation of wnt activity by rac1b occurs upstream of beta - catenin 24,25 ; this is similar to the activation of wnt activity by hdacis at the plasma membrane , resulting in enhanced levels of active beta - catenin 5 - 7 . therefore , one intriguing possibility is that upregulation of wnt activity by hdacis may be partially mediated by the action of rac1b . remarkably , rac1b - negative sw480 crc cells can be made to endogenously express levels of rac1b similar to that of the rac1b - positive ht-29 crc cell line , through a combination of asf / sf2 overexpression and sirna knockdown of srp20 22 . this suggests plasticity of rac1b phenotypes , perhaps extending to downstream effects of rac1b expression . manipulation of rac1b levels likely can induce rac1b - mediated cell survival pathways , including induction of moderate levels of wnt signaling activity . however , since increased wnt activity downregulates rac1b expression via srp20 , a negative feedback loop would likely occur . thus , low levels of wnt activity promotes increased expression of rac1b , which simulates wnt activity ; this increase in wnt signaling would in turn downregulate levels of rac1b through alternative rna splicing . our previous findings 5 - 7 are consistent with the hypothesis that moderate levels of wnt activity drive crc cell proliferation and survival , while wnt hyperactivation promotes crc cell death . thus , the negative wnt - rac1b feedback loop may serve to keep levels of wnt activity in the pro - proliferative range and below levels that would promote crc cell death . the overall relationship between wnt signaling , rac1b , and crc , with respect to alternative rna splicing , is outlined in fig . the development of other forms of cancer is also influenced by splicing factors whose expression is controlled by cancer - related cell signaling pathways . for example , the levels of asf / sf2 as well as of hnrnp a1 are increased in lung neoplasia ; this altered expression is associated with cancer - related changes in the alternative splicing of cd44 , which is involved in cell - cell and cell - matrix interactions 26 . thus , the effects of srp20 and asf / sf2 on rac1b may be part of a broader modulation of rna processing in tumorigenesis , responsible for altering the levels of protein variants in the neoplastic cell 22,26 . consistent with a significant role for rna metabolism in mediating the effects of cell signaling pathways in normal and neoplastic cells , analysis of beta - catenin - containing nuclear complexes showed the presence of a variety of proteins that bind rna and/or influence rna metabolism , such as hnrnp a2/b1 , hnrnp m , hnrnp g , hnrnp a1 , and hnrnp k , among others 27 . in addition , beta - catenin interacts with fusion ( fus)/translocated in liposarcoma ( fus / tls ) , a rna binding protein that influences mrna transcription and splicing 27 . overexpression of fus / tls repressed wnt activity in crc cells and repressed crc cell clonogenic growth ; in addition , fus / tls levels are increased in crc and may serve as negative feedback , limiting the effects of deregulated wnt activity 27 . this interaction further links rna metabolism to wnt activity , which in turn influences crc cell growth . thus , fus / tls and similar factors may integrate wnt - mediated rna transcription with effects on rna processing , to exert more fine - grained control of the expression of genes associated with tumorigenesis . statistical analyses demonstrated that naturally intronless genes are over - represented among genes coding for proteins with binding or signal transduction / receptor activities and under - represented among genes coding for proteins with catalytic functions 33 . signal transduction / receptor proteins are highly relevant to the signaling pathways involved in the development of cancer ; therefore , these data suggest the possibility that intronless rna metabolism is associated with tumorigenesis . rapid - response genes , which include c - jun , are often intronless ; rna metabolism of these genes reflects the requirement to bypass splicing processes for more efficient expression and function 34 . the protein product of the intronless human c - jun gene associates with c - fos , forming the transcription factor ap-1 , which participates in jnk signaling 35,36 . ap-1/jnk signaling plays an important role in the development of crc 37 , and a dominant negative mutant of c - jun has been proposed as a gene therapy approach for crc , through disruption of ap-1 activity 38 . supporting this view , c - jun is overexpressed in crc , and jnk signaling activates intestinal tumorigenesis in mouse models of crc ( 39 and refs . therein ) . jnk signaling stimulates progenitor cell proliferation in intestinal crypts ; importantly , expression of the wnt target genes axin 2 and lgr5 is upregulated by jnk activity 39 . further , expression of tcf4 , a key component of the wnt signaling complex , is upregulated by jnk activity , and chromatin immunoprecipitation analyses demonstrated that tcf4 is a direct c - jun target gene 39 . c - jun itself is a wnt target gene ; therefore , these findings suggest that positive feedback between wnt and jnk activity , mediated through c - jun , contributes to intestinal tumorigenesis 39 . in addition , c - jun , together with the wnt signaling factors beta - catenin and tcf , associate with a 3 ' enhancer region of the wnt - target gene c - myc , a proto - oncogene necessary for colorectal neoplasia 40,41 . with respect to rna metabolism , while intron - containing genes often require the presence of introns for proper mrna accumulation ; naturally intronless genes contain sequence elements which can substitute for introns to allow for efficient gene expression ( 42 and references therein ) . an example of such an element is the human c - jun gene enhancer ( cje ) element 42 . the rna metabolism of intronless c - jun , by influencing levels of c - jun protein , may alter signaling cross - talk ( e.g. , wnt - jnk ) and therefore contribute to the development of crc 41 . in addition , preliminary findings from our laboratory ( unpublished data ) indicate that wnt activity can enhance the activity of sequences that confer efficient intron - independent gene expression ; this suggests that wnt activity influences c - jun expression both at the level of transcription as well as at the level of post - transcriptional modulation of intronless rna metabolism ( e.g. , rna stability ) . further research into the effects of wnt activity on intronless rna metabolism is required to evaluate this possibility . importantly for crc , c - jun expression is upregulated by butyrate both in vitro and in vivo 
 43,44 , and our microarray data ( above ) show a marked upregulation of c - jun expression in butyrate - treated hct-116 cells . thus , while hdaci treatment is generally pro - apoptotic and hence therapeutic against cancer , enhanced wnt activity resulting from exposure to hdacis can also increase the expression of pro - tumorigenic genes . therefore , it is important to understand the various mechanisms whereby basal and hdaci - induced wnt activity influences the expression of c - jun , including effects on intronless rna metabolism . induction of c - jun expression by hdacis can be rapid ; for example , exposure of leukemic cells to the butyric acid prodrug pivaloyloxymethyl butyrate ( a-9 ) induces significant c - jun expression in only 15 minutes 45 . genes that contain introns , the interplay between rna processing and gene expression , including the kinetics of gene expression , likely differs in a gene specific manner . for example , the relative importance of the intronless condition for rapid gene expression may be influenced by factors such as promoter strength , chromosome location and surrounding chromatin structure , and/or the presence or absence of other regulatory sequences . certain intronless genes , such as c - jun , may require the intronless state for efficient and rapid expression ; this dependence on the intronless state influences the levels of the relevant gene products , with consequent effects on cell physiology , including neoplasia . therefore , differences of rna metabolism in intron - dependent vs. intron - independent pathways of expression may in part determine the degree to which induction of gene expression by wnt signaling contributes to tumorigenesis . further , a complete understanding of how cell signaling pathways influence intronless gene expression in tumorigenesis may lead to approaches to suppress expression of tumorigenic factors such as c - jun , which would enhance the efficacy of , e.g. , hdaci - based anti - cancer therapeutics . 3 ' end processing is also of relevance to cancer ; for example , alternative 3 ' end processing can enhance oncogene expression in cancer cells , and differential 3 ' end formation is associated with clinically relevant cancer subtypes 46 . altered gene expression in cancer , caused by alternative 3 ' end processing , often results from enhanced rna stability , in some cases involving shortened 3 ' untranslated regions ( 3 ' utrs ) and the consequent loss of sites mediating repression by mirnas 47 . several genes linked to neoplasia exhibit regulation of expression through control of 3 ' end processing . the wnt target cyclin d2 , involved in cell cycle regulation , is regulated through modulation of 3 ' end processing , and other cancer related genes ( e.g. , n - acetyltransferase 1 ) also exhibit regulation of expression through alternative polyadenylation ( 48,49 and refs . therein ) . further , alternative polyadenylation produces a version of the wnt factor tcf4 that inhibits wnt activity 50 , suggesting a direct link between 3 ' end processing , wnt activity , and colonic neoplasia . a classic example of a crc - related gene whose rna stability 51 and expression is controlled by alternative polyadenylation is the wnt target cyclooxygenase-2 ( cox-2 ) ( 52 and refs . ht-29 crc cells primarily use the cox-2 distal polyadenylation site , while hepg2 liver cancer cells use both the distal and proximal cox-2 polyadenylation sites 52 . this tissue - specific difference in polyadenylation usage has practical consequences , as the longer cox-2 mrna ( distal site usage ) tends to be more stable than the shorter form ( proximal site usage ) , resulting in higher steady - state levels of the cox-2 protein that may promote colonic neoplasia . thus , differential rna stability , such as that resulting from alternative 3 ' end processing of cox-2 rna , can influence neoplasia . further , since our preliminary microarray data ( see above ) demonstrated that wnt activity and butyrate influence the expression of factors involved in 3 ' end processing , levels of wnt signaling may influence alternative polyadenylation ( e.g. , of cox-2 rna ) . of particular relevance for crc , beta - catenin influences cox-2 rna stability 10,51 ; beta - catenin stabilizes cox-2 transcripts through interactions with au - rich elements in the 3 ' utr , again demonstrating cross - talk between wnt signaling and rna metabolism . the rna binding protein hur likely mediates these effects of beta - catenin ; the hur protein stabilizes transcripts correlated to the number of binding sites and level of association of hur with the targeted rna 50 , 53 , 54 . rnas containing hur binding sites in both intronic as well as 3 ' utr sequences exhibited greater stability than transcripts containing either intronic or 3 ' utr sites only ; these findings suggest that hur links pre - mrna processing with mature mrna stability 55 . therefore , wnt activity not only influences the levels of cox-2 at the transcriptional level , but also post - transcriptionally , at the level of rna stability . in addition , the association of hur with thrombospondin-1 mrna modulates the translation of the angiogenesis inhibitor tp1 , affecting neoplastic progression via altered tumor vascularity 56 . therefore , rna metabolism involving the 3 ' ends of transcripts can influence multiple points along the neoplastic continuum . at least 60% of human genes exhibit alternative splicing , and microarray methodologies have been developed to analyze the patterns of pre - mrna splicing in different cells and under varied conditions 16 - 18 . tumor - specific splicing variants have been identified and some of these variants influence wnt activity 19 - 22 . for example , the splicing factor srp20 is a wnt target gene ; srp20 upregulates crc cell proliferation and has been linked to tumorigenesis 14 . it is known that variants of tcf / lef factors such as tcf1 and tcf4 , which bind to beta - catenin and drive wnt transcriptional activity , can be generated by alternative splicing 23 . further , lee et al . 10 demonstrated that an rna aptamer that interferes with the interaction between beta - catenin and tcf4 in crc cells not only repressed wnt activity but also regulated the alternative splicing of rna from an adenovirus e1a minigene . conversely , alternative splicing influences wnt activity ; thus , while the full length form of sulfatase 1 ( sulf1a ) stimulates wnt signaling , a shorter sulf isoform ( sulf1b ) produced by alternative splicing inhibits wnt activity 19 . thus , cross - talk between rna splicing and wnt signaling activity is bidirectional , and is associated with colonic neoplasia . importantly , wnt activity is influenced not only by the expression of the splicing factor srp20 14,22 but also by that of asf / sf2 22,24 . thus , changes in the relative levels of srp20 and asf / sf2 results in altered rna splicing and differential expression of rac1b , a rac1 variant overexpressed in certain colon tumors that contributes to cell survival 14,22 , and which can enhance wnt signaling 25 . srp20 enhances skipping of the rac1 alternative exon 3b in crc cells , while asf / sf2 promotes exon 3b inclusion ; thus , asf / sf2 increases and srp20 decreases rac1b expression . increased levels of wnt activity enhance expression of srp20 , which in turn results in repressed expression of the rac1b variant 22 . rac1b is more highly expressed in crc cells with lower levels of wnt activity , promoting survival of these low - wnt activity cells 20 . conversely , the expression of asf / sf2 is blocked by phosphatidylinositol 3-kinase ( pi3-k ) signaling ; therefore , formation of rac1b is ultimately controlled by levels of wnt and pi3-k signaling 22,24 . crc cells characterized by low levels of wnt activity and pi3-k signaling utilize rac1b to promote survival ; however , crc cell types with higher levels of wnt and pi3-k signaling exhibit low or no expression of rac1b and utilize alternative signaling pathways to promote cell survival 20 . these findings are consistent with our observations that very high levels of wnt activity induced by hdacis promote apoptosis of crc cells , since lower levels of wnt signaling , associated with increased expression of rac1b , promote cell survival 5 - 7 . further , rac1b itself upregulates wnt activity when overexpressed in hct-116 cells , a cell line that normally expresses relatively low levels of rac1b 25 . this upregulation of wnt activity by rac1b occurs upstream of beta - catenin 24,25 ; this is similar to the activation of wnt activity by hdacis at the plasma membrane , resulting in enhanced levels of active beta - catenin 5 - 7 . therefore , one intriguing possibility is that upregulation of wnt activity by hdacis may be partially mediated by the action of rac1b . remarkably , rac1b - negative sw480 crc cells can be made to endogenously express levels of rac1b similar to that of the rac1b - positive ht-29 crc cell line , through a combination of asf / sf2 overexpression and sirna knockdown of srp20 22 . this suggests plasticity of rac1b phenotypes , perhaps extending to downstream effects of rac1b expression . manipulation of rac1b levels likely can induce rac1b - mediated cell survival pathways , including induction of moderate levels of wnt signaling activity . however , since increased wnt activity downregulates rac1b expression via srp20 , a negative feedback loop would likely occur . thus , low levels of wnt activity promotes increased expression of rac1b , which simulates wnt activity ; this increase in wnt signaling would in turn downregulate levels of rac1b through alternative rna splicing . our previous findings 5 - 7 are consistent with the hypothesis that moderate levels of wnt activity drive crc cell proliferation and survival , while wnt hyperactivation promotes crc cell death . thus , the negative wnt - rac1b feedback loop may serve to keep levels of wnt activity in the pro - proliferative range and below levels that would promote crc cell death . the overall relationship between wnt signaling , rac1b , and crc , with respect to alternative rna splicing , is outlined in fig . the development of other forms of cancer is also influenced by splicing factors whose expression is controlled by cancer - related cell signaling pathways . for example , the levels of asf / sf2 as well as of hnrnp a1 are increased in lung neoplasia ; this altered expression is associated with cancer - related changes in the alternative splicing of cd44 , which is involved in cell - cell and cell - matrix interactions 26 . thus , the effects of srp20 and asf / sf2 on rac1b may be part of a broader modulation of rna processing in tumorigenesis , responsible for altering the levels of protein variants in the neoplastic cell 22,26 . consistent with a significant role for rna metabolism in mediating the effects of cell signaling pathways in normal and neoplastic cells , analysis of beta - catenin - containing nuclear complexes showed the presence of a variety of proteins that bind rna and/or influence rna metabolism , such as hnrnp a2/b1 , hnrnp m , hnrnp g , hnrnp a1 , and hnrnp k , among others 27 . in addition , beta - catenin interacts with fusion ( fus)/translocated in liposarcoma ( fus / tls ) , a rna binding protein that influences mrna transcription and splicing 27 . overexpression of fus / tls repressed wnt activity in crc cells and repressed crc cell clonogenic growth ; in addition , fus / tls levels are increased in crc and may serve as negative feedback , limiting the effects of deregulated wnt activity 27 . this interaction further links rna metabolism to wnt activity , which in turn influences crc cell growth . thus , fus / tls and similar factors may integrate wnt - mediated rna transcription with effects on rna processing , to exert more fine - grained control of the expression of genes associated with tumorigenesis . statistical analyses demonstrated that naturally intronless genes are over - represented among genes coding for proteins with binding or signal transduction / receptor activities and under - represented among genes coding for proteins with catalytic functions 33 . signal transduction / receptor proteins are highly relevant to the signaling pathways involved in the development of cancer ; therefore , these data suggest the possibility that intronless rna metabolism is associated with tumorigenesis . rapid - response genes , which include c - jun , are often intronless ; rna metabolism of these genes reflects the requirement to bypass splicing processes for more efficient expression and function 34 . the protein product of the intronless human c - jun gene associates with c - fos , forming the transcription factor ap-1 , which participates in jnk signaling 35,36 . ap-1/jnk signaling plays an important role in the development of crc 37 , and a dominant negative mutant of c - jun has been proposed as a gene therapy approach for crc , through disruption of ap-1 activity 38 . supporting this view , c - jun is overexpressed in crc , and jnk signaling activates intestinal tumorigenesis in mouse models of crc ( 39 and refs . therein ) . jnk signaling stimulates progenitor cell proliferation in intestinal crypts ; importantly , expression of the wnt target genes axin 2 and lgr5 is upregulated by jnk activity 39 . further , expression of tcf4 , a key component of the wnt signaling complex , is upregulated by jnk activity , and chromatin immunoprecipitation analyses demonstrated that tcf4 is a direct c - jun target gene 39 . c - jun itself is a wnt target gene ; therefore , these findings suggest that positive feedback between wnt and jnk activity , mediated through c - jun , contributes to intestinal tumorigenesis 39 . in addition , c - jun , together with the wnt signaling factors beta - catenin and tcf , associate with a 3 ' enhancer region of the wnt - target gene c - myc , a proto - oncogene necessary for colorectal neoplasia 40,41 . with respect to rna metabolism , while intron - containing genes often require the presence of introns for proper mrna accumulation ; naturally intronless genes contain sequence elements which can substitute for introns to allow for efficient gene expression ( 42 and references therein ) . an example of such an element is the human c - jun gene enhancer ( cje ) element 42 . the rna metabolism of intronless c - jun , by influencing levels of c - jun protein , may alter signaling cross - talk ( e.g. , wnt - jnk ) and therefore contribute to the development of crc 41 . in addition , preliminary findings from our laboratory ( unpublished data ) indicate that wnt activity can enhance the activity of sequences that confer efficient intron - independent gene expression ; this suggests that wnt activity influences c - jun expression both at the level of transcription as well as at the level of post - transcriptional modulation of intronless rna metabolism ( e.g. , rna stability ) . further research into the effects of wnt activity on intronless rna metabolism is required to evaluate this possibility . importantly for crc , c - jun expression is upregulated by butyrate both in vitro and in vivo 
 43,44 , and our microarray data ( above ) show a marked upregulation of c - jun expression in butyrate - treated hct-116 cells . thus , while hdaci treatment is generally pro - apoptotic and hence therapeutic against cancer , enhanced wnt activity resulting from exposure to hdacis can also increase the expression of pro - tumorigenic genes . therefore , it is important to understand the various mechanisms whereby basal and hdaci - induced wnt activity influences the expression of c - jun , including effects on intronless rna metabolism . induction of c - jun expression by hdacis can be rapid ; for example , exposure of leukemic cells to the butyric acid prodrug pivaloyloxymethyl butyrate ( a-9 ) induces significant c - jun expression in only 15 minutes 45 . genes that contain introns , the interplay between rna processing and gene expression , including the kinetics of gene expression , likely differs in a gene specific manner . for example , the relative importance of the intronless condition for rapid gene expression may be influenced by factors such as promoter strength , chromosome location and surrounding chromatin structure , and/or the presence or absence of other regulatory sequences . certain intronless genes , such as c - jun , may require the intronless state for efficient and rapid expression ; this dependence on the intronless state influences the levels of the relevant gene products , with consequent effects on cell physiology , including neoplasia . therefore , differences of rna metabolism in intron - dependent vs. intron - independent pathways of expression may in part determine the degree to which induction of gene expression by wnt signaling contributes to tumorigenesis . further , a complete understanding of how cell signaling pathways influence intronless gene expression in tumorigenesis may lead to approaches to suppress expression of tumorigenic factors such as c - jun , which would enhance the efficacy of , e.g. , hdaci - based anti - cancer therapeutics . 3 ' end processing is also of relevance to cancer ; for example , alternative 3 ' end processing can enhance oncogene expression in cancer cells , and differential 3 ' end formation is associated with clinically relevant cancer subtypes 46 . altered gene expression in cancer , caused by alternative 3 ' end processing , often results from enhanced rna stability , in some cases involving shortened 3 ' untranslated regions ( 3 ' utrs ) and the consequent loss of sites mediating repression by mirnas 47 . several genes linked to neoplasia exhibit regulation of expression through control of 3 ' end processing . the wnt target cyclin d2 , involved in cell cycle regulation , is regulated through modulation of 3 ' end processing , and other cancer related genes ( e.g. , n - acetyltransferase 1 ) also exhibit regulation of expression through alternative polyadenylation ( 48,49 and refs . therein ) . further , alternative polyadenylation produces a version of the wnt factor tcf4 that inhibits wnt activity 50 , suggesting a direct link between 3 ' end processing , wnt activity , and colonic neoplasia . a classic example of a crc - related gene whose rna stability 51 and expression is controlled by alternative polyadenylation is the wnt target cyclooxygenase-2 ( cox-2 ) ( 52 and refs . ht-29 crc cells primarily use the cox-2 distal polyadenylation site , while hepg2 liver cancer cells use both the distal and proximal cox-2 polyadenylation sites 52 . this tissue - specific difference in polyadenylation usage has practical consequences , as the longer cox-2 mrna ( distal site usage ) tends to be more stable than the shorter form ( proximal site usage ) , resulting in higher steady - state levels of the cox-2 protein that may promote colonic neoplasia . thus , differential rna stability , such as that resulting from alternative 3 ' end processing of cox-2 rna , can influence neoplasia . further , since our preliminary microarray data ( see above ) demonstrated that wnt activity and butyrate influence the expression of factors involved in 3 ' end processing , levels of wnt signaling may influence alternative polyadenylation ( e.g. , of cox-2 rna ) . of particular relevance for crc , beta - catenin influences cox-2 rna stability 10,51 ; beta - catenin stabilizes cox-2 transcripts through interactions with au - rich elements in the 3 ' utr , again demonstrating cross - talk between wnt signaling and rna metabolism . the rna binding protein hur likely mediates these effects of beta - catenin ; the hur protein stabilizes transcripts correlated to the number of binding sites and level of association of hur with the targeted rna 50 , 53 , 54 . rnas containing hur binding sites in both intronic as well as 3 ' utr sequences exhibited greater stability than transcripts containing either intronic or 3 ' utr sites only ; these findings suggest that hur links pre - mrna processing with mature mrna stability 55 . therefore , wnt activity not only influences the levels of cox-2 at the transcriptional level , but also post - transcriptionally , at the level of rna stability . in addition , the association of hur with thrombospondin-1 mrna modulates the translation of the angiogenesis inhibitor tp1 , affecting neoplastic progression via altered tumor vascularity 56 . therefore , rna metabolism involving the 3 ' ends of transcripts can influence multiple points along the neoplastic continuum . we summarize the interactions between wnt activity and aspects of rna processing discussed in this paper in fig . wnt signaling interacts with various points of rna metabolism , and the gene products so influenced themselves feed back to affect wnt activity and , hence , influence neoplasia . analysis of cancer - specific rna metabolism is a relatively unexplored area of research , with potentially significant implications for the prevention and treatment of crc . rna metabolism can be altered by wnt signaling as well as by butyrate and other hdacis . rna metabolism is likely a major control point mediating the changes in gene expression that result from deregulated wnt signaling . in particular , intron - dependent expression of genes such as c - jun , and cross - talk between wnt signaling , rac1b expression , and mrna processing likely play fundamental roles in colonic neoplasia . determining how rna processing and intron - independent gene expression influences wnt - initiated crc may lead to novel interventions aimed at restoring normal gene expression for therapeutic benefit .