OBJECTIVES To investigate patterns of programmed death protein-1 (PD-L1) expression in microsatellite instability (MSI)-high intestinal carcinomas and correlate them with pathologic and molecular features. METHODS One hundred and fifteen MSI-high and 41 microsatellite stable carcinomas were included. Tumor sections were immunohistochemically labeled for PD-L1. The results were correlated with histologic subtypes, MSI, and BRAF status. RESULTS As expected, MSI status was associated with PD-L1 expression. Among 115 MSI-high tumors, PD-L1 expression was observed on tumor cells in 28 tumors and on tumor-associated inflammatory cells in 77 tumors. Medullary carcinoma demonstrated more frequent PD-L1 expression on tumor cells than mucinous and typical adenocarcinoma. PD-L1 expression was more frequent in medullary and typical adenocarcinoma than in mucinous adenocarcinoma based on combined positive scores. Tumors with more nucleotide shifts by PCR-based MSI testing were more likely to express PD-L1. CONCLUSIONS Expression of PD-L1 is different among different histologic subtypes of MSI-high intestinal carcinomas.

Primary central nervous system lymphoma (PCNSL) patients have a poorer prognosis than systemic lymphoma. Gain-of-function MYD88 c.794T > C (p. L265P) mutation and programed cell death-1 (PD-1) pathway alterations are potential targetable pathways. Our study objective was to determine the clinicopathologic correlates of MYD88 mutation and PD-1 alterations in PCNSL and the impact of Epstein-Barr virus (EBV) infection. We studied 53 cases including 13 EBV-associated (EBVpos) PCNSL, 49% harbored MYD88 mutation, none seen in EBVpos PCNSL. MYD88 protein expression did not correlate with MYD88 mutation. T-cell and macrophage infiltration was common. All PD-L1-positive tumors were EBVpos. Two PD-L1 positive tumors showed 9p24.1/PD-L1 locus alterations by Fluorescence In Situ Hybridization. T cells and macrophages expressed PD-1 and/or PD-L1 in 98% and 83% cases, respectively. MYD88 mutation or protein expression and PD-1 or PD-L1 expression did not predict outcome. We hypothesize that EBVpos PCNSL has a distinct activation mechanism, independent of genetic alterations.

Elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) are common in colorectal cancers (CRCs). The association between EMAST and classic mono/dinucleotide microsatellite instability (MSI) is unknown. We assessed the stability of 13 tetranucleotide and three pentanucleotide repeat markers in tumor and normal tissue from 22 MSI-high and 107 microsatellite-stable CRC samples. When present, instability was observed at tetra/pentanucleotide repeats and was defined as elevated microsatellite alterations at selected tetra/pentanucleotide repeats-high (EMASTP-H; ≥30% instability), -low (EMASTP-L; <30% instability), or -stable (EMASTP-S). EMASTP instability, including high and low, was observed in 50 of 123 CRCs (41%), including all MSI-high tumors and 28 of 101 microsatellite-stable tumors (28%). MSI-high CRCs were more likely to be EMASTP-H compared with microsatellite-stable tumors with EMASTP instability. Tetranucleotide markers VWA and D13S317 were the two most frequently altered loci. Loss of heterozygosity was more common in EMASTP-L/S than in EMASTP-H CRCs. Frequencies of loss of heterozygosity at three loci were different between EMASTP-L and EMASTP-S tumors. In addition, right-sided tumor site, large tumor size, high tumor grade, and the presence of Crohn-like reaction were significantly associated with EMASTP-H CRCs. However, there were no differences in clinicopathologic features between EMASTP-L and EMASTP-S tumors. In summary, more CRCs exhibited genomic instability as EMASTP than as MSI. EMASTP instability may prove to be an important prognostic/therapeutic indicator in CRCs.

We previously reported that single cells from a human colorectal cancer (CRC) cell line (HCA-7) formed either hollow single-layered polarized cysts or solid spiky masses when plated in 3D in type-I collagen. To begin in-depth analyses into whether clonal cysts and spiky masses possessed divergent properties, individual colonies of each morphology were isolated and expanded. The lines thus derived faithfully retained their parental cystic and spiky morphologies and were termed CC (cystic) and SC (spiky), respectively. Although both CC and SC expressed EGF receptor (EGFR), the EGFR-neutralizing monoclonal antibody, cetuximab, strongly inhibited growth of CC, whereas SC was resistant to growth inhibition, and this was coupled to increased tyrosine phosphorylation of MET and RON. Addition of the dual MET/RON tyrosine kinase inhibitor, crizotinib, restored cetuximab sensitivity in SC. To further characterize these two lines, we performed comprehensive genomic and transcriptomic analysis of CC and SC in 3D. One of the most up-regulated genes in CC was the tumor suppressor 15-PGDH/HPGD, and the most up-regulated gene in SC was versican (VCAN) in 3D and xenografts. Analysis of a CRC tissue microarray showed that epithelial, but not stromal, VCAN staining strongly correlated with reduced survival, and combined epithelial VCAN and absent HPGD staining portended a poorer prognosis. Thus, with this 3D system, we have identified a mode of cetuximab resistance and a potential prognostic marker in CRC. As such, this represents a potentially powerful system to identify additional therapeutic strategies and disease-relevant genes in CRC and possibly other solid tumors.

Colorectal (CRCs) and endometrioid (EMCs) cancers in patients with Lynch syndrome exhibit microsatellite instability (MSI) detected by PCR or immunohistochemistry (IHC). While both assays are equally sensitive for CRCs, some suggest that PCR has a higher false-negative rate than IHC in EMCs. We assessed the MSI profiles of 91 EMC and 311 CRC specimens using five mononucleotide repeat markers: BAT25, BAT26, NR21, NR24, and MONO27. EMCs with high MSI (MSI-H) showed a mean left shift of 3 nucleotides (nt), which was significantly different from 6 nt in CRCs. A shift of 1 nt was observed in multiple markers in 76% of MSI-H EMCs, whereas only 12% of MSI-H CRCs displayed a 1-nt shift in one of five markers. IHC against four mismatch repair proteins was performed in 78 EMCs. Loss of staining in one or more proteins was detected in 18 of 19 tumors that were MSI-H by PCR. When EMC tumor cell burden was diluted to <30%, MSI-H was no longer observed in two of three EMCs with a mean nucleotide shift of 1 nt. These results indicate that EMC and CRC MSI profiles are different and that caution should be exercised when interpreting the results, as subtle, 1-nt changes may be missed. These findings provide a potential cause of previously reported discordant MSI and IHC results in EMCs.

BACKGROUND & AIMS: Interstitial cells of Cajal (ICC) control intestinal smooth muscle contraction to regulate gut motility. ICC within the plane of the myenteric plexus (ICC-MY) arise from KIT-positive progenitor cells during mouse embryogenesis. However, little is known about the ontogeny of ICC associated with the deep muscular plexus (ICC-DMP) in the small intestine and ICC associated with the submucosal plexus (ICC-SMP) in the colon. Leucine-rich repeats and immunoglobulinlike domains protein 1 (LRIG1) marks intestinal epithelial stem cells, but the role of LRIG1 in nonepithelial intestinal cells has not been identified. We sought to determine the ontogeny of ICC-DMP and ICC-SMP, and whether LRIG1 has a role in their development. METHODS: Lrig1-null mice (homozygous Lrig1-CreERT2) and wild-type mice were analyzed by immunofluorescence and transit assays. Transit was evaluated by passage of orally administered rhodamine B-conjugated dextran. Lrig1-CreERT2 mice or mice with CreERT2 under control of an inducible smooth muscle promoter (Myh11-CreERT2) were crossed with Rosa26-LSL-YFP mice for lineage tracing analysis. RESULTS: In immunofluorescence assays, ICC-DMP and ICC-SMP were found to express LRIG1. Based on lineage tracing, ICC-DMP and ICC-SMP each arose from LRIG1-positive smooth muscle progenitors. In Lrig1-null mice, there was loss of staining for KIT in DMP and SMP regions, as well as for 2 additional ICC markers (anoctamin-1 and neurokinin 1 receptor). Lrig1-null mice had significant delays in small intestinal transit compared with control mice. CONCLUSIONS: LRIG1 regulates the postnatal development of ICC-DMP and ICC-SMP from smooth muscle progenitors in mice. Slowed small intestinal transit observed in Lrig1-null mice may be due, at least in part, to loss of the ICC-DMP population.

Leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) is a pan-ErbB negative regulator and intestinal stem cell marker down-regulated in many malignancies. We previously reported that 14 of 16 Lrig1-CreERT2/CreERT2 (Lrig1(-/-)) mice developed duodenal adenomas, providing the first in vivo evidence that Lrig1 acts as a tumor suppressor. We extended this study to a larger cohort and found that 49 of 54 Lrig1(-/-) mice develop duodenal adenomas beginning at 3 months. Most adenomas were histologically low grade and overlaid expanded Brunner glands. There was morphologic and biochemical blurring of the boundary between the epithelium and Brunner glands with glandular coexpression of ErbB2, which is normally restricted to the epithelium, and the Brunner gland marker Mucin6. Some adenomas were high grade with reduced Brunner glands. At age 4 to 5 weeks, before adenoma formation, we observed enhanced proliferation in Brunner glands and, at 2 months, an increase in the size of the Brunner gland compartment. Elevated expression of the epidermal growth factor receptor (Egfr) ligands amphiregulin and beta-cellulin, as well as Egfr and phosphorylated Egfr, was detected in adenomas compared with adjacent normal tissue. These adenomas expressed the gastric-specific genes gastrokine1 and mucin5ac, indicating gastric metaplasia. Moreover, we found that a subset of human duodenal tumors exhibited features of LRIG1(-/-) adenomas, including loss of LRIG1, gastric metaplasia (MUCIN5AC and MUCIN6), and increased amphiregulin and Egfr activity.

The epithelial-to-mesenchymal transition (EMT) transcriptional program is characterized by repression of E-cadherin (CDH1) and induction of N-cadherin (CDH2), and mesenchymal genes like vimentin (VIM). Placenta-specific 8 (PLAC8) has been implicated in colon cancer; however, how PLAC8 contributes to disease is unknown, and endogenous PLAC8 protein has not been studied. We analyzed zebrafish and human tissues and found that endogenous PLAC8 localizes to the apical domain of differentiated intestinal epithelium. Colon cancer cells with elevated PLAC8 levels exhibited EMT features, including increased expression of VIM and zinc finger E-box binding homeobox 1 (ZEB1), aberrant cell motility, and increased invasiveness. In contrast to classical EMT, PLAC8 overexpression reduced cell surface CDH1 and upregulated P-cadherin (CDH3) without affecting CDH2 expression. PLAC8-induced EMT was linked to increased phosphorylated ERK2 (p-ERK2), and ERK2 knockdown restored cell surface CDH1 and suppressed CDH3, VIM, and ZEB1 upregulation. In vitro, PLAC8 directly bound and inactivated the ERK2 phosphatase DUSP6, thereby increasing p-ERK2. In a murine xenograft model, knockdown of endogenous PLAC8 in colon cancer cells resulted in smaller tumors, reduced local invasion, and decreased p-ERK2. Using MultiOmyx, a multiplex immunofluorescence-based methodology, we observed coexpression of cytosolic PLAC8, CDH3, and VIM at the leading edge of a human colorectal tumor, supporting a role for PLAC8 in cancer invasion in vivo.

Introduction: Recently, it has been shown that cells expressing the glial marker GFAP can make enteric neurons, raising the possibility that enteric glia can function as enteric neural precursor cells (ENPC). However, it is not known whether all or only a sub-population of enteric glia are ENPC. Apart from glial markers, ENPC are also known to express Nestin and p75NTR. Hence, using a Nestin-GFP mouse, we studied whether ENPC, defined by the co-expression of Nestin and p75NTR, express glial cell markers and whether all enteric glial cells express these ENPC markers. Methods: Cells from the longitudinal muscle-myenteric plexus (LMMP) of Nestin-GFP mice were flow sorted into four populations based on expression of Nestin-GFP and p75NTR for clonal proliferation and differentiation assays. Differentiated cells were tested for the presence of neurons and glia using immunocytochemistry. In separate analyses, these four cell populations were analyzed by FACS for the presence, co-localization, and intensity of fluorescence of glial markers GFAP and S100B. Results: By clonal analyses, we observe that only cells that co-express Nestin and p75NTRHI proliferate (Mean ± S.E. of percentage proliferative cells: 10.2 ± 0.62) and differentiate to form neurons and glia (Mean ± S.E. of percentage of neurospheres that differentiate to neurons and glia: 78.26 ± 4.35). ENPC, defined now by Nestin-p75NTRHI co-expression, express the glial markers, GFAP and S100B. We observe that only 3.86% of enteric glial cells that co-express GFAP and S100B express Nestin-GFP and p75HI and hence qualify for ENPC. All other cells are presumably mature enteric glia. Discussion: We show that ENPC co-express Nestin and p75NTRHI apart from GFAP and S100B, confirming earlier results that ENPC express glial markers. However, ENPC only form a small fraction of the total enteric glial cells as only 3.86% of cells that co-express the glial markers express Nestin-GFP and p75NTRHI. Hence, ENPC either represent a sub-category of enteric glial cells or are non-glial cells expressing shared markers. Our data shows that not all enteric glia are ENPC. This work paves the way for understanding not only the true identity of ENPC, but also that of mature enteric glia.

Introduction: Recently, it has been shown that cells expressing the glial marker GFAP can make enteric neurons, raising the possibility that enteric glia can function as enteric neural precursor cells (ENPC). However, it is not known whether all or only a sub-population of enteric glia are ENPC. Apart from glial markers, ENPC are also known to express Nestin and p75NTR. Hence, using a Nestin-GFP mouse, we studied whether ENPC, defined by the co-expression of Nestin and p75NTR, express glial cell markers and whether all enteric glial cells express these ENPC markers. Methods: Cells from the longitudinal muscle-myenteric plexus (LMMP) of Nestin-GFP mice were flow sorted into four populations based on expression of Nestin-GFP and p75NTR for clonal proliferation and differentiation assays. Differentiated cells were tested for the presence of neurons and glia using immunocytochemistry. In separate analyses, these four cell populations were analyzed by FACS for the presence, co-localization, and intensity of fluorescence of glial markers GFAP and S100B. Results: By clonal analyses, we observe that only cells that co-express Nestin and p75NTRHI proliferate (Mean ± S.E. of percentage proliferative cells: 10.2 ± 0.62) and differentiate to form neurons and glia (Mean ± S.E. of percentage of neurospheres that differentiate to neurons and glia: 78.26 ± 4.35). ENPC, defined now by Nestin-p75NTRHI co-expression, express the glial markers, GFAP and S100B. We observe that only 3.86% of enteric glial cells that co-express GFAP and S100B express Nestin-GFP and p75HI and hence qualify for ENPC. All other cells are presumably mature enteric glia. Discussion: We show that ENPC co-express Nestin and p75NTRHI apart from GFAP and S100B, confirming earlier results that ENPC express glial markers. However, ENPC only form a small fraction of the total enteric glial cells as only 3.86% of cells that co-express the glial markers express Nestin-GFP and p75NTRHI. Hence, ENPC either represent a sub-category of enteric glial cells or are non-glial cells expressing shared markers. Our data shows that not all enteric glia are ENPC. This work paves the way for understanding not only the true identity of ENPC, but also that of mature enteric glia.

Transcriptional mechanisms governing hematopoietic stem cell (HSC) quiescence, self-renewal, and differentiation are not fully understood. Sequence-specific ssDNA-binding protein 2 (SSBP2) is a candidate acute myelogenous leukemia (AML) suppressor gene located at chromosome 5q14. SSBP2 binds the transcriptional adaptor protein Lim domain-binding protein 1 (LDB1) and enhances LDB1 stability to regulate gene expression. Notably, Ldb1 is essential for HSC specification during early development and maintenance in adults. We previously reported shortened lifespan and greater susceptibility to B cell lymphomas and carcinomas in Ssbp2(-/-) mice. However, whether Ssbp2 plays a regulatory role in normal HSC function and leukemogenesis is unknown. In this study, we provide several lines of evidence to demonstrate a requirement for Ssbp2 in the function and transcriptional program of hematopoietic stem and progenitor cells (HSPCs) in vivo. We found that hematopoietic tissues were hypoplastic in Ssbp2(-/-) mice, and the frequency of lymphoid-primed multipotent progenitor cells in bone marrow was reduced. Other significant features of these mice were delayed recovery from 5-fluorouracil treatment and diminished multilineage reconstitution in lethally irradiated bone marrow recipients. Dramatic reduction of Notch1 transcripts and increased expression of transcripts encoding the transcription factor E2a and its downstream target Cdkn1a also distinguished Ssbp2(-/-) HSPCs from wild-type HSPCs. Finally, a tendency toward coordinated expression of SSBP2 and the AML suppressor NOTCH1 in a subset of the Cancer Genome Atlas AML cases suggested a role for SSBP2 in AML pathogenesis. Collectively, our results uncovered a critical regulatory function for SSBP2 in HSPC gene expression and function.

Lrig1 is an intestinal stem cell marker important for epithelial homeostasis. However, the position of the Lrig1+ population in the intestinal crypt has been debated, largely due to discrepant staining patterns using two Lrig1 antibodies. Here, we set out to decipher the differences between these Lrig1 antibodies to clarify their use for Lrig1-related studies. We confirmed that the commercially available Lrig1-R&D antibody stained the bottom third of the colonic crypt, whereas an independently generated Lrig1-VU antibody recognized a subset of anti-Lrig1-R&D+ cells. Biochemically, we found that anti-Lrig1-VU recognized a non-glycosylated form of Lrig1; in contrast, anti-Lrig1-R&D recognized both glycosylated and non-glycosylated forms of Lrig1. In addition, we generated a reporter mouse (Lrig1-Apple) as an independent readout of Lrig1 transcriptional activity. Flow cytometry of isolated colonic epithelial cells from Lrig1-Apple mice demonstrated anti-Lrig1-R&D recognized mostly RFP-hi cells, while anti-Lrig1-VU recognized cells that were largely RFP-mid. Of note, by qRT-PCR, Lgr5 was expressed in the RFP-hi population, but not in the RFP-mid population. We conclude that anti-Lrig1-R&D appears to recognize all Lrig1+ cells, while anti-Lrig1-VU recognizes a subpopulation of Lrig1+ cells.

Lineage mapping has identified both proliferative and quiescent intestinal stem cells, but the molecular circuitry controlling stem cell quiescence is incompletely understood. By lineage mapping, we show Lrig1, a pan-ErbB inhibitor, marks predominately noncycling, long-lived stem cells that are located at the crypt base and that, upon injury, proliferate and divide to replenish damaged crypts. Transcriptome profiling of Lrig1+ colonic stem cells differs markedly from the profiling of highly proliferative, Lgr5+ colonic stem cells; genes upregulated in the Lrig1+ population include those involved in cell cycle repression and response to oxidative damage. Loss of Apc in Lrig1+ cells leads to intestinal adenomas, and genetic ablation of Lrig1 results in heightened ErbB1-3 expression and duodenal adenomas. These results shed light on the relationship between proliferative and quiescent intestinal stem cells and support a model in which intestinal stem cell quiescence is maintained by calibrated ErbB signaling with loss of a negative regulator predisposing to neoplasia.

Whether proliferative and quiescent intestinal stem cells coexist in normal and cancerous tissue is controversial. Due to heightened transcript expression in the intestinal epithelial stem cell compartment and detection in quiescent hair follicle stem cells, we hypothesized that Leucine-rich repeats and immunoglobulin-like domain protein 1 (Lrig1) would mark a novel population of intestinal stem cells and may be important for intestinal homeostasis and in cancer. To this end, we generated Lrig1-CreERT2/+; R26R-LacZ mice for Lrig1 intestinal epithelial cell lineage analysis. Examining adult mice at baseline and after irradiation damage, we show that Lrig1 labels both quiescent and proliferative stem cells and these cells are distinct from the well-defined Lgr5-expressing proliferative stem cell population. In addition, in a regenerative response to gut injury, these singly-labeled Lrig1-expressing cells proliferated and gave rise to clusters of labeled daughter cells, demonstrating the ability of these quiescent stem cells to become activated. Interestingly, loss of Lrig1 resulted in heightened expression of ErbB1-3 in the normal intestine, duodenal adenomas and carcinoma, supporting a role for Lrig1 in the maintenance of intestinal epithelial homeostasis and its ability to act as a tumor suppressor. To directly compare the role of the Lrig1and Lgr5expressing cell populations in tumorigenesis, we used both Lrig1-CreERT2/+;Apcfl/fl and Lgr5-EGFP-IRES-CreERT2;Apcfl/fl mice to delete Apc. Five days after deletion, Lrig1CreERT2/+;Apcfl/fl mice have increased duodenal size and marked histological changes, which are absent in the Lgr5-EGFP-IRES-CreERT2;Apcfl/fl mice, further supporting a distinct and important role for the Lrig1 population in maintaining intestinal homeostasis. Finally, stochastic loss of Apc in Lrig1-expressing cells in Lrig1-CreERT2/+;Apcfl/+ mice results in multiple, large distal colonic tumors and less frequent, smaller intestinal tumorscommon features of human familial polyposis4.5 months after Cre activation. In summary, we show that Lrig1 is a distinct marker of both proliferative and quiescent intestinal stem cells, is important for intestinal homeostasis and acts as a tumor suppressor.

Abstract Whether proliferative and quiescent intestinal stem cells coexist in normal and cancerous tissue is controversial. Due to heightened transcript expression in the intestinal epithelial stem cell compartment, detection in quiescent hair follicle stem cells and frequent loss in breast cancer, we hypothesized that Leucine-rich repeats and immunoglobulin-like domain protein 1 (Lrig1) would mark a novel population of intestinal stem cells and may be important for intestinal homeostasis and in cancer. To study this, we generated Lrig1-CreERT2/+; R26R-LacZ mice to study the role of Lrig1. One day after Lrig1-CreERT2/+; R26R-LacZ mice received a single injection of tamoxifen, 1-2 cells were labeled in the progenitor zone. Three months after injection, 10% of colonic crypts were entirely labeled and 8% of crypts contained singly labeled, non-proliferative, Lgr5-negative cells at the crypt-base. In a regenerative response to gut injury, these singly labeled cells proliferated and gave rise to clusters of labeled daughter cells, demonstrating the ability of these quiescent stem cells to become activated. Loss of Lrig1 resulted in heightened expression of ErbB1-3 in the normal intestine, duodenal adenomas and carcinoma, supporting a functional role for Lrig1 in the maintenance of intestinal epithelial homeostasis and its ability to act as a tumor suppressor. In addition, driving stochastic loss of Apc in Lrig1-expressing cells results in multiple, large distal colonic tumors and less frequent, smaller intestinal tumors, which are common features of human familial polyposis. In summary, we show that Lrig1, a cell surface negative regulator of ErbB1-4, marks both proliferative and quiescent intestinal stem cells and acts as a tumor suppressor. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5188. doi:10.1158/1538-7445.AM2011-5188

Background: Truncating mutations in the tumor suppressor gene APC (Adenomatous Polyposis Coli) are thought to initiate the majority of colorectal cancers. The 15- and 20-amino acid repeat regions of APC bind beta-catenin and have been widely studied for their role in the negative regulation of canonical Wnt signaling. However, functions of APC in other important cellular processes, such as cell cycle control or aneuploidy, are only beginning to be studied. Our previous investigation implicated the 15- amino acid repeat region of APC (M2-APC) in the regulation of the G2/M cell cycle transition through interaction with topoisomerase II alpha (topo II alpha).Methodology/Principal Findings: We now demonstrate that the 20-amino acid repeat region of APC (M3-APC) also interacts with topo II alpha in colonic epithelial cells. Expression of M3-APC in cells with full-length endogenous APC causes cell accumulation in G2. However, cells with a mutated topo II alpha isoform and lacking topo II beta did not arrest, suggesting that the cellular consequence of M2- or M3-APC expression depends on functional topoisomerase II. Both purified recombinant M2- and M3-APC significantly enhanced the activity of topo II alpha. Of note, although M3-APC can bind beta-catenin, the G2 arrest did not correlate with beta-catenin expression or activity, similar to what was seen with M2- APC. More importantly, expression of either M2- or M3-APC also led to increased aneuploidy in cells with full-length endogenous APC but not in cells with truncated endogenous APC that includes the M2- APC region.Conclusions/Significance: Together, our data establish that the 20-amino acid repeat region of APC interacts with topo II alpha to enhance its activity in vitro, and leads to G2 cell cycle accumulation and aneuploidy when expressed in cells containing full-length APC. These findings provide an additional explanation for the aneuploidy associated with many colon cancers that possess truncated APC.