Tumor Suppressor Smad4 Research Articles

Abstract 1908: 19q13.2 amplification occurs in a subset of SMAD4-deficient pancreatic cancers.

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease. The PDAC genomes show evidence of high genomic instability leading to complex genomic rearrangements and copy number alterations. Major genetic events involved in PDAC progression have been characterized, but the subsequent alterations that contribute to different tumor behavior or patient outcomes are not well understood. Smad4 tumor suppressor gene mutation or deletion in pancreatic cancer correlates with metastatic disease and poorer patient outcome. We found that in a subset of PDACs with Smad4 expression loss, the chromosomal region 19q13.2 is focally amplified. Genomic amplifications found in cancers have been shown to contain driver genes that contribute to tumorigenesis. We are currently investigating the contribution of 19q13.2 amplicon resident genes in pancreatic cancer cell growth. Our characterization of a cancer amplicon in relation to known major genetic events in pancreatic cancer revealed that the 19q13.2 amplicon is closely associated with a subset of SMAD4-deficient tumors. Through additional in-depth tumor analyses we may better understand the heterogeneity and complexity of PDAC biology. Citation Format: Bosun Min, Albert Lai, Nabeel Bardeesy, Vikram Deshpande. 19q13.2 amplification occurs in a subset of SMAD4-deficient pancreatic cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1908. doi:10.1158/1538-7445.AM2013-1908

Genomic profiles and CRTC1–MAML2 fusion distinguish different subtypes of mucoepidermoid carcinoma

Mucoepidermoid carcinoma is the most common salivary gland malignancy, and includes a spectrum of lesions ranging from non-aggressive low-grade tumors to aggressive high-grade tumors. To further characterize this heterogeneous group of tumors we have performed a comprehensive analysis of copy number alterations and CRTC1–MAML2 fusion status in a series of 28 mucoepidermoid carcinomas. The CRTC1–MAML2 fusion was detected by RT-PCR or fluorescence in situ hybridization in 18 of 28 mucoepidermoid carcinomas (64%). All 15 low-grade tumors were fusion-positive whereas only 3 of 13 high-grade tumors were fusion-positive. High-resolution array-based comparative genomic hybridization revealed that fusion-positive tumors had significantly fewer copy number alterations/tumor compared with fusion-negative tumors (1.5 vs 9.5; P=0.002). Twelve of 18 fusion-positive tumors had normal genomic profiles whereas only 1 out of 10 fusion-negative tumors lacked copy number alterations. The profiles of fusion-positive and fusion-negative tumors were very similar to those of low- and high-grade tumors. Thus, low-grade mucoepidermoid carcinomas had significantly fewer copy number alterations/tumor compared with high-grade mucoepidermoid carcinomas (0.7 vs 8.6; P<0.0001). The most frequent copy number alterations detected were losses of 18q12.2-qter (including the tumor suppressor genes DCC, SMAD4, and GALR1), 9p21.3 (including the tumor suppressor genes CDKN2A/B), 6q22.1-q23.1, and 8pter-p12.1, and gains of 8q24.3 (including the oncogene MAFA), 11q12.3-q13.2, 3q26.1-q28, 19p13.2-p13.11, and 8q11.1-q12.2 (including the oncogenes LYN, MOS, and PLAG1). On the basis of these results we propose that mucoepidermoid carcinoma may be subdivided in (i) low-grade, fusion-positive mucoepidermoid carcinomas with no or few genomic imbalances and favorable prognosis, (ii) high-grade, fusion-positive mucoepidermoid carcinomas with multiple genomic imbalances and unfavorable prognosis, and (iii) a heterogeneous group of high-grade, fusion-negative adenocarcinomas with multiple genomic imbalances and unfavorable outcome. Taken together, our studies indicate that molecular genetic analysis can be a useful adjunct to histologic scoring of mucoepidermoid carcinoma and may lead to development of new clinical guidelines for management of these patients.

Abstract IA5: Genetics of clonal progression in pancreatic cancer

Abstract The goal of our laboratory is to understand the extent to which genetic features of a neoplasm influence its metastatic progression. Towards this goal we have focused on human pancreatic ductal adenocarcinoma (PDAC) as it represents an ideal tumor type for study of the metastatic process. For example, the precursor lesions that give rise to PDAC have been described in detail (pancreatic intraepithelial neoplasia, or PanIN), exomic sequencing has elucidated the major genetic alterations of this tumor type, and the accumulation of these genetic alterations during PanIN formation underlies the well accepted genetic progression model of pancreatic ductal carcinogenesis. Finally, PDAC is a tumor type that is often diagnosed in the advanced stages of disease and thus most patients have their primary carcinoma in situ for comparison to matched metastatic disease. Such is not the case for many other common human tumor types. We have taken the unique approach of performing rapid autopsies for acquisition of primary and metastatic cancer tissues from a large number of individuals, and have used this resource to make several novel observations related to the timing of acquisition of metastatic traits. First, not all pancreatic cancers are metastatic; of those that do form metastases the range of deposits seen at autopsy ranges from few (&amp;lt;10, oligometastatic disease) to &amp;gt;100 (widely metastatic). Moreover, loss of the SMAD4 tumor suppressor gene that occurs during carcinogenesis is highly correlated with widespread metastatic disease. Second, using a whole exome sequencing approach we have defined the clonal evolution of PDAC. This has shown that the majority of genetic alterations are founder mutations in that they are encompassed with the parental clone of cells that give rise to PDAC, upon which are superimposed additional genetic alterations that accumulate during subclonal evolution. Geographic mapping of subclones based on their unique genetic signatures indicates that metastatic subclones originate within the primary carcinoma; moreover, several years are required for development of metastatic subclones beyond initiation of the infiltrating carcinoma by the parental clone. Thus, even in carcinomas with SMAD4 loss additional events are needed for successful metastasis to occur. Most recently, we have investigated pancreatic cancer progression by utilizing a mathematical framework of metastasis formation together with comprehensive data of PDAC patients obtained at autopsy. This work indicates that primary and metastatic PDAC growth is initially exponential, and that patients with seemingly early stage disease likely harbor metastases at diagnosis. Furthermore, based on the estimating rates of PDAC growth and dissemination, we analyzed the effects of different treatment modalities and found that therapies which efficiently reduce the growth rate of cells earlier in the course of treatment appear to be superior to upfront tumor resection. These lines of investigation not only shed light on this difficult tumor type but also have implications for most other major tumor types for which metastatic progression is common. Citation Format: Christine A. Iacobuzio-Donahue. Genetics of clonal progression in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr IA5.

TIAF1 self-aggregation in peritumor capsule formation, spontaneous activation of SMAD-responsive promoter in p53-deficient environment, and cell death.

Self-aggregation of transforming growth factor β (TGF-β)1-induced antiapoptotic factor (TIAF1) is known in the nondemented human hippocampus, and the aggregating process may lead to generation of amyloid β (Aβ) for causing neurodegeneration. Here, we determined that overexpressed TIAF1 exhibits as aggregates together with Smad4 and Aβ in the cancer stroma and peritumor capsules of solid tumors. Also, TIAF1/Aβ aggregates are shown on the interface between brain neural cells and the metastatic cancer cell mass. TIAF1 is upregulated in developing tumors, but may disappear in established metastatic cancer cells. Growing neuroblastoma cells on the extracellular matrices from other cancer cell types induced production of aggregated TIAF1 and Aβ. In vitro induction of TIAF1 self-association upregulated the expression of tumor suppressors Smad4 and WW domain-containing oxidoreductase (WOX1 or WWOX), and WOX1 in turn increased the TIAF1 expression. TIAF1/Smad4 interaction further enhanced Aβ formation. TIAF1 is known to suppress SMAD-regulated promoter activation. Intriguingly, without p53, self-aggregating TIAF1 spontaneously activated the SMAD-regulated promoter. TIAF1 was essential for p53-, WOX1- and dominant-negative JNK1-induced cell death. TIAF1, p53 and WOX1 acted synergistically in suppressing anchorage-independent growth, blocking cell migration and causing apoptosis. Together, TIAF1 shows an aggregation-dependent control of tumor progression and metastasis, and regulation of cell death.

TIAF1 self‐aggregation causes spontaneous activation of SMAD‐responsive promoter in p53‐deficient environment and cell death

Self‐aggregation of TGF‐beta‐induced antiapoptotic factor (TIAF1) is known in the nondemented human hippocampus, and the aggregating process may lead to generation of amyloid beta (Abeta) for causing neurodegeneration. Here, we determined that solid tumors frequently overexpress TIAF1, which exhibits as aggregates together with Smad4 and Abeta in the cancer stroma and peritumor capsules. Also, TIAF1/Abeta aggregates are shown on the interface between neural cells and the metastatic cancer cell mass. In vitro induction of TIAF1 self‐association upregulated the expression of tumor suppressors Smad4 and WOX1 (or WWOX), and WOX1 in turn increased the TIAF1 expression. TIAF1/Smad4 interaction further generated Abeta. TIAF1 suppresses SMAD‐regulated promoter activation. Intriguingly, without p53, TIAF1 self‐association spontaneously activated the SMAD‐regulated promoter. TIAF1 was shown to be essential for p53‐, WOX1‐ and dominant negative JNK1‐induced cell death. Indeed, TIAF1, p53 and WOX1 acted synergistically in suppressing anchorage‐independent growth and causing apoptosis. Together, TIAF1 shows an aggregation‐dependent control of tumor progression and metastasis, and regulation of cell death. (supported by NSC and NHRI, Taiwan and DoD, USA)

Reduced BMP signaling due to BMPR1A germline mutations in juvenile polyposis

Introduction Juvenile polyposis (JP) is characterized by the development of hamartomatous polyps that collectively carry a high risk of malignant transformation. Germline mutations in the gene for bone morphogenetic protein receptor 1A (BMPR1A) and the tumor suppressor SMAD4 predispose to JP. We set out to evaluate the effect of JP BMPR1A mutations on downstream signaling through the bone morphogenetic protein (BMP) pathway. Methods Mutations found in JP patients were individually recreated in a wild-type (WT) BMPR1A expression vector using a PCR-based site directed mutagenesis (SDM) approach. SDM vectors were then cotransfected with a BMP responsive element luciferase vector (BRE-Luc) and a Renilla internal control into HEK-293T cells. Light units reflecting downstream signaling strength were then quantified after 48 hours. Student's t-test was used for statistical analysis. Results Twenty-two SDM JP mutants were created (14 nonsense and 8 missense). After adjusting for transfection efficiency, BMP signaling was found to be altered for 19 mutations, 17 of which were lower than WT. Two missense and 1 nonsense mutation did not lead to a significant change in activity. Compared to WT, the mean decrease in luciferase for nonsense mutations was 73.1% (p Conclusions To date, JP BMPR1A mutations have not been systematically studied in an in vitro model or been shown to affect downstream signaling. This study confirms that the BMPR1A mutations found in JP patients lead to loss of BMP signaling, which is the likely mechanism leading to this phenotype.

Antagonistic regulation of EMT by TIF1γ and Smad4 in mammary epithelial cells

TGF-β is a potent inducer of epithelial-to-mesenchymal transition (EMT), a process involved in tumour invasion. TIF1γ participates in TGF-β signalling. To understand the role of TIF1γ in TGF-β signalling and its requirement for EMT, we analysed the TGF-β1 response of human mammary epithelial cell lines. A strong EMT increase was observed in TIF1γ-silenced cells after TGF-β1 treatment, whereas Smad4 inactivation completely blocked this process. Accordingly, the functions of several TIF1γ target genes can be linked to EMT, as shown by microarray analysis. As a negative regulator of Smad4, TIF1γ could be crucial for the regulation of TGF-β signalling. Furthermore, TIF1γ binds to and represses the plasminogen activator inhibitor 1 promoter, demonstrating a direct role of TIF1γ in TGF-β-dependent gene expression. This study shows the molecular relationship between TIF1γ and Smad4 in TGF-β signalling and EMT.

Smad4 Inactivation Promotes Malignancy and Drug Resistance of Colon Cancer

SMAD4 is localized to chromosome 18q21, a frequent site for loss of heterozygosity in advanced stage colon cancers. Although Smad4 is regarded as a signaling mediator of the TGFβ signaling pathway, its role as a major suppressor of colorectal cancer progression and the molecular events underlying this phenomenon remain elusive. Here, we describe the establishment and use of colon cancer cell line model systems to dissect the functional roles of TGFβ and Smad4 inactivation in the manifestation of a malignant phenotype. We found that loss of function of Smad4 and retention of intact TGFβ receptors could synergistically increase the levels of VEGF, a major proangiogenic factor. Pharmacologic inhibition studies suggest that overactivation of the TGFβ-induced MEK-Erk and p38-MAPK (mitogen-activated protein kinase) auxiliary pathways are involved in the induction of VEGF expression in SMAD4 null cells. Overall, SMAD4 deficiency was responsible for the enhanced migration of colon cancer cells with a corresponding increase in matrix metalloprotease 9 enhanced hypoxia-induced GLUT1 expression, increased aerobic glycolysis, and resistance to 5'-fluoruracil-mediated apoptosis. Interestingly, Smad4 specifically interacts with hypoxia-inducible factor (HIF) 1α under hypoxic conditions providing a molecular basis for the differential regulation of target genes to suppress a malignant phenotype. In summary, our results define a molecular mechanism that explains how loss of the tumor suppressor Smad4 promotes colorectal cancer progression. These findings are also consistent with targeting TGFβ-induced auxiliary pathways, such as MEK-ERK, and p38-MAPK and the glycolytic cascade, in SMAD4-deficient tumors as attractive strategies for therapeutic intervention.

Distinguishing between cancer driver and passenger gene alteration candidates via cross-species comparison: a pilot study

BackgroundWe are developing a cross-species comparison strategy to distinguish between cancer driver- and passenger gene alteration candidates, by utilizing the difference in genomic location of orthologous genes between the human and other mammals. As an initial test of this strategy, we conducted a pilot study with human colorectal cancer (CRC) and its mouse model C57BL/6J ApcMin/+, focusing on human 5q22.2 and 18q21.1-q21.2.MethodsWe first performed bioinformatics analysis on the evolution of 5q22.2 and 18q21.1-q21.2 regions. Then, we performed exon-targeted sequencing, real time quantitative polymerase chain reaction (qPCR), and real time quantitative reverse transcriptase PCR (qRT-PCR) analyses on a number of genes of both regions with both human and mouse colon tumors.ResultsThese two regions (5q22.2 and 18q21.1-q21.2) are frequently deleted in human CRCs and encode genuine colorectal tumor suppressors APC and SMAD4. They also encode genes such as MCC (mutated in colorectal cancer) with their role in CRC etiology unknown. We have discovered that both regions are evolutionarily unstable, resulting in genes that are clustered in each human region being found scattered at several distinct loci in the genome of many other species. For instance, APC and MCC are within 200 kb apart in human 5q22.2 but are 10 Mb apart in the mouse genome. Importantly, our analyses revealed that, while known CRC driver genes APC and SMAD4 were disrupted in both human colorectal tumors and tumors from ApcMin/+ mice, the questionable MCC gene was disrupted in human tumors but appeared to be intact in mouse tumors.ConclusionsThese results indicate that MCC may not actually play any causative role in early colorectal tumorigenesis. We also hypothesize that its disruption in human CRCs is likely a mere result of its close proximity to APC in the human genome. Expanding this pilot study to the entire genome may identify more questionable genes like MCC, facilitating the discovery of new CRC driver gene candidates.

Uncoupled responses of Smad4-deficient cancer cells to TNFα result in secretion of monomeric laminin-γ2

BackgroundFunctional loss of the tumor suppressor Smad4 is involved in pancreatic and colorectal carcinogenesis and has been associated with the acquisition of invasiveness. We have previously demonstrated that the heterotrimeric basement membrane protein laminin-332 is a Smad4 target. Namely, Smad4 functions as a positive transcriptional regulator of all three genes encoding laminin-332; its loss is thus implicated in the reduced or discontinuous deposition of the heterotrimeric basement membrane molecule as evident in carcinomas. Uncoupled expression of laminin genes, on the other hand, namely overexpression of the laminin-γ2 chain is an impressive marker at invasive edges of carcinomas where tumor cells are maximally exposed to signals from stromal cell types like macrophages. As Smad4 is characterized as an integrator of multiple extracellular stimuli in a strongly contextual manner, we asked if loss of Smad4 may also be involved in uncoupled expression of laminin genes in response to altered environmental stimuli. Here, we address Smad4 dependent effects of the prominent inflammatory cytokine TNFα on tumor cells.ResultsSmad4-reconstituted colon carcinoma cells like adenoma cells respond to TNFα with an increased expression of all three chains encoding laminin-332; coincubation with TGFβ and TNFα leads to synergistic induction and to the secretion of large amounts of the heterotrimer. In contrast, in Smad4-deficient cells TNFα can induce expression of the γ2 and β3 but not the α3 chain. Surprisingly, this uncoupled induction of laminin-332 chains in Smad4-negative cells rather than causing intracellular accumulation is followed by the release of γ2 into the medium, either in a monomeric form or in complexes with as yet unknown proteins. Soluble γ2 is associated with increased cell migration.ConclusionsLoss of Smad4 may lead to uncoupled induction of laminin-γ2 in response to TNFα and may therefore represent one of the mechanisms which underlie accumulation of laminin-γ2 at the invasive margin of a tumor. The finding, that γ2 is secreted from tumor cells in significant amounts and is associated with increased cell migration may pave the way for further investigation to better understand its functional relevance for tumor progression.

A catalog of genes homozygously deleted in human lung cancer and the candidacy of PTPRD as a tumor suppressor gene

A total of 176 genes homozygously deleted in human lung cancer were identified by DNA array-based whole genome scanning of 52 lung cancer cell lines and subsequent genomic PCR in 74 cell lines, including the 52 cell lines scanned. One or more exons of these genes were homozygously deleted in one (1%) to 20 (27%) cell lines. These genes included known tumor suppressor genes, e.g., CDKN2A/p16, RB1, and SMAD4, and candidate tumor suppressor genes whose hemizygous or homozygous deletions were reported in several types of human cancers, such as FHIT, KEAP1, and LRP1B/LRP-DIP. CDKN2A/p16 and p14ARF located in 9p21 were most frequently deleted (20/74, 27%). The PTPRD gene was most frequently deleted (8/74, 11%) among genes mapping to regions other than 9p21. Somatic mutations, including a nonsense mutation, of the PTPRD gene were detected in 8/74 (11%) of cell lines and 4/95 (4%) of surgical specimens of lung cancer. Reduced PTPRD expression was observed in the majority (>80%) of cell lines and surgical specimens of lung cancer. Therefore, PTPRD is a candidate tumor suppressor gene in lung cancer. Microarray-based expression profiling of 19 lung cancer cell lines also indicated that some of the 176 genes, such as KANK and ADAMTS1, are preferentially inactivated by epigenetic alterations. Genetic/epigenetic as well as functional studies of these 176 genes will increase our understanding of molecular mechanisms behind lung carcinogenesis.

Identification and Characterization of a Novel Anticancer Agent With Selectivity Against Deleted in Pancreatic Cancer Locus 4 (DPC4)-Deficient Pancreatic and Colon Cancer Cells

The deleted in pancreatic cancer locus 4 (DPC4)/SMAD4 tumor suppressor gene is frequently inactivated in pancreatic (approximately 55%) and colorectal cancers (approximately 30%). Like other tumor suppressor genes, the loss-of-function mutations found in the DPC4 gene are specific to cancer cells. This provides an attractive and unique opportunity for therapeutic intervention. The aim of this study was to identify and characterize small molecules that selectively kill DPC4-deficient cancer cells. An unbiased chemical screening using isogenic cell lines that only differ in the DPC4 gene was carried out to identify positive hits. Selected hits were further verified in additional isogenic cell lines and characterized in cancer cells using several different cellular assays. A lead molecule, UA62784, was identified to be selectively cytotoxic against cancer cells with deficient DPC4. UA62784 preferentially induces cell cycle arrest and apoptosis in cells with deficient DPC4. It also selectively reduces the clonogenicity of DPC4-deficient cells on soft agar when compared with cells with wild type DPC4. We further demonstrate that UA62784 induces CDC2 kinase activity preferentially in DPC4-negative cells. UA62784 is a small molecule that selectively kills DPC4-deficient cancer cells. Its potent activity and relatively low molecular weight make it a decent candidate for further lead optimization.

Jab1/CSN5 induces the cytoplasmic localization and degradation of RUNX3

Runt-related (RUNX) transcription factors play pivotal roles in neoplastic development and have tissue-specific developmental roles in hematopoiesis (RUNX1), osteogenesis (RUNX2), as well as neurogenesis and thymopoiesis (RUNX3). RUNX3 is a tumor suppressor in gastric carcinoma, and its expression is frequently inactivated by DNA methylation or its protein mislocalized in many cancer types, including gastric and breast cancer. Jun-activation domain-binding protein 1 (Jab1/CSN5), a component of the COP9 signalosome (CSN), is critical for nuclear export and the degradation of several tumor suppressor proteins, including p53, p27(Kip1), and Smad4. Here, we find that Jab1 facilitates nuclear export of RUNX3 that is controlled by CSN-associated kinases. RUNX3 sequestered in the cytoplasm is rapidly degraded through a proteasome-mediated pathway. Our results identify a novel mechanism of regulating nuclear export and protein stability of RUNX3 by the CSN complex.

Divergent mechanisms underlie Smad4-mediated positive regulation of the three genes encoding the basement membrane component laminin-332 (laminin-5)

BackgroundFunctional inactivation of the tumor suppressor Smad4 in colorectal and pancreatic carcinogenesis occurs coincident with the transition to invasive growth. Breaking the basement membrane (BM) barrier, a prerequisite for invasive growth, can be due to tumor induced proteolytic tissue remodeling or to reduced synthesis of BM molecules by incipient tumor cells. Laminin-332 (laminin-5), a heterotrimeric BM component composed of α3-, β3- and γ2-chains, has recently been identified as a target structure of Smad4 and represents the first example for expression control of an essential BM component by a tumor and invasion suppressor. Biochemically Smad4 is a transmitter of signals of the TGFβ superfamily of cytokines. We have reported previously, that Smad4 functions as a positive transcriptional regulator of constitutive and of TGFβ-induced transcription of all three genes encoding Laminin-332, LAMA3, LAMB3 and LAMC2.MethodsPromoter-reporter constructs harboring 4 kb upstream regions, each of the three genes encoding Laminin-322 as well as deletion and mutations constructs were established. Promoter activities and TGFβ induction were assayed through transient transfections in Smad4-negative human cancer cells and their stable Smad4-positive derivatives. Functionally relevant binding sites were subsequently confirmed through chromatin immunoprecipitation.ResultsHerein, we report that Smad4 mediates transcriptional regulation through three different mechanisms, namely through Smad4 binding to a functional SBE site exclusively in the LAMA3 promoter, Smad4 binding to AP1 (and Sp1) sites presumably via interaction with AP1 family components and lastly a Smad4 impact on transcription of AP1 factors. Whereas Smad4 is essential for positive regulation of all three genes, the molecular mechanisms are significantly divergent between the LAMA3 promoter as compared to the LAMB3 and LAMC2 promoters.ConclusionWe hypothesize that this divergence in modular regulation of the three promoters may lay the ground for uncoupled regulation of Laminin-332 in Smad4-deficient tumor cells in response to stromally expressed cytokines acting on budding tumor cells.

Aberrant Crypt Foci in the Adenoma Prevention with Celecoxib Trial

Aberrant crypt foci (ACF) are the earliest visible neoplastic lesions in the colorectum. The natural history of these lesions and their role in the adenoma-carcinoma sequence are unknown. We studied ACF in a subset of patients randomized to placebo (n = 17), celecoxib (200 mg twice daily; n = 15), or celecoxib (400 mg twice daily; n = 13) in the Adenoma Prevention with Celecoxib (APC) trial. Magnification chromoendoscopy was done to identify, count, and biopsy ACF within the rectum at baseline and after 8 to 12 months of treatment. A total of 655 ACF were identified in 45 patients. We examined 70 of these ACF histologically, and all 70 were nondysplastic. Cohort characteristics and APC trial treatment results for substudy patients were similar to those of the overall APC trial. There was no significant modulation of ACF by celecoxib (versus placebo; P = 0.77). Immunohistochemical comparison of ACF with adjacent normal mucosa showed that ACF had an increased proliferative index as determined by Ki-67 (P < 0.0001), but lacked other features of neoplasia such as increased cyclooxygenase-2 expression and microvessel density, nuclear localization of beta-catenin, or decreased expression of the tumor suppressors SMAD4, Estrogen Receptor alpha, or MGMT. Only baseline SMAD4 expression in ACF correlated with posttreatment adenoma recurrence (independent of treatment arm; P = 0.01). The presence or number of nondysplastic ACF did not correlate with a higher risk of synchronous advanced or recurrent adenomas. Our overall results indicated that nondysplastic ACF were not accurate surrogate endpoint biomarkers of recurrent colorectal adenomas in the APC trial.

Abstract LB-242: Aberrant crypt foci (ACF) are not surrogate biomarkers of sporadic adenoma prevention with celecoxib: Results from the Adenoma Prevention with Celecoxib (APC) trial

Abstract Aberrant crypt foci (ACF) are the earliest visible neoplastic lesions in the colorectum. The natural history of these lesions and their role in the adenoma-carcinoma sequence are unknown. We studied ACF in a subset of patients enrolled in the Adenoma Prevention with Celecoxib (APC) Trial to determine whether the presence of ACF in the rectum correlated with that of adenomas in the entire large intestine. Participants were randomized to placebo (n=17), celecoxib 200 mg bid (n=15), or celecoxib 400 mg bid (n=13). Magnification chromoendoscopy (MCE) was performed to identify and biopsy ACF within the rectum at baseline and after 8-12 months of medication use. A total of 670 ACF were identified in 45 patients, and 70 of these were examined histologically. All of the ACF examined were non-dysplastic. Analysis showed no differences in ACF number between baseline and post-treatment studies (8.3±1.0 vs. 6.3±1.1) and no modulation by treatment (celecoxib vs placebo; p=0.77). Immunohistochemical studies showed that ACF contained cells exhibiting increased proliferation, but lacked other features of neoplasia such as increased Cox-2 expression and microvascular density, nuclear localization of β-catenin, or decreased expression of the tumor suppressors SMAD4, estrogen receptor (ERα) or MGMT. Of the secondary markers assessed at baseline, only SMAD4 expression correlated with post-treatment adenoma recurrence. Taken together, these data showed that non-dysplastic ACF are not accurate surrogate endpoint biomarkers of colorectal adenoma risk or NSAID chemoprevention response.

BCL6 Represses Smad Signaling in Transforming Growth Factor-β Resistance

Transforming growth factor-beta (TGF-beta) controls a wide spectrum of cellular processes. Deregulation of TGF-beta signaling contributes to the pathogenesis of many diseases including cancer and autoimmune diseases. TGF-beta signaling is generally mediated through intracellular signal transducers and transcription factors called Smads. Herein, we have identified the oncoprotein BCL6 as a transcriptional corepressor of tumor suppressor Smad4. BCL6 physically interacts with Smad3 and Smad4, disrupts the Smad-p300 interaction, and represses the transcriptional activity of Smad4. In accordance, B-cell lymphoma cells with a high expression level of BCL6 were found to be refractory to TGF-beta antiproliferative response, whereas knockdown of BCL6 expression in B-cell lymphoma cells partially restores the TGF-beta responses. This study provides strong evidence that overexpression of BCL6 contributes to TGF-beta resistance in B-cell lymphoma.

Normal erythropoiesis but severe polyposis and bleeding anemia in Smad4-deficient mice

AbstractThe tumor suppressor Smad4 mediates signaling by the transforming growth factor beta (TGF-β) superfamily of ligands. Previous studies showed that several TGF-β family members exert important functions in hematopoiesis. Here, we studied the role of Smad4 in adult murine hematopoiesis using the inducible Mx-Cre/loxP system. Mice with homozygous Smad4 deletion (Smad4Δ/Δ) developed severe anemia 6 to 8 weeks after induction (mean hemoglobin level 70 g/L). The anemia was not transplantable, as wild-type mice reconstituted with Smad4Δ/Δ bone marrow cells had normal peripheral blood counts. These mice did not develop an inflammatory disease typical for mice deficient in TGF-β receptors I and II, suggesting that the suppression of inflammation by TGF-β is Smad4 independent. The same results were obtained when Smad4 alleles were deleted selectively in hematopoietic cells using the VavCre transgenic mice. In contrast, lethally irradiated Smad4Δ/Δ mice that received wild-type bone marrow cells developed anemia similar to Smad4Δ/Δ mice that did not receive a transplant. Liver iron stores were decreased and blood was present in stool, indicating that the anemia was due to blood loss. Multiple polyps in stomach and colon represent a likely source of the bleeding. We conclude that Smad4 is not required for adult erythropoiesis and that anemia is solely the consequence of blood loss.

BMP7: A New Bone Metastases Prevention?

Prostate cancer is the most frequently diagnosed cancer in men and a leading cause of cancer death. More than 218,000 cases will occur in 2007, and prostate cancer will lead to more than 27,000 deaths.1 Although the 5-year survival rate is excellent for localized stages (100%), the relative survival rapidly decreases to 33% when prostate cancer metastasizes.1 Cancer cells from prostate primary tumors commonly colonize bones, and postmortem analyses show that 65 to 75% of patients with advanced disease have bone metastases.2 Scientific knowledge of bone metastasis pathophysiology has increased in recent years, but effective therapy for this devastating complication of cancer remains suboptimal. In this issue of The American Journal of Pathology, Buijs and colleagues3 shed more light into pathophysiology of bone metastases. More importantly, they apply this knowledge to develop novel therapy for bone metastases using bone morphogenetic protein 7 (BMP7).

Smad4 Regulates Claudin-1 Expression in a Transforming Growth Factor-β–Independent Manner in Colon Cancer Cells

We have recently reported that the expression of a tight junction protein, claudin-1, is increased during colon carcinogenesis and particularly metastatic colorectal cancer. Manipulation of claudin-1 levels in colon cancer cells showed a positive correlation between claudin-1 expression and tumor growth and metastasis. However, the mechanisms underlying the increased claudin-1 expression in colorectal cancer remains unknown. The tumor suppressor Smad4 is a central intracellular signal transduction component of the transforming growth factor-beta (TGF-beta) family of cytokines. Loss of Smad4 protein expression is correlated with poor prognosis and is frequently observed in invasive and metastatic colorectal carcinoma. In the present study, we report an inverse relationship between Smad4 and claudin-1 expression in human colorectal carcinoma tumor samples and in human colon cancer cell lines. We found that the expression of Smad4 in Smad4-deficient but claudin-1-positive SW480 or HT29 colon cancer cell lines down-regulates claudin-1 expression through transcriptional repression by modulating beta-catenin/T-cell factor/lymphocyte enhancer factor activity. Furthermore, this Smad4-dependent inhibition of claudin-1 expression is independent of TGF-beta signaling because Smad4 expression alone is insufficient to restore TGF-beta signaling in the SW480 cells, and the selective TGF-beta receptor kinase inhibitor LY364947 did not prevent the Smad4 suppression of claudin-1 protein expression in either SW480 or HT29 cells. Taken together, these findings suggest a novel mechanism underlying Smad4 tumor-suppressive function through regulation of a potential metastatic modulator, claudin-1, in a TGF-beta-independent manner.