Basement membrane component laminin-5 is a target of the tumor suppressor Smad4.

The tumor suppressor Smad4 is involved in carcinogenesis mainly of the pancreas and colon. Functional inactivation of Smad4 is a genetically late event that occurs upon transition from premalignant stages to invasive and metastatic growth. Smad4 encodes an intracellular messenger common to all signalling cascades induced by members of the transforming growth factor-beta (TGF-beta) superfamily of cytokines. Despite extensive knowledge about the mechanisms of TGF-beta/Smad signal transduction, little is known about Smad4 targets involved in the transition to malignancy. The hallmark of invasive growth is a breakdown of the basement membrane (BM), a specialized sheet of extracellular matrix produced through cooperation of epithelial and stromal cells. Laminin-5, a heterotrimeric epithelial-derived BM component, is commonly lost in carcinomas but not in premalignant tumors. Herein, we report that in human colon and pancreatic tumor cells, Smad4 functions as a positive transcriptional regulator of all three genes encoding laminin-5. Coordinate re-expression of the three laminin-5 chains induced by reconstitution of Smad4 leads to secretion and deposition of the heterotrimeric molecule in BM-like structures. These data define the expression control of an essential BM component as a novel function for the tumor suppressor Smad4.

Mutations of the DPC4/Smad4 gene in biliary tract carcinoma.

A candidate tumor suppressor gene, DPC4, located at 18q21.1, has recently been shown to be inactivated in half of pancreatic adenocarcinomas. The close developmental relationship of the pancreas and biliary tract prompted us to determine the role of DPC4 in the multistep carcinogenesis of biliary tract carcinoma. A search for mutations in the genomic sequence of the highly conserved COOH-terminal domain of DPC4 (exons 8-11) was performed by single-strand conformational polymorphism analysis. Five of 32 (16%) primary biliary tract carcinomas had point mutations in the DPC4 sequence. Interestingly, inactivation of DPC4 was especially common in carcinomas originating from the common bile duct (four of eight specimens analyzed), suggesting an important role for DPC4 in the development of this subtype of biliary tract tumor.

Abrogation of the Rb/p16 tumor-suppressive pathway in virtually all pancreatic carcinomas.

The Rb/p16 tumor-suppressive pathway is abrogated frequently in human tumors, either through inactivation of the Rb or p16INK4a/CDKN2/MTS1 tumor-suppressor proteins, or through alteration or overexpression of the cyclin D1 or cyclin-dependent kinase 4 oncoproteins. We reported previously that the p16 gene was genetically inactivated in 82% of pancreatic carcinomas. Nearly half of these inactivations were by intragenic mutation of p16, and the remainder were by homozygous deletion of the gene. Here, we analyzed pancreatic carcinomas for additional mechanisms by which the Rb/p16 pathway might be inactivated. Transcriptional silencing of the p16 gene in association with methylation of its 5'-CpG island was examined by methylation-specific PCR in 18 pancreatic carcinomas. Nine of these were known to harbor an intragenic mutation in p16, and nine had a wild-type p16 coding sequence. Seven of the 18 tumors were hypermethylated, and all 7 were p16 wild-type (P = 0.001). Complete silencing of transcription from methylated wild-type gene sequences was demonstrated. Immunohistochemical analysis revealed normal expression levels of the Rb protein in all carcinomas studied. None of the carcinomas had genomic amplification of the cyclin D1 or CDK4 genes, and none had mutation of the p16-binding domain of CDK4. An additional p16 mutation was identified. In total, the Rb/p16 pathway was abrogated in 49 of the 50 carcinomas (98%) studied, all through inactivation of the p16 gene. Similar results were obtained in an independently analyzed series of 19 pancreatic carcinomas. These data demonstrate the central role of the Rb/p16 pathway in the development of pancreatic carcinoma.

Functional identification and molecular cloning of a rat gene mediating growth inhibition and programmed cell death in normal and transformed rat cell lines.

We wished to identify DNA sequences conferring suppression of proliferation and transformed phenotypes. Thus, we have transfected DNA from normal rat cells, covalently linked to neo DNA coding for neomycin resistance into a tumorigenic, HRAS transformed rat cell line. Phenotypic revertants were selected after the first cycle of transfection by enrichment procedures that served to eliminate transformed cells. The revertant clones continued to express the HRAS oncogene, but exhibited a lower tumorigenicity, loss of anchorage-independent proliferation, flat morphology, and retardation of growth in monolayer culture. The reverted phenotype could be transferred in a second cycle of transfection into the HRAS transformed rat cells. Neo DNA ligated to genomic donor DNA was used as a tagging sequence to molecularly clone the transferred DNA sequence in a recombinant phage. Fragments of the cloned DNA detect a 2.5 kb transcript in parental cells and revertants. Thus, the recombinant phage harbors a putative growth inhibitory gene, designated trg, that is expressed at a higher level in rat embryo fibroblasts and in the REF52 cell line. Introduction of recombinant phage DNA into established 208F and Rat-2 cells and into HRAS-, v-fgr-, v-fms- and v-raf-transformed rat cell lines resulted in inhibition of growth and induction of programmed cell death.

Evidence for human DNA-mediated transfer of the suppressed phenotype into malignant Chinese hamster cells.

Genetic suppression of the neoplastic phenotype has been demonstrated in somatic cell hybrids between tumor and normal cells. Suppression in whole-cell and microcell hybrids cannot, as yet, be attributed to specific elements defined at the molecular level. To identify a gene capable of suppressing the neoplastic phenotype, we have introduced DNA of normal human cells into tumorigenic Chinese hamster Wg3-h-o cells. Primary and secondary transfectants which exhibit the suppressed phenotype similar to Wg3-h-o x embryonic fibroblast hybrids were selected. The cells require serum growth factors and anchorage for proliferation in vitro and show a reduced tumorigenicity in nude mice. Transferred human DNA segments were molecularly cloned from a secondary transfectant. Indirect evidence suggests that the cloned human DNA is associated with the expression of the suppressed phenotype.

Expression of ril, a novel LIM domain gene, is down-regulated in Hras-transformed cells and restored in phenotypic revertants.

Several candidate genes involved in the maintenance of normal growth control (H-rev) were identified by differential expression cloning on the assumption that they are expressed in phenotypically normal rat cells and repressed in closely related H-ras transformed cells. Previously the genes coding for lysyl oxidase (H-rev142) and for an 18K-protein of unknown function (H-rev107) were recovered as cDNAs by subtraction cloning. Here we describe the identification and expression pattern of ril, a novel member of the heterogeneous group of genes encoding proteins with LIM/double zinc finger domains. The ril gene is expressed in normal fibroblasts and down-regulated in H-ras-transformed derivatives. Expression is restored in several independent phenotypic revertants derived from H-ras transformed cells. The predicted protein product of ril harbors a single LIM domain but lacks a homeodomain. The ril gene is highly conserved during evolution and is transcribed in various normal cell lines. Northern blot analysis and in situ hybridization studies showed that ril is expressed in meiotic spermatocytes, in somites of developing mice, and in a wide variety of tissues of adult mice.

DPC4/SMAD4 mediated tumor suppression of colon carcinoma cells is associated with reduced urokinase expression.

We recently identified DPC4/Smad4 as a candidate tumor suppressor gene mutated or lost in one half of pancreatic carcinomas and in a subset of colon and biliary tract carcinomas. DPC4 plays a key role in signal transduction of the TGF-beta superfamily of molecules and inactivation of TGF-beta mediated growth inhibition is supposed to be the driving force for DPC4 inactivation in human tumors. However, DPC4 mediated tumor suppression by reconstitution of defective cells has not yet been reported. Here we show suppression of tumorigenicity in nude mice by stable reexpression of DPC4 in SW480 colon carcinoma cells. In vitro growth of DPC4-transfected cells was not affected and resistance towards TGF-beta mediated growth inhibition was retained. Instead, cells exhibited morphological alterations and adhesion and spreading were accelerated. These phenotypic changes were associated with reduced expression levels of the endogenous urokinase-type plasminogen activator (uPA) and plasminogen-activator-inhibitor-1 (PAI-1) genes, the products of which are implicated in the control of cell adhesion and invasion. In patients, high expression levels of uPA and PAI-1 correlate with poor prognosis. Thus, reduced expression of uPA and PAI-1 is consistent with suppression of tumorigenicity in DPC4 reconstituted cells. These results demonstrate DPC4's tumor suppressive function and suggest a potential role for DPC4 as a modulator of cell adhesion and invasion.

P53 mutations and loss of heterozygosity on chromosomes 8p, 16q, 17p, and 18q are confined to advanced prostate cancer.

We analysed 39 prostatic carcinomas for loss of heterozygosity on chromosomal arms 8p, 10q, 16q, 17p and 18q and for mutations in the p53 anti-oncogene. Loss of heterozygosity (LOH) on 8p was detected in one out of 5 informative tumors, LOH on 16q in 3 out of 21 tumors, LOH on 17p in 2 out of 18 tumors, and LOH on 18q in 2 out of 17 tumors. No deletions were observed on 10q in 14 informative tumors. p53 alterations occurred in 3 out of 38 examined tumors, comprising two point mutations and a small deletion. Chromosomal deletions and p53 mutations were confined to locally invasive prostatic carcinomas, suggesting that they are associated with the progression of some prostate cancers rather than with tumor initiation.

Antitumor activity of the HER2 dimerization inhibitor pertuzumab on human colon cancer cells in vitro and in vivo.

PURPOSE: The monoclonal antibody pertuzumab represents the first HER2 dimerization inhibitor with unknown activity in colon cancer treatment. We examined the antitumor activity of pertuzumab as a single agent or in combination with erlotinib or irinotecan in human colon cancer cells in vitro and in vivo. METHODS: Colon cancer cell lines were tested for HER1/HER2 expression by western blot analysis. The effect of pertuzumab on cell cycle distribution was analyzed by FACS. Nude mice bearing xenograft tumors were treated with pertuzumab alone, or in combination either with irinotecan or with erlotinib. Tumor volume was measured repeatedly. Tumor histology was analyzed for necrosis. RESULTS: Six of nine cell lines showed high expression of HER1/HER2. Pertuzumab inhibited cell cycle progression in various cell lines. Pertuzumab showed minor antitumor activity in xenograft tumors, but significantly inhibited tumor growth when combined with erlotinib (P < 0.001). Combination of pertuzumab with irinotecan had no additional effect on growth of additional tumors. Pertuzumab treated DLD-1 xenograft tumors did not show enhanced necrosis, which, however, was found in HCT116 derived xenografts. CONCLUSIONS: Pertuzumab has some antitumor activity on human colon cancer cells in vitro and in vivo, in particular when combined with erlotinib. In vivo, pertuzumab combination treatment was not superior to irinotecan monotherapy. These data warrant further investigation of simultaneous HER1/EGFR TKI inhibition and HER1/HER2 dimerization inhibition for colorectal cancer therapy.

Functional interaction of wild-type and mutant p53 transfected into human tumor cell lines carrying activated ras genes.

We have analyzed the antiproliferative activity of the p53 tumor suppressor gene in human tumor cell lines harboring activated ras genes. The levels of p53 protein and incorporation of bromodeoxyuridine in transiently transfected cells were determined simultaneously by flow cytometry. The human HT1080 fibrosarcoma, EJ bladder carcinoma, and SW480 colon carcinoma cell lines were equally sensitive toward wild-type p53-mediated inhibition of DNA synthesis, independent of the state of the endogenous p53 protein. Overexpression of p53 genes mutated at amino acid codon 143 resulted in increased proliferation of SW480 cells, which have two mutated endogenous p53 alleles. To mimic the genetic constitution of an evolving tumor cell that has sustained a mutation in one p53 allele, we coexpressed both wild-type and mutant p53 genes controlled by strong viral promoters in HT1080 cells. Transiently transfected cells showed a reduced bromodeoxyuridine uptake similar to cells into which only wild-type p53 had been introduced. The wild-type p53 gene is a dominant growth suppressor over the mutant in all three different cell lines analyzed. By immunoprecipitation with antibodies PAb 122, PAb 420, and PAb 1620, we demonstrate the presence of both the mutant and wild-type conformations of the p53 protein in the transfected cells.

Mesalazine causes a mitotic arrest and induces caspase-dependent apoptosis in colon carcinoma cells.

Non-steroidal anti-inflammatory drugs (NSAID) may inhibit colon cancer development through affecting proliferation and apoptosis. However, their use in cancer chemoprevention is still limited due to toxicities. There is longstanding clinical experience with the aminosalicylate mesalazine in the treatment of patients with inflammatory bowel disease with very few side effects. So far, most studies on the cellular effects of mesalazine were focused on its anti-inflammatory properties. Recent data, however, indicate that mesalazine may also reduce cell growth in vivo. We therefore investigated the growth inhibitory effect of mesalazine on human colon cancer cells in vitro compared with established chemopreventive agents. We also wished to determine the underlying cellular mechanisms of the effect. Here we show that mesalazine dose- and time-dependently inhibited the proliferation of colon cancer cells. Mesalazine was less potent in reducing cell growth than sulindac sulfide or indomethacin but growth effective mesalazine concentrations were comparable with concentrations achievable in vivo under standard mesalazine treatment. While other NSAID induced a robust G(1) arrest, mesalazine specifically blocked cells in mitosis although microtubule polymerization or spindle orientation was not affected. In addition, mesalazine induced apoptosis in colon cancer cells possibly through activation of caspase-3 whereas the levels of bcl-2 family proteins were not altered. We conclude that mesalazine inhibits growth of colon cancer cells largely through a mitotic arrest, which has not been reported for NSAID so far. Mesalazine also induces apoptosis through partial activation of caspases similar to, although weaker than, established chemopreventive agents. These findings may suggest a potential of mesalazine as a chemopreventive agent for colorectal cancer.

Allelic loss is often the first hit in the biallelic inactivation of the p53 and DPC4 genes during pancreatic carcinogenesis.

The presumed precursor lesions of pancreatic ductal adenocarcinoma were recently classified according to their increasing grade of dysplasia and were designated as pancreatic intraepithelial neoplasia (PanIN) 1 through 3. In this study, we tested whether molecular genetic alterations can be correlated with this classification and may help to further categorize the various PanIN grades. We determined the frequencies of allelic loss at chromosomal arms 9p, 17p, and 18q in 81 microdissected duct lesions of various PanIN grades, using a combination of whole genome amplification and microsatellite analysis. In addition we examined the p53 and Dpc4 protein expression patterns by immunohistochemical analysis. In PanIN-1, we did not detect allelic losses. In PanIN-2, allelic losses were found in increasing frequency, and were particularly high in those lesions with moderate-grade dysplasia (low grade, 20, 33, and 17%, loss at 9p, 17p, and 18q, respectively; moderate grade, 46, 77, and 58%). PanIN-3 and invasive carcinomas exhibited abundant losses. Abnormal p53 and Dpc4 protein expression was only rarely identified in PanIN-2 lesions, but occurred frequently in PanIN-3 lesions and invasive carcinomas. The combined genetic and protein expression data support a model in which allelic loss is the first hit in the biallelic inactivation of the p53 and DPC4 tumor suppressor genes. In addition, our data indicate that allelic loss analysis may be useful in separating PanIN-2 lesions with low-grade dysplasia from those PanIN-2 lesions with moderate-grade dysplasia, each potentially representing a distinct progression step toward invasive carcinoma.

Smad4/DPC4-mediated tumor suppression through suppression of angiogenesis.

Smad4/DPC4 (deleted in pancreatic carcinoma, locus 4) is a tumor suppressor gene lost at high frequency in cancers of the pancreas and other gastrointestinal organs. Smad4 encodes a key intracellular messenger in the transforming growth factor beta (TGF-beta) signaling cascade. TGF-beta is a potent inhibitor of the growth of epithelial cells; thus, it has been assumed that loss of Smad4 during tumor progression relieves this inhibition. Herein, we show that restoration of Smad4 to human pancreatic carcinoma cells suppressed tumor formation in vivo, yet it did not restore sensitivity to TGF-beta. Rather, Smad4 restoration influenced angiogenesis, decreasing expression of vascular endothelial growth factor and increasing expression of thrombospondin-1. In contrast to the parental cell line and to control transfectants that produced rapidly growing tumors in vivo, Smad4 revertants induced small nonprogressive tumors with reduced vascular density. These data define the control of an angiogenic switch as an alternative, previously unknown mechanism of tumor suppression for Smad4 and identify the angiogenic mediators vascular endothelial growth factor and thrombospondin-1 as key target genes.