Indian Hedgehog is an antagonist of Wnt signaling in colonic epithelial cell differentiation.

Wnt signaling defines the colonic epithelial progenitor cell phenotype, and mutations in the gene adenomatous polyposis coli (APC) that activate the Wnt pathway cause the familial adenomatous polyposis coli (FAP) syndrome and most sporadic colon cancers. The mechanisms that regulate the transition of epithelial precursor cells into their differentiated derivatives are poorly characterized. We report that Indian hedgehog (Ihh) is expressed by mature colonocytes and regulates their differentiation in vitro and in vivo. Hedgehog (Hh) signaling restricts the expression of Wnt targets to the base of the colonic crypt in vivo, and transfection of Ihh into colon cancer cells leads to a downregulation of both components of the nuclear TCF4-beta-catenin complex and abrogates endogenous Wnt signaling in vitro. In turn, expression of Ihh is downregulated in polyps of individuals with FAP and expression of doxycycline-inducible dominant negative TCF4 (dnTCF4) restores Ihh expression in APC mutant DLD-1 colon cancer cells. These data identify a new Wnt-Hh axis in colonic epithelial renewal.

The bone morphogenetic protein pathway is inactivated in the majority of sporadic colorectal cancers.

BACKGROUND & AIMS: The finding of bone morphogenetic protein (BMP) receptor 1a mutations in juvenile polyposis suggests that BMPs are important in colorectal cancer (CRC). We investigated the BMP pathway in sporadic CRC. METHODS: We investigated BMP receptor (BMPR) expression using immunoblotting and sequenced BMPR2 in CRC cell lines. We assessed the expression of BMPRs, SMAD4, and pSMAD1/5/8 in 72 sporadic CRCs using a tissue microarray and immunohistochemistry. We assessed the effect of reintroduction of wild-type BMPR2 on BMP pathway activity and the effect of wild-type or mutated BMPR2 3' untranslated region (UTR) sequences on protein expression by attachment to pCMV-Luc. RESULTS: BMPR2 and SMAD4 protein expression is abrogated in microsatellite unstable (MSI) and microsatellite stable (MSS) cell lines, respectively. BMPR2 3'UTR is mutated in all MSI and in none of the MSS cell lines. Mutant BMPR2 3'UTR sequences reduced luciferase expression 10-fold compared with wild-type BMPR2 3'UTR. BMPR2 expression is impaired more frequently in MSI CRCs than MSS (85% vs 29%; P < .0001) and shows a mutually exclusive pattern of impaired expression compared with SMAD4. Nine of 11 MSI cancers with impaired expression of BMPR2 have microsatellite mutations. The BMP pathway is inactivated, as judged by nuclear pSMAD1/5/8 expression, in 70% of CRCs, and this correlates with BMPR and SMAD4 loss. CONCLUSIONS: Our data suggest that the BMP pathway is inactivated in the majority of sporadic CRCs. In MSI CRC this is associated predominantly with impaired BMPR2 expression and in MSS CRC with impaired SMAD4 expression.

Bone morphogenetic protein signaling suppresses tumorigenesis at gastric epithelial transition zones in mice.

Bone morphogenetic protein (BMP) signaling is known to suppress oncogenesis in the small and large intestine of mice and humans. We examined the role of Bmpr1a signaling in the stomach. On conditional inactivation of Bmpr1a, mice developed neoplastic lesions specifically in the squamocolumnar and gastrointestinal transition zones. We hypothesized that the regulation of epithelial cell fate may be less well defined in these junctional zones than in the adjacent epithelium and found that the mucosa at the squamocolumnar junction in mice shows a lack of differentiated fundic gland cell types and that foveolar cells at the gastrointestinal junctional zone lack expression of the foveolar cell marker Muc5ac. Precursor cell proliferation in both transition zones was higher than in the surrounding epithelium. Our data show that BMP signaling through Bmpr1a suppresses tumorigenesis at gastric epithelial junctional zones that are distinct from the adjacent gastric epithelium in both cellular differentiation and proliferation.

Tetrahydroxyquinone induces apoptosis of leukemia cells through diminished survival signaling.

OBJECTIVE: Tetrahydroxyquinone is a molecule best known as a primitive anticataract drug but is also a highly redox active molecule that can take part in a redox cycle with semiquinone radicals, leading to the formation of reactive oxygen species (ROS). Its potential as an anticancer drug has not been investigated. METHODS: The effects of tetrahydroxyquinone on HL60 leukemia cells are investigated using fluorescein-activated cell sorting-dependent detection of phosphatidylserine exposure combined with 7-amino-actinomycin D exclusion, via Western blotting using phosphospecific antibodies, and by transfection of constitutively active protein kinase B. RESULTS: We observe that in HL60 leukemia cells tetrahydroxyquinone causes ROS production followed by apoptosis through the mitochondrial pathway, whereas cellular physiology of normal human blood leukocytes was not affected by tetrahydroxyquinone. The antileukemic effect of tetrahydroxyquinone is accompanied by reduced activity of various antiapoptotic survival molecules including the protein kinase B pathway. Importantly, transfection of protein kinase B into HL60 cells and thus artificially increasing protein kinase B activity inhibits tetrahydroxyquinone-dependent cytotoxicity. CONCLUSION: Tetrahydroxyquinone provokes cytotoxic effects on leukemia cells by reduced protein kinase B-dependent survival signaling followed by apoptosis through the mitochondrial pathway. Thus, tetrahydroxyquinone may be representative of a novel class of chemotherapeutic drugs, inducing apoptosis in cancer cells through diminished survival signaling possibly as a consequence of ROS generation.

The bone morphogenetic protein pathway is active in human colon adenomas and inactivated in colorectal cancer.

BACKGROUND: Transforming growth factor beta (TGFbeta) is important in colorectal cancer (CRC) progression. Bone morphogenetic proteins (BMPs), a subgroup within the TGFbeta superfamily, recently also have been implicated in CRC, but their precise role in CRC has yet to be investigated. METHODS: The authors used a tissue microarray and immunohistochemistry of BMP receptors and signal transduction elements in adenomas and CRC specimens to elucidate the role of BMP signaling in CRC carcinogenesis. RESULTS: The adenoma specimens expressed all 3 BMP receptors (BMPRs) (BMPR type 1a [BMPR1a], BMPR1b, and BMPR2) and expressed SMAD family member 4 (SMAD4); and 20 of 22 adenomas (90.9%) exhibited active BMP signaling, as determined by nuclear phosphorylated SMAD1,5,8 (pSMAD1,5,8) expression. In contrast, pSMAD1,5,8 nuclear staining was present in 5 CRC specimens (22.7%) but was lost in 17 CRC specimens (77.3%; cancer vs adenoma; P< .0001). The earliest loss of pSMAD1,5,8 nuclear staining was detected in regions of high-grade dysplasia/carcinoma in situ within adenomas. CRCs showed frequent loss of BMPR2 (P< .0001) and SMAD4 (P< .01) compared with adenomas. Negative expression of BMPR2 was observed more frequently in earlier stage cancers (Dukes stage B) than in advanced cancers (Dukes stage C; P< .05). CONCLUSIONS: Taken together, the current results indicated that loss of BMP signaling correlates tightly with progression of adenomas to cancer and occurs relatively early during cancer progression.

The effect of statins in colorectal cancer is mediated through the bone morphogenetic protein pathway.

BACKGROUND & AIMS: Epidemiological evidence suggests that statins prevent colorectal cancer (CRC), but the biological mechanism remains obscure. Statins induce bone morphogenetic protein (BMP) expression in bone cells. We have previously shown that BMPs act as tumor suppressors in CRC. We hypothesized that the action of statins in CRC involves the induction of BMPs. METHODS: We investigated the effects of statins on CRC cell lines using immunoblotting, measurements of apoptosis and cell proliferation, and luciferase reporter assays. The effect of statins was confirmed in a xenograft mouse model. RESULTS: CRC cell lines show widely differing sensitivities to statin treatment. Sensitive cell lines show induction of BMP2 protein levels and a BMP2 reporter construct, activation of the BMP pathway, and induction of the BMP target gene ID-2, whereas resistant cell lines do not. The addition of the specific inhibitor of BMPs, noggin, completely prevents lovastatin-induced apoptosis in sensitive cells. Sensitive cell lines express the central BMP pathway element SMAD4, whereas the resistant cell lines do not. Targeted knockout of SMAD4 leads to the loss of statin sensitivity and reconstitution with SMAD4, to the restoration of statin sensitivity. In a xenograft mouse model, tumors from sensitive and insensitive cell lines were treated with oral simvastatin. Significant inhibition of tumor growth using sensitive cells but increased tumor growth when using insensitive cells was observed. CONCLUSIONS: Statins induce apoptosis in CRC cells through induction of BMP2. Statin therapy may only be effective in SMAD4-expressing CRCs and may have adverse effects in SMAD4-negative tumors.

Bone morphogenetic protein 2 is expressed by, and acts upon, mature epithelial cells in the colon.

BACKGROUND AND AIMS: The recent findings of bone morphogenetic protein (BMP) receptor Ia mutations in juvenile polyposis and frequent Smad4 mutations in colon cancer suggest a role for BMPs in the colonic epithelium and colon cancer. We investigated the role of BMP2 in the colon. METHODS: We assessed BMP receptor expression in cell lines using the reverse-transcribed polymerase chain reaction and immunoblotting. We investigated the effect of BMP2 on cell lines using the MTT assay and by immunoblotting for markers of differentiation, proliferation, and apoptosis. We assessed the expression of BMP2, its receptors, and signal transduction elements in mouse and human colon tissue using immunohistochemistry. We also investigated the effect of the BMP antagonist noggin in vivo in mice by assessing colon tissue with immunohistochemistry and immunoblotting. Finally, we investigated the expression of BMP2 in microadenomas from familial adenomatous polyposis patients. RESULTS: BMP receptors (BMPR) Ia, BMPR Ib, and BMPR II are all expressed in colonic epithelial cell lines. BMP2 inhibits colonic epithelial cell growth in vitro, promoting apoptosis and differentiation and inhibiting proliferation. BMP2, BMPRIa, BMPRIb, BMPRII, phosphorylated Smad1, and Smad4 are expressed predominantly in mature colonocytes at the epithelial surface in normal adult human and mouse colon. Noggin inhibits apoptosis and proliferation in mouse colonic epithelium in vivo. BMP2 expression is lost in the microadenomas of familial adenomatous polyposis patients. CONCLUSIONS: These data suggest that BMP2 acts as a tumor suppressor promoting apoptosis in mature colonic epithelial cells.

Tumor suppressor pRB functions as a co-repressor of the CCAAT displacement protein (CDP/cut) to regulate cell cycle controlled histone H4 transcription.

The CCAAT displacement protein (CDP-cut/CUTL1/cux) performs a key proliferation-related function as the DNA binding subunit of the cell cycle controlled HiNF-D complex. HiNF-D interacts with all five classes (H1, H2A, H2B, H3, and H4) of the cell-cycle dependent histone genes, which are transcriptionally and coordinately activated at the G(1)/S phase transition independent of E2F. The tumor suppressor pRB/p105 is an intrinsic component of the HiNF-D complex. However, the molecular interactions that enable CDP and pRB to form a complex and thus convey cell growth regulatory information onto histone gene promoters must be further defined. Using transient transfections, we show that CDP represses the H4 gene promoter and that pRB functions with CDP as a co-repressor. Direct physical interaction between CDP and pRB was observed in glutathione-S-transferase (GST) pull-down assays. Furthermore, interactions between these proteins were established by yeast and mammalian two-hybrid experiments and co-immunoprecipitation assays. Confocal microscopy shows that subsets of each protein are co-localized in situ. Using a series of pRB mutants, we find that the CDP/pRB interaction, similar to the E2F/pRB interaction, utilizes the A/B large pocket (LP) of pRB. Thus, several converging lines of evidence indicate that complexes between CDP and pRB repress cell cycle regulated histone gene promoters.