Mutated in Colorectal Cancer, a Putative Tumor Suppressor for Serrated Colorectal Cancer, Selectively Represses B-Catenin-Dependent Transcription

Home , Lamin, P73

Oncogene (2008) 27, 6044–6055 & 2008 Macmillan Publishers Limited All rights reserved 0950-9232/08 $32.00 www.nature.com/onc ORIGINAL ARTICLE Mutated in colorectal cancer, a putative tumor suppressor for serrated colorectal cancer, selectively represses b-catenin-dependent transcription

R Fukuyama1,5, R Niculaita1,2,KPNg1,6, E Obusez3, J Sanchez4, M Kalady4, PP Aung1, G Casey1,3,4 and N Sizemore1,2,3,4

1Department of Cancer Biology, Cleveland Clinic, Cleveland, OH, USA; 2School of Biomedical Sciences, Kent State University, Kent, OH, USA; 3Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, OH, USA and 4Department of Colorectal Surgery, Cleveland Clinic, Cleveland, OH, USA

Mutated in colorectal cancer (MCC )was originally Introduction identified as a candidate gene for familial adenomatous polyposis (FAP)but further study identified adenomatous The mutated in colorectal cancer (MCC ) gene was polyposis coli (APC)as responsible for FAP and the originally isolated as a candidate tumor suppressor for physiologic/pathologic roles of MCC remained poorly familial adenomatous polyposis (FAP) (Ashton-Rickardt understood. Recently, MCC promoter methylation was et al., 1991; Kinzler et al., 1991b). However, further discovered as a frequent early event in a distinct subset of studies revealed that the mutation of the adenomatous precursor lesions and colorectal cancer (CRC)associated polyposis coli (APC) gene and not MCC is responsible with the serrated CRC pathway. Here we provide the first for FAP and the initiation of the majority of sporadic evidence of the biological significance of MCC loss in colorectal cancer (CRC) (Groden et al., 1991; Nishisho CRC and the molecular pathways involved. We show et al., 1991; Kinzler et al., 1991a). Subsequently, few MCC expression is dramatically decreased in many studies on the physiologic and/or pathologic roles of CRC cell lines and the distinct subset of sporadic CRC MCC were carried out. characterized by the CpG island methylator phenotype MCC codes for a protein of 829 amino acids that is and BRAFV600E mutation due to promoter methylation as highly conserved across many species but has little reported previously. Importantly, we find MCC interacts homology to other known proteins. The three previous with b-catenin and that reexpression of MCC in CRC studies hinted that MCC may be important in several cells specifically inhibits Wnt signaling, b-catenin/T-cell cellular processes involved in the development of cancer factor/lymphoid-enhancer factor-dependent transcription (Matsumine et al., 1996; Senda et al., 1999; Bouwmee- and cellular proliferation even in the presence of oncogenic ster et al., 2004). MCC was previously found to be mutant APC. We also show that MCC is localized in the localized to the cytoplasm and membrane-cytoskeletal nucleus and identify two functional nuclear localization components (Senda et al., 1999). Also both MCC and signals. Taken together, MCC is a nuclear, b-catenin- MCC2, the only MCC homologous gene isolated, interacting protein that can act as a potential tumor contain a C-terminal PSD-95/Discs-large/ZO-1 (PDZ) suppressor in the serrated CRC pathway by inhibiting domain-binding ligand sequence (Senda et al., 1999; Wnt/b-catenin signal transduction. Ishikawa et al., 2001; Nourry et al., 2003). MCC was Oncogene (2008) 27, 6044–6055; doi:10.1038/onc.2008.204; also found to negatively regulate cell cycle in NIH3T3 published online 30 June 2008 cells (Matsumine et al., 1996), suggesting a role of MCC in cell growth regulation. Moreover, MCC is highly Keywords: colorectal cancer; MCC; Wnt; b-catenin; expressed in murine colonic surface epithelial cells and serrated other types of well-differentiated cells (Matsumine et al., 1996; Senda et al., 1999) indicating a potential general physiologic role for MCC in cell differentiation. Most recently, MCC was found to play a possible role in the negative regulation of the nuclear factor-k B (NF-kB) pathway (Bouwmeester et al., 2004) which is known to be important in CRC pathology (Greten et al., 2004; Luo et al., 2004; Agarwal et al., 2005; Fukuyama Correspondence: Dr N Sizemore, Department of Cancer Biology ND- et al., 2006). 50, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195, USA. Little is known of a specific role of MCC in CRC. E-mail: [email protected] Although an presumed inactivating MCC mutation in 5Current address: Aichi Cancer Center, Nagoya 464-8682, Japan. the mouse alone failed to induce any evident CRC, the 6Current address: Department of Ophthalmic Research, Cole Eye Institute, Cleveland Clinic, Cleveland, OH, USA. homozygous mice displayed a slightly higher prolifera- Received 14December 2007; revised 2 May 2008; accepted 23 May 2008; tion rate of the epithelial crypt cells (Heyer et al., published online 30 June 2008 1999). In earlier studies, loss of heterozygosity (LOH), MCC inhibits b-catenin-dependent transcription R Fukuyama et al 6045 deletions and mutations of the MCC gene were found in analyse MCC promoter methylation quantitatively in a human CRC (Ashton-Rickardt et al., 1991; Kinzler separate independent cohort of 24CIMP-positive et al., 1991a, b; Nishisho et al., 1991). Most recently, (CIMP þ ) and 24CIMP-negative (CIMP À) colorectal promoter methylation of MCC was found to be a tumors from a consecutive series of CRC patients with frequent early event in CRC. MCC promoter methyla- known CIMP and BRAF/KRAS mutation status. MCC tion was highly associated with a distinct subset of CRC methylation was detected in 28 out of 48 sporadic cancer characterized by the CpG island methylator phenotype specimens (58.3%). MCC methylation was detected in (CIMP) and BRAFV600E mutation (Kohonen-Corish all but 2 CIMP þ tumors (Po0.0001), in all 13 tumors et al., 2007) which constitute the serrated CRC pathway. with the BRAFV600E mutation (P ¼ 0.0001), and in all but Serrated CRC comprises a morphologically and geneti- 1 high microsatellite instability (MSI-H) tumors cally distinct group of CRC and precursor lesions in (P ¼ 0.0005; Table 1). There was no correlation with which CRC development and progression appear driven the presence of KRAS mutation. Our results are in by an alternative molecular pathway (Jass et al., 2002). strong agreement with the previous study and add Cumulatively, this evidence highly favors a role of MCC further support of the strong association of MCC in CRC carcinogenesis. However, currently almost promoter methylation with this distinct type of CRC nothing is known about the signal transduction path- characterized as CIMP þ , MSI-H, with BRAFV600E ways that MCC participates in that are biologically mutation. significant to MCC loss in CRC. From these previous Six of eight human CRC lines tested including RKO, reports and our observation that many CRC cell lines DLD-1, Caco-2 and HT29 (Figure 1a) as well as Ls174T lack MCC expression, we hypothesized that MCC might and SW48 (data not shown) had little detectable possess significant, undiscovered physiological/patholo- expression of MCC irrespective of the mutational or gical roles in colorectal epithelial cell biology and CRC LOH status of the APC gene (Figure 1a). Only the tumorigenesis. Here for the first time, we show that HCT116 and SW480 CRC cells express detectable levels MCC is present in the nucleus of cells and interacts with of MCC (Figure 1a). As, MCC promoter methylation b-catenin. Importantly, we provide the first evidence is responsible for MCC loss in CIMP þ and BRAF that MCC can function as a tumor suppressor in CRC mutated cases of primary CRC (Kohonen-Corish et al., by suppressing Wnt signaling, b-catenin/T-cell factor 2007), we investigated whether MCC promoter methy- (TCF)/lymphoid-enhancer factor (LEF)-dependent lation was responsible for the loss of MCC expression transcription and cellular proliferation even in the seen in our panel of CRC cell lines. Indeed, MCC presence of mutation of the master negative regulator promoter methylation correlated well to loss MCC of Wnt signaling in the intestinal epithelium, APC. protein expression in the CRC cell lines consistent with the previous study (Figure 1b). It is noteworthy that MCC expression was lost in both the CRC cell lines, RKO and HT29, which have the BRAFV600E mutation Results mirroring the association seen in clinical samples. MCC is lost by promoter methylation in CIMP þ sporadic CRC tumors and cell lines and is associated with Nuclear localization of MCC BRAF mutation Interestingly in cells that express MCC, we detected As a previous study had found a significant correlation MCC in both the cytoplasm and nucleus (Figure 1a and of MCC promoter methylation to CIMP and BRAFV600E Supplementary Figure 1). The nuclear localization of mutation (Kohonen-Corish et al., 2007), we decided to MCC had not been previously reported. A motif search

Table 1 Comparison of MCC promoter methylation with CIMP, BRAF mutation and MSI in a set of 24CIMP+ and 24CIMP À tumors MCC (methylated) MCC (unmethylated) Total no. of patients P

CIMP+ 22 (91.7) 2 (8.3) 24 CIMPÀ 6 (25) 18 (75) 24 o0.0001

BRAF V600E mutation Yes 13 (100) 0 (0) 13 No 14(40) 21 (60) 35 0.0001

KRAS codon 12/13 or 61 mutation Yes 4(66.7) 2 (33.3) 6 No 23 (54.8) 19 (45.2) 42 0.6830

Microsatellite instability MSI-H 14(93.3) 1 (6.7) 15 MSI-L/MSS 13 (39.4) 20 (60.6) 33 0.0005

Abbreviations: CIMP, CpG island methylator phenotype; MCC, mutated in colorectal cancer; MSI-H, high microsatellite instability; MSI-L, low microsatellite instability; MSS, microsatellite stable.

Oncogene MCC inhibits b-catenin-dependent transcription R Fukuyama et al 6046

SW480 RKO DLD1MB231 HCT116 Caco2 HT29 Cy N Cy N Cy N Cy N Cy N Cy N Cy N

MCC

Lamin B - APC MT WT MT WT WT MT MT BRAF V600E --+ ---+

Cell: H2O RKO HT29 HCT116 SW480 DLD1 SW48 PCR: M U M U M U M U M U M U

MCC low low ++ + low low

∆C11 ∆C67 ∆C266 full ∆N536 pcDNA 279-536 Cy N Cy N Cy N Cy N Cy N Cy N Cy N

MCC MCC

HDAC3 LaminB

1 100 200 300 400 500 600 700 829 PDZ-ligand TRAF2 serine rich

NLS NES NLS NES NES

Figure 1 Mutated in colorectal cancer (MCC) is found in both the cytoplasm and nucleus but lost in many colorectal cancer (CRC) cell lines by promoter methylation. (a) MCC protein levels were assessed by western blot analysis in cytoplasmic (Cy) and nuclear (N) fractions of CRC and other cell lines. The nuclear protein lamin B was used to assess the purity of the cytoplasmic and nuclear fractions. The mutational status of adenomatous polyposis coli (APC) is indicated, MT stands for mutant and WT stands for wild-type APC. b-Actin was used for loading control (data not shown). (b) Methylation-specific PCR using primers specific for the methylated (M, 99 bp) and unmethylated (U, 94bp) MCC promoter in bisulfite-treated DNA samples. ( c) Determination of Cy and N localization of full-length and MCC deletion constructs expressed in COS-7 cells by western blot analysis. DC and DN equals truncation of MCC’s C terminus and N terminus, respectively. For detection of DN536 and 279–536 MCC segments, Xpress-tag antibody was used, as the commercially available MCC antibody only reacts with the N terminus. The nuclear proteins lamin B and HDAC3 were used to assess the purity of the cytoplasmic and nuclear fractions. b-Actin was used for loading control (data not shown). (d) (Upper panel) A schematic presentation of the putative NLS (nuclear localization signal, grey boxes), NES (nuclear exclusion signal, open boxes), serine rich, TRAF2 binding and PSD-95/Discs-large/ZO-1 (PDZ) ligand-binding sequences in MCC with the reference number of amino acid above. (Lower panel) Schematic summary of functional assignment of NLS and NES sequences to MCC.

of MCC revealed intriguing amino acid stretches (Figure 1c). These data overall indicate that the including several potential nuclear localization signals N- and C termini of MCC likely include both NLS (NLSs) and nuclear exclusion signals (NESs), as well as, and NES sequences whereas the central 1/3 of MCC has a typical tumor necrosis factor (TNF) receptor-asso- no NLS sequence (Figure 1d). Immunoﬂuorescent ciated factor 2 (TRAF2)-binding sequence, a serine-rich staining conﬁrmed the subcellular localization of the domain and a PDZ domain-binding motif (Figure 1d). various truncated MCC proteins in COS and RKO cells First, we determined the domains of MCC responsible (data not shown). for its nuclear localization. Both N-terminally (DN279 The potential NLS sequences of MCC include a (not shown), DN536) and C-terminally (DC11, DC67 classical bipartite NLS with an amino acid sequence and DC266) truncated MCC proteins were able to quite similar to that of p53 and other known nuclear localize to both the cytoplasmic and nuclear fractions localized proteins (Figure 2a). Moreover, several more (Figure 1c). However, the central domain (279–536 a.a.) NLS-like and possible NES-like sequences are present in of MCC containing a putative NES but no putative MCC (Figure 2a). We tested whether the putative NLSs NLSs, is found exclusively in cytoplasmic fraction in MCC are in fact functional. Both the bipartite NLS

Oncogene MCC inhibits b-catenin-dependent transcription R Fukuyama et al 6047 (766–782) and N-terminal arginine-rich stretch (R-rich (Figure 3a and data not shown). In addition, stable NLS, 267–278) of MCC confer strong nuclear localiza- expression of MCC significantly reduces the strong basal tion in the RKO (Figure 2b) and COS7 cells (data not b-catenin/TCF/LEF-dependent promoter activity in the shown) when linked to enhanced green fluorescent SW480 MCC-overexpressing cell lines as well as the protein (EGFP). Furthermore, treatment of RKO cells Wnt3a-stimulated b-catenin/TCF/LEF-dependent pro- expressing the full-length MCC with the active nuclear moter activity of the MEF MCC-overexpressing cell export inhibitor, leptomycin B (LMB), dramatically lines as compared to parental control cells (Figure 3c increases nuclear localization of MCC (Figure 2c). and data not shown). Transient and stable expression of These studies strongly suggest that besides being a MCC also suppressed the basal and Wnt3a-stimulated cytoplasmic protein, endogenous MCC also resides in b-catenin/TCF/LEF-dependent promoter activity of the nucleus of the cell, contains two functional NLS RKO and HT29 CRC (Figures 3a and b and data not sequences, and that MCC is likely shuttled in and out of shown). The MCC-mediated suppression of b-catenin/ nucleus by the basic importin–exportin-dependent TCF/LEF-dependent promoter activity was specifically mechanism. relieved by MCC siRNA treatment of both parental and MCC-overexpressing SW480 cells that exclusively MCC suppresses both oncogenic and Wnt-stimulated suppressed MCC protein levels and correspondingly b-catenin/TCF/LEF-dependent transcription in CRC cells increased b-catenin/TCF/LEF-dependent promoter ac- As MCC had previously been described to be a tivity as well as the b-catenin transcriptional targets, potential inhibitor of the NF-kB transcription factor c-myc and cyclin D (Figure 3d and data not shown). (Bouwmeester et al., 2004), we decided to investigate the potential MCC regulation of several important transcription factors in CRC including of NF-kBand MCC interacts with b-catenin, blocks b-catenin/TCF/ b-catenin. Transient MCC reexpression significantly LEF-DNA binding, and can affect endogenous b-catenin suppressed both the low basal and the high Wnt3a- localization/levels stimulated b-catenin/TCF/LEF-dependent promoter Stable expression of MCC specifically suppresses activity in RKO cells in a dose-dependent manner b-catenin/TCF/LEF but not SRE and AP-1 DNA- (Figures 3a and b). Importantly, MCC also significantly binding activity in CRC cells (Figure 4a, left panel). suppressed even the strong basal b-catenin/TCF/LEF Also, as expected from the unaltered NF-kB-dependent activity in DLD-1 (Figure 3a) and SW480 (Figure 3c) promoter activity, the basal and TNF-a-induced cells caused by oncogenic APC mutations. This suppres- NF-kB-DNA-binding activity in MCC-overexpressing sion of b-catenin/TCF/LEF-dependent promoter stable cell clones is not significantly changed (data not activity is specific, as surprisingly the high constitutive shown). Western analysis demonstrated that stable NF-kB-dependent promoter activity was not altered by expression of MCC does not significantly reduce nuclear transient MCC expression in any of the CRC cell lines b-catenin levels in SW480 cells but does increase the

bipartite NLS of known nuclear proteins P53 305 KRALPNNTSSSPQPKKKP nucleoplasmin 155 KRPAATKKAGQAKKKKLD P73 327 KRAFKQSPPAVPALGAGVKKRR Nrf2 494 RRRGKQKVAANQCRKRK MCC 766 KRANSNLVAAYEKAKKKH

putative NLS in MCC: putative NES in MCC: 171-173: RKK consensus; LX(2-3)LX(2)LXL 267-278: REERDRLRRRVR 158-170: LX(3)LX(3)LX(2)L 729-733: RREKKIK 461-474: LX(3)LX(6)LXL 766-782: KRANSNLVAAYEKAKKKH 611-623: LXLX(4)LX(3)L

pEGFP bipartite NLS R-rich NLS -LMB +LMB

Figure 2 Functional confirmation of the biNLS (nuclear localization signal) and arginine-rich NLS sequences of mutated in colorectal cancer (MCC). (a) Amino acid sequences of the putative NLSs and nuclear exclusion signals (NESs) in MCC and the comparison of MCC’s biNLS to the defined biNLSs of several other nuclear proteins. (b) Detection of subcellular localization of MCC’s biNLS and arginine-rich NLS linked with enhanced green fluorescent protein (EGFP) protein in RKO cells. (c) Immunofluorescent staining of MCC in MCC-overexpressing RKO cells either untreated or treated overnight with leptomycin B (LMB) at 5 nM.

Oncogene MCC inhibits b-catenin-dependent transcription R Fukuyama et al 6048 cytoplasmic levels of b-catenin (Figure 4b). In addition, AP-1 DNA binding is dramatically increased in both confocal analysis of MCC and b-catenin localization in the parental and MCC-overexpressing SW480 cells the MCC-overexpressing SW480 and RKO cells demon- (Figure 4a, right panel) as expected from the increase strated that MCC is localized in both the cytoplasm and b-catenin/TCF/LEF-dependent promoter activity ob- nucleus and that b-catenin is mostly excluded from the served with MCC siRNA (Figure 3d). We found that nucleus in these cells (Supplementary Figure 1). As endogenous MCC interacts speciﬁcally with endogenous expected, when MCC mRNA and protein levels are b-catenin with interaction enhanced upon Wnt stimula- reduced by siRNA, the b-catenin/TCF/LEF but not the tion in MEF cells (Figure 4c). Finally, transient over-

TCF/LEF NF-κB RKO DLD-1 RKO DLD-1 0.8 16 60 0.6 12

40 0.4 8

** 0.2 4 ** 20 ** *** 0 0 pcDNA MCC pcDNA MCC pcDNA MCC pcDNA MCC pcDNA MCC pcDNA MCC Wnt3a Wnt3a

RKO pcDNA MCC 0.5 200 200 25 100 200 Wnt3a: - + + + + 0.4

0.3 ** MCC 0.2 *** 0.1 ERK 0 200 20025 100 200 Wnt3a: -+ + + + pcDNA MCC

SW480 MEF 6 0.4

5 0.3 4 3 0.2 2 *** *** *** 0.1 1 0 0 L Wnt L Wnt L Wnt L Wnt control MCC-A control E5

SW480 MCC-A *** SW480 MCC-A 8 iCo iMCC iCo iMCC iCo iMCC iCo iMCC MCC days 3 1 2 3 3 1 2 3 6 *** 4 c-myc MCC 2 CyclinD1 β actin β 0 actin iCo iMCC iCo iMCC SW480 MCC-A

Oncogene MCC inhibits b-catenin-dependent transcription R Fukuyama et al 6049 expression of MCC but not p65 NF-kB in COS-7 cells in NIH3T3 murine ﬁbroblast cells. Stable expression of decreases total b-catenin by 35–52% depending on MCC in MEFs, MBA-MD-231 breast cancer, as well as MCC expression levels while not altering the level of RKO, SW480 and HT29 CRC cells suppresses basal extracellular signal-regulated kinase (ERK) or b-actin proliferation (Figures 6a and b and data not shown). (Figure 4d). These results indicate that MCC interacts Stable expression of MCC also suppressed Wnt3a- with b-catenin and speciﬁcally suppresses b-catenin/ stimulated proliferation of SW480, MEFs, MBA-MD- TCF/LEF-dependent transcriptional activity down- 231 and RKO cells (Figure 6c). Intriguingly, both the stream to the destruction complex by interfering SW480 and HT29 CRC cells have constitutively with the DNA-binding activity of b-catenin/TCF/LEF transcriptionally active b-catenin as a result of onco- in the nucleus potentially through relocalization of b- genic mutations in APC indicating that MCC can act as catenin to the cytoplasm and indirectly increasing its a suppressor of cellular proliferation even in the degradation. presence of these oncogenic Wnt pathway mutations.

MCC domain/s responsible for suppression of b-catenin/ TCF/LEF-dependent transcription Discussion N-terminal deletions of MCC uncovered both a significant (^^^) domain (130–278 aa segment) and a In contrast to APC (Gregorieff and Clevers, 2005), the minor (^) domain (279–536 aa segment) necessary for molecular mechanism and signaling pathways of MCC repressing b-catenin/TCF/LEF-dependent transcription important to colorectal epithelial cell biology and CRC (Figures 5a and b). Suppression of b-catenin/TCF/LEF- remain largely unknown. Here we provide the first dependent promoter activity by the removal of the C- evidence of the molecular signaling pathways that MCC terminal segments (DC293 and DC65) is not as extensive participates in and the biological significant functions of as the full-length or the DN130 MCC constructs MCC in CRC. (Figures 5a and b). These data indicate that there is a We find that MCC expression is absent or dramati- second major cooperating domain (###) at MCC’s C cally decreased in many CRC cell lines regardless of terminus that is necessary along with the N-terminally their APC mutational status (Figure 1a). The loss of located domain (^^^) to suppress b-catenin/TCF/LEF- MCC protein correlates to the methylation of the MCC dependent promoter activity. The most significant promoter in these CRC cell lines (Figure 1b). These functional segments of MCC, 130–278 aa (^^^) and data may indicate a selective advantage of MCC loss 536–829 aa (###), for repression of b-catenin/TCF/ independent to APC loss in CRC cells. Recently, MCC LEF-dependent promoter activity contain putative NES promoter methylation has been found as a frequent sequences as well as the arginine-rich NLS-like stretch early event in 45–52% of 187 cases of primary CRC. (striped box) and the biNLS (speckled box), respec- MCC promoter methylation was highly significantly tively. These data indicate that MCC repression of associated with cyclin-dependent kinase inhibitor type 2A b-catenin/TCF/LEF transcriptional activity may require (CDKN2A) methylation, CIMP and BRAFV600E muta- MCC functionally capable of proper nuclear-cytoplas- tion but was not associated with either APC methylation mic shuttling. or KRAS mutations (Kohonen-Corish et al., 2007). This group also found that MCC methylation was more MCC inhibits basal and Wnt3a-stimulated CRC cell common in serrated polyps than adenomas thus proliferation associating MCC methylation with a distinct spectrum Matsumine et al. (1996) previously demonstrated that of precursor lesions from those with APC methylation transient MCC over-expression suppressed cell growth which give rise to CRC through the alternative serrated

Figure 3 Mutated in colorectal cancer (MCC) speciﬁcally suppresses b-catenin/T-cell factor (TCF)/lymphoid-enhancer factor (LEF) transcriptional activity. (a) RKO and DLD-1 cells were transiently co-transfected with either 200 ng of MCC or pcDNA control plasmid together with either 1 mg of the b-catenin/TCF/LEF or the nuclear factor-kB (NF-kB) reporter construct. Cells were stimulated for 16 h with either L or Wnt3a medium prior to luciferase reporter assay. The Y axis is relative normalized luciferase activity, data are represented as mean±s.d., n ¼ 3. (b) RKO cells were co-transfected with 25, 100 or 200 ng of MCC or 200 ng of pcDNA control plasmid together with 1 mg of the b-catenin/TCF/LEF reporter construct. Cells were stimulated for 16 h with either L or Wnt3a medium prior to luciferase reporter assay. The Y axis is relative normalized luciferase activity, data are represented as mean±s.d., n ¼ 3. Lysates not used for luciferase assay were pooled and electrophoresed on a 10% SDS–PAGE (polyacrylamide gel) and analysed by western blotting for MCC and total extracellular signal-regulated kinase (ERK) protein which was used as a loading control. (c) Parental and MCC- overexpressing clones of SW480 and MEF were transiently transfected with 1 mg of the b-catenin/TCF/LEF reporter construct. Cells were stimulated for 16 h with either L or Wnt3a medium prior to luciferase reporter assay. MCC-A and E5 indicate the corresponding stable MCC-overexpressing clones of the SW480 and MEF parental cells, respectively. The Y axis is relative normalized luciferase activity, data are represented as mean±s.d., n ¼ 3. (d) (Left panel) Time-responsive suppression of MCC expression by MCC siRNA (iMCC) treatment at 20 nM in SW480 parental cells and MCC-A, the stable SW480 MCC-overexpressing clone. Control siRNA (iCo) was used as a control. (Middle panel) SW480 and MCC-A cells were co-transfected with 20 nM iMCC or iCo together with 1 mg of the b-catenin/TCF/LEF reporter construct. Cells were incubated for 2 days prior to luciferase reporter assay. The Y axis is relative normalized luciferase activity, data are represented as mean±s.d., n ¼ 3. (Right panel) Lysates not used for luciferase assay were pooled and electrophoresed on a 10% SDS–PAGE and analysed by western blot analysis of MCC, c-myc, cyclin D and b-actin which was used as a loading control. ** and *** indicate Po0.001 and 0.0001, respectively.

Oncogene MCC inhibits b-catenin-dependent transcription R Fukuyama et al 6050 SW480 RKO SW480 A SW480 A co A J co B2 C5 si: Co MCC Co MCC Cy N Cy N

TCF TCF /LEF /LEF MCC

LEF-1 AP-1 SRE βcatenin MCC p65 LaminB AP-1 β catenin LaminB

cyclinD1 MCC MCC

GAPDH βactin βactin

IP pcD pME #5 #13 p65 WB IgG MCC MCC β-catenin p65 NFκB MCC --+Wnt IκBα

β -catenin

ERK

β actin 1.0 0.65 0.48 0.53 0.97 Figure 4 Mutated in colorectal cancer (MCC) interacts with b-catenin and inhibits b-catenin/T-cell factor (TCF)/lymphoid-enhancer factor (LEF)-DNA binding. (a) (Left panel) Electrophoretic mobility shift assay (EMSA) for b-catenin/TCF/LEF, SRE and AP-1 of SW480, RKO and their MCC-overexpressing clones (SW480: A and J, RKO: B2 and C5). Western blot analysis of MCC with b-actin used as a loading control. (Right panel) EMSA for b-catenin/TCF/LEF and AP-1 of parental SW480 and MCC-stable overexpressing SW480 clone MCC-A (A) that were transfected with 20 nM MCC siRNA (MCC) or control siRNA (Co). Cells were incubated for 2 days prior to EMSA. Western blot analysis of MCC and b-catenin in the nuclear fraction with reference to the nuclear protein, lamin B. Levels of MCC mRNA by reverse transcriptase (RT)–PCR with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) used as a loading control. (b) Western blot analysis of MCC, LEF-1, b-catenin, p65 NF-kB (nuclear factor-kB) and cyclin D1 in parental SW480 and MCC-stable overexpressing SW480 clone MCC-A (A). Lamin B and b-actin were used as a loading control for nuclear and total protein, respectively. (c) Immunopreciptation (IP)/western blot (WB) analysis of the interaction between endogenous MCC and b-catenin in MEF cells either treated with L (À) or Wnt3a ( þ ) medium for 30 min. (d) COS-7 cells were transiently co-transfected with either 200 ng of pcDNA control plasmid (pcD), several different full-length MCC constructs (pME, #5, #13), or p65 NF-kB. At 18 h following transfection, total cell lysates were analysed by western blot analysis of MCC, p65 NF-kB, IkBa, b-catenin, extracellular signal-regulated kinase (ERK) and b-actin which was used as a loading control.

CRC neoplasia pathway (Jass, 2005; Kohonen-Corish distinct spectrum of CRC that rarely demonstrates APC et al., 2007). Our study of MCC promoter methylation loss. We demonstrate that analogous to APC, MCC can in an independent cohort of CRC patients solidly be important as a potential tumor suppressor in CRC by supports the finding of MCC promoter methylation in inhibiting both Wnt/b-catenin signal transduction and this distinct subset of sporadic CRC that characterizes cellular proliferation (Figures 3–6). We show MCC not the serrated CRC pathway. only inhibits murine fibroblast proliferation as had been Here we present the first evidence of the potential previously demonstrated (Matsumine et al., 1996), but biological significance of the MCC loss seen in this also inhibits both basal and Wnt3a-stimulated MEF,

Oncogene MCC inhibits b-catenin-dependent transcription R Fukuyama et al 6051 R-Rich bi- TCF/LEF NLS NLS PDZ Supression His +++ ∆N130 His +++ ∆ N278 His + ∆ N536 His - His ∆C65 + His ∆ C293 + ### ^^^ ^ consensus

pcDNA ∆N ∆C TCF/LEF-dependent luciferase activity Full ∆C65 80 ∆ N130 ∆ N278 ∆C293 60 ** ** ∆ unit) ** N536 Anti-His 40 *** *** Anti-MCC Full ∆C65 (relative 20 ∆C293

0 β pcDNA full ∆N130 ∆N278 ∆N536 ∆C293 ∆C65 actin

Figure 5 Mutated in colorectal cancer (MCC) domains necessary for b-catenin/T-cell factor (TCF)/lymphoid-enhancer factor (LEF) transcriptional suppression. (a) Schematic summary of the domain-dependent suppression of b-catenin/TCF/LEF-dependent activity. ^^^ and ### indicate major domains on the N terminus and C terminus, respectively, of MCC necessary for suppression of b-catenin/ TCF/LEF transcriptional activity. ^ indicates a minor domain of MCC necessary for suppression of b-catenin/TCF/LEF transcriptional activity. The locations of the arginine-rich nuclear localization signal (NLS)-like stretch (striped box), the biNLS (speckled box), and the PSD-95/Discs-large/ZO-1 (PDZ) domain (black box) are indicated. (b) Full-length and several truncated forms of MCC were transiently expressed in COS-7 cells and the b-catenin/TCF/LEF-dependent transcriptional activity was assayed. (Left panel) COS-7 cells were transiently co-transfected with 200 ng of either the indicated MCC construct or pcDNA control plasmid together with either 1 mg of the b-catenin/TCF/LEF reporter construct. Cells were incubated for 16 h prior to luciferase reporter assay. The Y axis is relative normalized luciferase activity, data are represented as mean±s.d., n ¼ 3. ** and *** indicate Po0.001 and 0.0001, respectively. (Right panel) The expression of MCC segments was assessed by western blot analysis with either anti-His-tag antibody that detects all constructs or anti-MCC antibody that detects only the full-length and DC MCC constructs using b-actin as a loading control.

CRC and breast cancer cellular proliferation (Figure 6). the b-catenin/TCF/LEF transcriptional complex indir- Previously MCC was reported as a potential negative ectly by modulating the activity of these kinases. regulator of the transcription factor NF-kB (Bouwmee- Further study will be needed to address the potential ster et al., 2004). However, we saw no effect of MCC regulation of these kinases and the phosphorylation of expression on either the constitutive or TNF-a-induced b-catenin/TCF/LEF transcriptional complex by MCC. NF-kB activity seen in our CRC cell lines (Figure 3 and Another more plausible hypothesis based on our current data not shown). Perhaps MCC is unable to inhibit the data is that MCC can suppress b-catenin/TCF/LEF- activation of NF-kB in CRC cells, which is constitu- DNA binding directly perhaps by interacting with tively activated by a number of oncogenic mechanisms. b-catenin and affecting its subcellular localization thus Nevertheless, we do demonstrate that expression of indirectly its degradation. We found that MCC does MCC in CRC cells dramatically inhibits Wnt signaling interact with b-catenin but failed to coimmunoprecipi- and b-catenin/TCF/LEF-dependent promoter activity tate with APC, TCF-4or LEF-1 (Figure 4cand data not even in the presence of mutated oncogenic APC shown). MCC also causes an increase in the cytoplasmic (Figure 3). However, unlike APC, MCC does not seem levels of b-catenin and a decrease in total b-catenin to directly facilitate the destabilization of b-catenin but levels (Figures 4b and c; Supplementary Figure 1). The instead interacts with b-catenin and specifically inter- studies of the direct interactions of MCC with b-catenin feres with the binding of the b-catenin/TCF/LEF and potentially other Wnt pathway members in regulat- transcriptional complex to DNA in the nucleus ing the subcellular localization, b-catenin stability and (Figure 4). b-Catenin/TCF/LEF-DNA binding is suppression of DNA binding by the b-catenin/TCF/LEF known to be suppressed by specific phosphorylation of transcriptional complex are on-going. TCF/LEF by Nemo-like kinase (NLK), which is Our evidence indicates that nuclear-cytoplasmic activated by transforming growth factor-b-activated shuttling of MCC may be important for suppressing kinase 1 (Ishitani et al., 1999, 2003). Hence, MCC could b-catenin/TCF/LEF-dependent transcription. We possibly promote phosphorylation and inactivation of demonstrate for the first time that MCC is localized,

Oncogene MCC inhibits b-catenin-dependent transcription R Fukuyama et al 6052 SW480 J 14 SW480 J 12 SW480 10 J A MCC 8 6 4 Actin 2 0 053 (day) 30 MEF E5 MEF E5 25 MEF E5 20 MCC H4 15 10 Actin 5 0 0 3 5 (day) MB231 B12 MB231 B12 35 30 MB231 MCC 25 B12 20 15 Actin 10 5 0 0 3 5 (day) RKO B2B2 RKO B2B2 100 RKO MCC 80 B2A10 60 B2B2 40 Actin 20 0 0 3 5 (day) 50 L Wnt3a 40

0 480 J MEF E5 231 B12 RKO B2B2

Figure 6 Mutated in colorectal cancer (MCC) inhibits both basal and Wnt-stimulated cellular proliferation. (a) (Left panel) Immunoﬂuorescent staining of MCC in stable MCC-overexpressing clones from various cell lines. (Right panel) Western blotting of MCC expression in stable MCC-overexpressing clones from various cell lines. b-Actin was used for loading control. (b) Cell growth of several stable MCC-overexpressing clones from various cell lines was monitored by Trypan blue exclusion method on 3 and 5 days after plating 1 Â 104 cells on day 0. (c) Cell growth of the parental and stable MCC-overexpressing clones from various cell lines was monitored by Trypan blue exclusion method 3 days after plating 1 Â 104 cells on day 0 and treatment with either L or Wnt3a medium on day 1. J, E5, B12 and B2B2 represent the MCC-overexpressing clones of SW480(480), MEF, MB231(231) and RKO parental cells, respectively. The Y axis is the cell number in 1 Â 104, data are represented as mean±s.d., n ¼ 3.

not only to cytoplasmic and membrane fractions as arginine-rich NLS at the N terminus as well as several indicated by previous studies (Senda et al., 1999), but potential NES sequences are located in the two most also to the nucleus likely through two functional NLSs signiﬁcant segments of MCC necessary to repress we identify at the termini of MCC (Figures 1 and 2). b-catenin/TCF/LEF-dependent promoter activity Signiﬁcantly, the biNLS at the C terminus and the (Figure 4). The tumor suppressor APC has several

Oncogene MCC inhibits b-catenin-dependent transcription R Fukuyama et al 6053 functional NES and NLS sequences which have been in which MCC is silenced but further investigation is shown to affect the nuclear-cytoplasmic shuttling of needed. b-catenin (Henderson, 2000; Rosin-Arbesfeld et al., Finally, the concurrent MCC silencing and the 2000; Zhang et al., 2000). However, additional b-catenin BRAFV600E mutation seen in MSI-H/CIMP þ sporadic nuclear-cytoplasmic shuttling mechanisms are possible as CRC and its precursor lesions does not seem to be a even in APC-mutant CRC cells, b-catenin is efficiently chance association and supports the theory that MCC, exported from the nucleus (Eleftheriou et al., 2001). in addition to BRAF, may be a key gene in activating Collectively our data that show that MCC interacts with molecular pathways related to development of sporadic b-catenin and blocks b-catenin/TCF/LEF-DNA binding MSI-H/CIMP þ CRC through the serrated neoplasia as well as the potential importance of MCC nuclear- pathway. Therefore, further study is needed to deter- cytoplasmic shuttling in suppression of b-catenin-depen- mine the role of MCC in normal colorectal epithelial dent transcription provide evidence of a novel regulatory cell biology and how loss of MCC interacts with pathway for b-catenin by MCC. Our future studies will BRAFV600E in the serrated CRC neoplasia pathway. address the potential importance of MCC subcellular localization and nuclear-cytoplasmic shuttling in suppressing Wnt signaling. Materials and methods In summary, we found MCC suppresses cell proliferation and the Wnt/b-catenin pathway in CRC cells. Construction of plasmids Like APC, MCC interacts with b-catenin and inhibits Plasmid harboring the full-length MCC cDNA (pME18S) was Wnt/b-catenin-dependent transcriptional activity although obtained from Dr Kazuo Maruyama (University of Tokyo, through a distinct mechanism. Our data combined with Tokyo, Japan). Using this as a template, the full-length and the frequent epigenetic loss of MCC seen in a distinct set truncated MCC cDNAs were amplified by PCR with appro- of CRCs and precursor lesions independent of APC loss priate primer sets and subcloned into the pcDNA3/4A vector (Kohonen-Corish et al., 2007) indicate that MCC could (Invitrogen Corp., Carlsbad, CA, USA). Double-stranded oligonucleotides for the bipartite NLS and the arginine-rich play a significant tumor suppressor role in this distinct NLS sequences of MCC gene were subcloned upstream to the subset of CRCs, perhaps equivalent to that of APC in EGFP in the pEGFPN3 vector (Clontech, Mountain View, the traditional adenoma-carcinoma CRC sequence, by CA, USA). All plasmids were verified by DNA sequencing. inhibiting b-catenin/Wnt signaling and restraining colorectal epithelial cell proliferation when it is vitally Establishment of MCC stable transformants important early during CRC initiation. MCC inactiva- The empty pcDNA3/4A vector or pcDNA3/4A containing tion therefore could be an initiating Wnt pathway- full-length MCC was transfected into each cell line and stable activating event in this group of CRC arising through pools and clones were selected with G418. Multiple clones for the alternative serrated CRC neoplasia pathway. As our each cell line were isolated and the MCC level was verified by in vitro data indicate that MCC represses b-catenin western blotting and reverse transcriptase–PCR (RT–PCR). signaling in CRC cells and as MCC epigenetic silencing is particularly frequent in sporadic CIMP þ , MSI-H Transfection and promoter assays CRC tumors, one would predict to observe robust b-Catenin/TCF/LEF- or NF-kB-dependent transcriptional b-catenin-dependent effects in these tumors. Predisposi- activity was determined as described previously (Agarwal tion to tumor budding at the invasive margin of CRC et al., 2005). Exponentially growing cells in 24-well plates were has been strongly linked to upregulation of b-catenin transfected with either the b-catenin/TCF/LEF- (pOT) or signaling (Conacci-Sorrell et al., 2003; Zlobec et al., NF-kB-dependent (NF-kB) luciferase reporter construct alone 2007). However, tumor budding is often low or or co-transfected with the indicated MCC plasmid with Lipofectamin 2000 (Invitrogen Corp.). Luciferase reporter completely absent in sporadic CIMP þ , MSI-H CRC plasmids for pOT and NF-kB were gifts from Dr Bert Vogelstein tumors (Jass et al., 2003; Jass, 2007; Prall and Ostwald, (John Hopkins, Baltimore, MD, USA) and Dr Bryan Williams 2007; Zlobec et al., 2007), where MCC epigenetic (Monash Institute of Medical Research, Clayton, Victoria, silencing is most often seen. This inconsistency between Australia), respectively. The Renilla luciferase construct pRL- our in vitro data and the pathology of this subset of TK (Promega, Madison, WI, USA) was co-transfected as a CRC may be explained by the striking peritumoural normalization control. After an overnight incubation, the cells lymphocytic infiltration (PTL) seen frequently in spora- were stimulated with either control or Wnt3a medium, dic CIMP þ , MSI-H CRC tumors (Jass, 2007). Speci- harvested, and lysed. Each lysate (20 ml) was incubated with fically, an inverse relation is seen between tumor luciferase substrates for firefly and Renilla luciferase assays were budding and PTL and it has been suggested that the performed with the dual luciferase reporter assay system (Promega). The b-catenin/TCF/LEF- or NF-kB-dependent relative lack of tumor budding observed in this CRC firefly luciferase activity was normalized with the value of the subset may well be due to the high PTL frequently seen corresponding Renilla-dependent luciferase activity and the ratio in CIMP þ , MSI-H CRC tumors (Jass et al., 2003; (n ¼ 3, mean±s.d.) was statistically analysed. Shinto et al., 2005). Findings in DNA mismatch repair-proficient CRC suggest that PTL and their Electrophoretic mobility shift assay associated tumor infiltrating lymphocytes may destroy The b-catenin/TCF/LEF and NF-kB binding sites from the budding tumor cells (Jass, 2007; Zlobec et al., 2007). cyclin D1 and the immunoglobulin gene promoters, respectively, Therefore, this could also be true for the lack of tumor were synthesized by ITD Technology Inc. (Coralville, IA, USA) budding seen in sporadic CIMP þ , MSI-H CRC tumors and used as probes. Oligonucleotides for SRE and AP-1 were

Oncogene MCC inhibits b-catenin-dependent transcription R Fukuyama et al 6054 purchased from Santa Cruz (Santa Cruz, CA, USA). Electro- Tumor specimens phoretic mobility shift assay (EMSA) and the supershift assays A consecutive set consisting of 24CIMP þ and 24CIMP— were performed on nuclear lysates as previously described colorectal tumors were chosen from an existing biobank of (Sizemore et al., 2002). colorectal tumors. CIMP status was determined in bisulfite- treated DNA by MethyLight quantitative PCR with recently MCC suppression by siRNA defined CIMP markers (CACNA1G, IGF2, NEUROG1, A Smart Pool Set of siRNAs for MCC from Dharmacon RUNX3 and SOCS1) (Weisenberger et al., 2006). The cutoff (Chicago, IL, USA) was used to suppress MCC expression. A for a CIMP þ call in the MethyLight quantitative PCR was set control siRNA pool was to control for nonspecific effects of at percent methylated reference (PMR) X10 at X3 markers. transfection and siRNA. The MCC or control siRNA pools MSI status was determined using PCR-based analysis of 11 were transfected with SiLentFect Lipid (Bio-Rad, Hercules, microsatelite markers (BAT25, BAT26, D17S250, D5S346, CA, USA) according to manufacturer’s instructions into cells ACTC, BAT40, MYC-L, BAT34C4, D10S197, D18S55 and growing in 24-well plates and harvested after the indicated D25123) that define MSI status as previously reported (Lindor number of days incubation. et al., 2006). Mutation analysis of KRAS codons 11/12 and 61 and BRAF codons 12/13, was analysed as previously reported Reverse transcriptase–PCR (Ogino et al., 2006). Patient samples were obtained through Cells were harvested, lysed and cleared by brief centrifugation institutionally approved protocols. as previously described (Sizemore et al., 2002). Total RNA was isolated from 6 or 24-well culture plates using Trizol (Invitrogen Corp.). Approximately 1 mg of total RNA from Methylation-specific and methylation-specific quantitative PCR each sample was reverse-transcribed as described by the DNA from CRC cancer cell lines or tumor specimens was manufacturer, using the iScript cDNA Synthesis Kit from bisulfite treated using standard methods (Herman et al., 1996). Bio-Rad. Gene sequences available from the National Center Both the methylation-specific and methylation-specific quanti- for Biotechnology Information GenBank and Unigene data- tative PCR was performed on the MCC promoter from bases were selected to design primers. Optimum primer bisulfite-treated DNA using the primers, the methylated sequences were selected after verification for gene-specific amplicon-specific fluorogenic hybridization probe and meth- complementation using the National Center for Biotechnology ods as reported previously (Kohonen-Corish et al., 2007). The Information Blast program. PCR primers for glyceraldehyde cutoff for a positive call in the methylation-specific quantita- 3-phosphate dehydrogenase (GAPDH) are previously reported tive PCR was set at PMR X10. (Fukuyama et al., 2006). The PCR primers for MCC is as follows: MCC (GenBank accession number, NM_002387), forward, 50-TACGAATCCAATGCCACA-30, reverse, Statistics 50-AGCTTCATGAGCAGGGCCTT-30 (248 bp). cDNA Statistical analysis was carried out with one-way analysis of concentrations for each sample were normalized by using variance with Bonferroni’s multiple correction using Statview. GAPDH as a control gene. Numbers of samples were n ¼ 3 þ s.d. for cell proliferation, dual luciferase and in vitro migration. ** and *** indicate Immunostaining Po0.001 and 0.0001, respectively. The Fisher’s exact test was Cells were grown on glass coverslips to 50–60% confluency. used to evaluate differences in the contingency table with the Prior to staining, cells were washed two times with phosphate- two-tailed P-value reported. The level for statistical signifi- buffered saline (PBS), fixed for 10 min in methanol. Cells were cance was set at Pp0.05. permeabilized with 0.1% Triton X-100 in PBS for 10 min. After blocking with 1% bovine serum albumin (BSA) in PBS for 60 min, the samples were stained with the indicated Acknowledgements antibody in 1% BSA/PBS for 2 h at room temperature. After 3 Â washing with PBS, the samples were stained with the We thank Dr Kenneth W Kinzler, Dr Kazuo Maruyama, Dr appropriate fluorescein isothiocyanate-conjugated secondary Inder Verma, Dr Bert Vogelstein and Dr Bryan Williams for antibody (Invitrogen Corp.) for 1 h at room temperature. the various reagents used for this work as well as Lisa Nuclei were stained with 40,6-diamidino-2-phenylindole con- Krumroy and Alona Merkulova for technical assistance. This tained in mounting solution (Vector Laboratories, Burlin- work was supported by grants to NS from the Cleveland Clinic game, CA, USA). Cells were analysed with a Leica confocal Taussig Cancer Center/Scott Hamilton CARES Initiative and microscope (Leica Microsystems, Exton, PA, USA). the National Cancer Institute Grant CA 100748.

References

Agarwal A, Das K, Lerner N, Sathe S, Cicek M, Casey G et al. (2005). Conacci-Sorrell M, Simcha I, Ben-Yedidia T, Blechman J, Savagner P, The AKT/I kappa B kinase pathway promotes angiogenic/ Ben-Ze’ev A. (2003). Autoregulation of E-cadherin expression by metastatic gene expression in colorectal cancer by activating nuclear cadherin-cadherin interactions: the roles of beta-catenin signaling, factor-kappa B and beta-catenin. Oncogene 24: 1021–1031. Slug, and MAPK. J Cell Biol 163: 847–857. Ashton-Rickardt PG, Wyllie AH, Bird CC, Dunlop MG, Steel CM, Eleftheriou A, Yoshida M, Henderson BR. (2001). Nuclear export of Morris RG et al. (1991). MCC, a candidate familial polyposis gene human beta-catenin can occur independent of CRM1 and the in 5q.21, shows frequent allele loss in colorectal and lung cancer. adenomatous polyposis coli tumor suppressor. J Biol Chem 276: Oncogene 6: 1881–1886. 25883–25888. Bouwmeester T, Bauch A, Ruffner H, Angrand PO, Bergamini G, Fukuyama R, Ng KP, Cicek M, Kelleher C, Niculaita R, Casey G Croughton K et al. (2004). A physical and functional map of the et al. (2006). Role of IKK and oscillatory NFkappaB kinetics in human TNF-alpha/NF-kappa B signal transduction pathway. Nat MMP-9 gene expression and chemoresistance to 5-ﬂuorouracil in Cell Biol 6: 97–105. RKO colorectal cancer cells. Mol Carcinog 46: 402–413.

Oncogene MCC inhibits b-catenin-dependent transcription R Fukuyama et al 6055 Gregorieff A, Clevers H. (2005). Wnt signaling in the intestinal experience of a six-laboratory consortium. Cancer Biomarkers 2: epithelium: from endoderm to cancer. Genes Dev 19: 877–890. 5–9. Greten FR, Eckmann L, Greten TF, Park JM, Li ZW, Egan LJ et al. Luo JL, Maeda S, Hsu LC, Yagita H, Karin M. (2004). Inhibition of (2004). IKKbeta links inflammation and tumorigenesis in a mouse NF-kappaB in cancer cells converts inflammation- induced tumor model of colitis-associated cancer. Cell 118: 285–296. growth mediated by TNFalpha to TRAIL-mediated tumor regres- Groden J, Thliveris A, Samowitz W, Carlson M, Gelbert L, Albertsen sion. Cancer Cell 6: 297–305. H et al. (1991). Identification and characterization of the familial Matsumine A, Senda T, Baeg GH, Roy BC, Nakamura Y, Noda M adenomatous polyposis coli gene. Cell 66: 589–600. et al. (1996). MCC, a cytoplasmic protein that blocks cell Henderson BR. (2000). Nuclear-cytoplasmic shuttling of APC cycle progression from the G0/G1 to S phase. J Biol Chem 271: regulates beta-catenin subcellular localization and turnover. Nat 10341–10346. Cell Biol 2: 653–660. Nishisho I, Nakamura Y, Miyoshi Y, Miki Y, Ando H, Horii A et al. Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB. (1996). (1991). Mutations of chromosome 5q21 genes in FAP and colorectal Methylation-specific PCR: a novel PCR assay for methylation status cancer patients. Science 253: 665–669. of CpG islands. Proc Natl Acad Sci USA 93: 9821–9826. Nourry C, Grant SG, Borg JP. (2003). PDZ domain proteins: plug and Heyer J, Yang K, Lipkin M, Edelmann W, Kucherlapati R. (1999). play! Sci STKE. http://stke.sciencemag.org/cgi/content/full/sig- Mouse models for colorectal cancer. Oncogene 18: 5325–5333. trans;2003/179/re7. Ishikawa S, Kobayashi I, Hamada J, Tada M, Hirai A, Furuuchi K Ogino S, Kawasaki T, Kirkner GJ, Loda M, Fuchs CS. (2006). CpG et al. (2001). Interaction of MCC2, a novel homologue of MCC island methylator phenotype-low (CIMP-low) in colorectal cancer: tumor suppressor, with PDZ-domain protein AIE-75. Gene 267: possible associations with male sex and KRAS mutations. J Mol 101–110. Diagn 8: 582–588. Ishitani T, Ninomiya-Tsuji J, Matsumoto K. (2003). Regulation of Prall F, Ostwald C. (2007). High-degree tumor budding and podia- lymphoid enhancer factor 1/T-cell factor by mitogen-activated formation in sporadic colorectal carcinomas with K-ras gene protein kinase-related Nemo-like kinase-dependent phosphorylation mutations. Hum Pathol 38: 1696–1702. in Wnt/beta-catenin signaling. Mol Cell Biol 23: 1379–1389. Rosin-Arbesfeld R, Townsley F, Bienz M. (2000). The APC tumour Ishitani T, Ninomiya-Tsuji J, Nagai S, Nishita M, Meneghini M, suppressor has a nuclear export function. Nature 406: 1009–1012. Barker N et al. (1999). The TAK1-NLK-MAPK-related pathway Senda T, Matsumine A, Yanai H, Akiyama T. (1999). Localization of antagonizes signalling between beta-catenin and transcription factor MCC (mutated in colorectal cancer) in various tissues of mice and TCF. Nature 399: 798–802. its involvement in cell differentiation. J Histochem Cytochem 47: Jass JR. (2005). Serrated adenoma of the colorectum and the DNA- 1149–1158. methylator phenotype. Nat Clin Pract Oncol 2: 398–405. Shinto E, Tsuda H, Ueno H, Hashiguchi Y, Hase K, Tamai S et al. Jass JR. (2007). Classification of colorectal cancer based on correlation (2005). Prognostic implication of laminin-5 gamma 2 chain of clinical, morphological and molecular features. Histopathology expression in the invasive front of colorectal cancers, disclosed by 50: 113–130. area-specific four-point tissue microarrays. Lab Invest 85: 257–266. Jass JR, Barker M, Fraser L, Walsh MD, Whitehall VL, Gabrielli B Sizemore N, Lerner N, Dombrowski N, Sakurai H, Stark GR. (2002). et al. (2003). APC mutation and tumour budding in colorectal Distinct roles of the Ikappa B kinase alpha and beta subunits in cancer. J Clin Pathol 56: 69–73. liberating nuclear factor kappa B (NF-kappa B) from Ikappa B and Jass JR, Whitehall VL, Young J, Leggett BA. (2002). Emerging in phosphorylating the p65 subunit of NF-kappa B. J Biol Chem concepts in colorectal neoplasia. Gastroenterology 123: 862–876. 277: 3863–3869. Kinzler KW, Nilbert MC, Su LK, Vogelstein B, Bryan TM, Levy DB Weisenberger DJ, Siegmund KD, Campan M, Young J, et al. (1991a). Identification of FAP locus genes from chromosome Long TI, Faasse MA et al. (2006). CpG island methylator 5q21. Science 253: 661–665. phenotype underlies sporadic microsatellite instability and is tightly Kinzler KW, Nilbert MC, Vogelstein B, Bryan TM, Levy DB, Smith associated with BRAF mutation in colorectal cancer. Nat Genet 38: KJ et al. (1991b). Identification of a gene located at chromosome 787–793. 5q21 that is mutated in colorectal cancers. Science 251: 1366–1370. Zhang F, White RL, Neufeld KL. (2000). Phosphorylation near Kohonen-Corish MR, Sigglekow ND, Susanto J, Chapuis PH, Bokey nuclear localization signal regulates nuclear import of adenomatous EL, Dent OF et al. (2007). Promoter methylation of the mutated in polyposis coli protein. Proc Natl Acad Sci USA 97: 12577–12582. colorectal cancer gene is a frequent early event in colorectal cancer. Zlobec I, Lugli A, Baker K, Roth S, Minoo P, Hayashi S et al. Oncogene 26: 4435–4441. (2007). Role of APAF-1, E-cadherin and peritumoral lymphocytic Lindor NM, Smalley R, Barker M, Bigler J, Krumroy LM, Lum-Jones infiltration in tumour budding in colorectal cancer. J Pathol 212: A et al. (2006). Ascending the learning curve—MSI testing 260–268.

Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

Oncogene