Distribution of Transforming Growth Factor-β and Its Receptors in Gastric Carcinoma Tissue

The distribution of the three mammalian isoforms of transforming growth factor (TGF)-β (TGF-β1,-β2, and -β3) as well as their signaling receptors, TGF-β type I and type II receptors (TβR-I and TβR-II, respectively), in gastric carcinoma tissue was examined by immunohistochemistry using specific antibodies. Tissue specimens were obtained from 25 cases of gastric carcinoma, which were classified into two groups according to Lauren's classification, i.e. 15 cases of diffuse carcinoma and 10 cases of intestinal carcinoma. In normal gastric mucosa apart from carcinoma nests, all of TGF-β1, -β2, -β3, TβR-I and TβR-II were clearly demonstrated in fundic glands. In sharp contrast, none of them was detectable in surface mucous cells. In carcinoma cells, strong staining for TGF-β1, -β2 and β3 was obtained only in diffuse-type carcinoma. In particular, carcinoma cells scattered as single cells or small nests had a tendency to show strong staining for TGF-βs. The receptors tended to be distributed concomitantly with the ligands, and diffuse-type carcinoma showed stronger receptor staining than intestinal-type carcinoma. In cancer stroma, TGF-βs and receptors were detected in both diffuse and intestinal types, but the area with positive staining was wider and more dispersed in diffuse-type carcinoma than in intestinal carcinoma. These results suggest that TGF-β may contribute in part to the variety of histogenesis and mode of progression of gastric carcinoma.     

Reduced levels of transforming growth factor-beta type I receptor in human gastric carcinomas.

The expressions of transforming growth factor beta (TGF-beta) and its receptor and TGF-beta inhibitory element (TIE)-binding protein were examined on human gastric carcinomas by Northern blot hybridization, immunohistochemistry, affinity labeling and gel retardation analysis. TGF-beta mRNA was expressed in tumor and normal tissues at various levels. Immunohistochemically, TGF-beta expression was confirmed to be present within tumor cells. Out of the 17 human gastric carcinoma tissues, 14 (82%) showed a reduction in the level of type I receptor (65 kDa) for TGF-beta when compared to corresponding normal mucosas. Interestingly, in seven of the 14 tumors the level of TIE-binding protein in the tumor tissue was lower than that in normal mucosa. Human gastric carcinoma cell line TMK-1, whose growth was inhibited by TGF-beta, had only type I receptor for TGF-beta and showed a high level of TIE-binding protein. Conversely, MKN-1, a TGF-beta-resistant cell line, exhibited an extremely low level of TGF-beta receptor and had no TIE-binding protein. These results overall indicate that although human gastric carcinoma cells produced TGF-beta, they showed a reduction in TGF-beta type I receptor and a low level of TIE-binding protein, resulting in escape from growth inhibition by TGF-beta.     

c-Ski overexpression promotes tumor growth and angiogenesis through inhibition of transforming growth factor-β signaling in diffuse-type gastric carcinoma

c-Ski, originally identified as a proto-oncogene product, is an important negative regulator of transforming growth factor (TGF)-β family signaling through interaction with Smad2, Smad3, and Smad4. High expression of c-Ski has been found in some cancers, including gastric cancer. We previously showed that disruption of TGF-β signaling by dominant-negative TGF-β type II receptor in a diffuse-type gastric carcinoma model accelerated tumor growth through induction of tumor angiogenesis by decreased expression of the anti-angiogenic factor thrombospondin (TSP)-1. Here, we examined the function of c-Ski in human diffuse-type gastric carcinoma OCUM-2MLN cells. Overexpression of c-Ski inhibited TGF-β signaling in OCUM-2MLN cells. Interestingly, c-Ski overexpression resulted in extensive acceleration of the growth of subcutaneous xenografts in BALB/c nu/nu female mice (6 weeks of age). Similar to tumors expressing dominant-negative TGF-β type II receptor, histochemical studies revealed less fibrosis and increased angiogenesis in xenografted tumors expressing c-Ski compared to control tumors. Induction of TSP-1 mRNA by TGF-β was attenuated by c-Ski in vitro , and expression of TSP-1 mRNA was decreased in tumors expressing c-Ski in vivo . These findings suggest that c-Ski overexpression promotes the growth of diffuse-type gastric carcinoma through induction of angiogenesis. ( Cancer Sci  2009; 100: 1809–1816)     

Mutations and Reduced Expression of the Transforming Growth Factor-β Receptor II Gene in Rat Lung Adenocarcinomas Induced by N-Nitrosobis-(2-hydroxypropyl)amine

Mutations and expression of the transforming growth factor-β receptor type II (TGF-βRII) gene were investigated in lung adenocarcinomas induced by N -nitrosobis(2-hydroxypropyl)amine (BHP) in rats. Males of the Wistar strain, 6 weeks old, were given 2000 ppm of BHP in their drinking water for 12 weeks and then maintained without further treatment until killed at week 25. Total RNA was extracted from 12 adenocarcinomas and mutations in TGF-βRII were investigated by RT-PCR-restriction-SSCP analysis followed by sequencing analysis. Two out of 12 adenocarcinomas showed band shifts, indicative of mutations (16.7%). One was a CTG-to-TTG (Leu to Leu) transition at codon 308 without amino acid alteration and the other a frameshift deletion of one of two guanines at nucleotides 1434 to 1435 (codon 477 to 478). Semi-quantitative RT-PCR analysis demonstrated significantly reduced TGF-βRII expression in adenocarcinomas, as compared with normal lung tissue. These results suggest that TGF-βRII alterations may play a role in the acquisition of growth advantage by lung adenocarcinomas induced by BHP in rats.     

Improvement of Macrophage Dysfunction by Administration of Anti-transforming Growth Factor-β Antibody in EL4-bearing Hosts

An experimental therapy for improvement of macrophage dysfunction caused by transforming growth factor-β (TGF-β) was tried in EL4 tumor-bearing mice. TGF-β was detected in cell-free ascitic fluid from EL4-bearers, but not in tbat from normal mice, by western blot analysis. The ascites also showed growth-suppressive activity against MvlLn cells, and the suppressive activity was potentiated by transient acidification. To investigate whether the functions of peritoneal macrophagcs were suppressed in EL4-bearers, the abilities to produce nitric oxide and tumor necrosis factor-α (TNF-α) upon lipopolysaccharide (LPS) stimulation were measured. Both abilities of macrophages in EL4-bearing mice were suppressed remarkably on day 9, and decreased further by day 14, compared with non-tumor-bearing controls. TGF-β activity was abrogated by administration of anti-TGF-α antibody to EL4-bearing mice. While a large amount of TGF-β was detected in ascitic fluid from control EL4-bearers, little TGF-β was detectable in ascites from EL4-bearers given anti-TGF-β antibody. Furthermore, while control macrophages exhibited little or no production of nitric oxide and TNF-α on LPS stimulation in vitro , macrophages from EL4-bearers administered with anti-TGF-β antibody showed the same ability as normal macrophages. These results clearly indicate that TGF-β contributes to macrophage dysfunction and that the administration of specific antibody for TGF-β reverses macrophage dysfunction in EL4-bearing hosts.     

Inhibitory Effect of 8-Chloro-cyclic Adenosine 3',5'-Monophosphate on Cell Growth of Gastric Carcinoma Cell Lines

A cAMP analogue, 8-chloro-cAMP (8-Cl-cAMP), selectively binds to site 1 receptor of type II regulatory subunit (RII) of cAMP-dependent protein kinase. The effects of 8-Cl-cAMP on human gastric carcinoma cell lines were studied. Twenty μM 8-CI-cAMP clearly inhibited cell growth in six cell lines (TMK-1, KATO-III, MKN-7, -28, -45, and -74) but not in MKN-1. Cell population in the G₁ phase was increased in KATO III cells, which were most responsive to 8-Cl-cAMP, while cell cycle progression in TMK-1 and MKN-1 cells was apparently not influenced by 8-Cl-cAMP. The various changes induced by 8-Cl-cAMP were further analyzed in TMK-1 cells. Decrease of type I regulatory subunit (RI) of cAMP-dependent protein kinase and translocation of RII from cytosol to nucleus were induced by 8-Cl-cAMP treatment. 8-CI-cAMP increased the level of cAMP-response element (CRE) binding protein in addition to inducing FOS mRNA, whose promoter contains CRE. 8-Cl-cAMP decreased the expression of mRNA for transforming growth factor-α (TGF-α), while the expression of epidermal growth factor receptor was not changed. Expression of HRAS and MYC mRNAs was slightly increased, whereas the amounts of HRAS and MYC proteins remained unchanged. Our results overall suggest that 8-Cl-cAMP might be a useful tool for antitnmor therapy of gastric cancers and that cell growth inhibition by 8-Cl-cAMP might account for the decrease of TGF-α expression by tumor cells.     

Close Correlation between the Dephosphorylation of p53 and Growth Suppression by Transforming Growth Factor-β1 in Nasopharyngeal Carcinoma Cells Transduced with Adenovirus Early Region Genes

The mechanism of growth inhibition by transforming growth factor (TGF)-β1 was investigated. We examined the growth inhibitory effects of TGF-β1 on human nasopharyngeal carcinoma (KB) cells which constitutively expressed p53. TGF-β1 suppressed the DNA synthesis of KB cells in a dose-dependent manner. It had minimal effect on adenovirus-2-transduced KB cells expressing either adenovirus early region 1B (E1B) or 1A (E1A) product, which respectively binds to p53 or Rb product and inhibits its function, and no growth inhibition at all was observed with KB cells expressing both E1B and E1A products. Dephosphorylation of the p53 was promoted by TGF-β1 stimulation in KB cells, but not in E1B-producing KB cells, which sequestrate the function of p53. The growth inhibition of KB cells by TGF-β1 was significantly reduced by treatment with okadaic acid. These results suggest that p53 transduces the antiproliferative signal of TGF-β1 possibly through its dephosphorylation.     

Elevated Levels of Plasma Transforming Growth Factor-β in Patients with Hepatocellular Carcinoma

We measured the plasma transforming growth factor-β (TGF-β) concentration in 14 patients with human hepatocellular carcinoma (HCC) and 9 age-matched normal subjects using growth inhibition assay of mink lung epithelial cells. The calculated plasma TGF-β concentration in the patients with HCC was 28.6 ± 27.9 ng/ml (mean± SE), showing significant elevation compared with that in 9 normal subjects (5.3 ± 3.3 ng/ml, P<0.01). In three cases, we could measure plasma TGF-β levels before and after their treatment for HCC. The plasma TGF-β levels decreased from 59.0 to 18.2 ng/ml after hepatic resection in one case, and from 24.0 to 10.7 ng/ml and from 12.4 to 3.4 ng/ml after transhepatic arterial embolization in the other two cases. These data indicate that plasma TGF-β level is elevated in patients with HCC, probably due to release from HCC tissues.     

Detection of Active Form of Transforming Growth Factor-β in Cerebrospinal Fluid of Patients with Glioma

We examined transforming growth factor-β (TGF-β) activity in cerebrospinal fluid of 39 patients with various brain tumors, and found it in 10 glioma cases that had lesions related to subarachnoid space or ventricle. In one glioma case, TGF-β detected on admission disappeared after radiation and chemotherapy. We confirmed that five glioma cell lines produced TGF-β, and that four of them produced active form of TGF-β directly. The active form of TGF-β was also identified from cerebrospinal fluid before the acidification treatment in two cases. The calculated contents were 110 ng/ml and 18 ng/ml. These results indicate that active form of TGF-β is directly produced by tumor cells in patients with glioma, and may contribute to immunodeficiency of the host.     

Induction of Growth Factor-receptor and Metalloproteinase Genes by Epidermal Growth Factor and/or Transforming Growth Factor-α in Human Gastric Carcinoma Cell Line MKN-28

We examined the effects of epidermal growth factor (EGF) and transforming growth factor-α (TGF-α) on EGF receptor (EGFR) phosphorylation and the expression of mRNAs for oncogenes, growth factors, their receptors and metalloproteinase genes by MKN-28 gastric carcinoma cells which express EGF, TGF-α and EGFR genes. Both EGF and TGF-α stimulated EGFR phosphorylation. EGF and TGF-α induced FOS, MYC and ERBB-2 oncogene expression. Interestingly, EGF increased the expression of mRNAs for TGF-α and EGFR. On the other hand, TGF-α increased TGF-α mRNA but decreased the expression of mRNAs for EGFR and TGF-β. Furthermore, mRNAs for interstitial collagenase, stromelysin and procollagen type I genes were also enhanced after treatment with EGF and TGF-α. These results indicate that EGF and TGF-α successively evoke cascade phenomena which favor tumor progression, invasion and extracellular matrix formation, acting as autocrine growth regulators for gastric carcinomas.     

Expression of Integrin α3 in Gastric and Colorectal Cancers: Its Relation to Wall Contraction and Mode of Invasion

We macroscopically classified 25 gastric and 23 colorectal advanced cancers into "contracted" and "uncontracted" types, and found immunohistochemically that integrin subunit α3 was more frequently expressed in the extracellular matrix (ECM) in the former than in the latter (75%:9/12 vs. 38%: 5/13 in gastric and 86%:6/7 vs. 25%:4/16 in colorectal cancers, respectively). Integrin subunit α3 was also expressed more frequently in cancers producing transforming growth factor-beta (TGF-β), which is related to ECM deposition, integrin expression and cell mobility, than in those which did not produce TGF-β (67%:10/15 vs. 40%:4/10 in gastric and 57%:4/7 vs. 38%:6/16 in colorectal cancers, respectively). In addition, integrin subunit α3 was not expressed in 2 benign gastric ulcers combined with gastric cancer elsewhere in the stomach. On the other hand, a retrospective analysis of 107 cases of rectal cancer which recurred after a curative operation revealed that local recurrence was more frequent in "contracted" than "uncontracted" types (44%:ll/25 vs. 26%:21/82). These results may suggest that the abundant interstitial fibrosis which leads to remarkable gastric or colorectal wall contraction is a result of the interaction between cancer cells and ECM, along with the expression of integrin and/or the production of TGF-β, This fibrosis may also be closely related to the mode of gastric and colorectal cancer invasion.     

Estrogen Inhibits the Growth of MCF-7 Cell Variants Resistant to Transforming Growth Factor-beta

Human breast cancer MCF-7 cells containing estrogen receptor are killed by transforming growth factor-beta (TGF-β). We isolated variants of MCF-7 highly resistant to TGF-β. Variants ES-1 and ES-4 were cloned, and the growth of ES-1 and ES-4 was found to be inhibited by estradiol, whereas estradiol stimulated the growth of the parental MCF-7 cells. ES-1 cells contained about 2-fold higher level of estradiol receptor than MCF-7 cells. Addition of estradiol to the culture medium for MCF-7 and the variant changed the expression of several secreted proteins. The repertoire of secreted proteins was markedly altered in the variant. Polypeptides of molecular weight 52,000 (52 K), 65 K and 160 K were increased about 10- to 50-fold in both estradiol-treated MCF-7 and ES-1 cells. Polypeptide of 130 K was decreased in estradiol-treated ES-1 cells while this polypeptide was increased about 4-fold in estradiol-treated MCF-7, as compared with untreated MCF-7. Polypeptide of 100 K was specifically secreted in ES-1 whether or not estradiol was present, but there appeared to be no significant amount of the 100 K protein in MCF-7. The estradiol-hypersensitive phenotype is discussed in relation to its aberrant expression of secreting proteins.     

Wakame Seaweed Suppresses the Proliferation of 7,12-Dimethylbenz(a)-anthracene-induced Mammary Tumors in Rats

We examined the anti-tumor proliferation effects of wakame seaweed on 7,12-dimethylbenz(a)-anthracene (DMBA)-induced rat mammary tumor. DMBA was administered to 8-week-old female Sprague-Dawley rats, and rats which developed mammary tumors were assigned randomly to three groups. Commercial rat feed was used in a control group (group I-A), and two feed mixtures were prepared, which contained commercial rat feed blended with wakame at 1.0% (group I-B) and 5.0% (group I-C) by weight. The respective feeds were given to each group for 8 weeks, and changes in mammary tumor size were compared. At the end of the experiment, mammary tumors and thyroid glands were resected to compare their weights. Serum total iodine and thyroxin (T4) levels were measured. Immunohistochemical studies for bromodeoxyuridine (BrdU) labeling, transforming growth factor (TGF)-β, and apoptosis were carried out in the resected tumor. Significant suppression of tumor growth was observed in groups I-B and I-C compared with I-A. In groups I-B and I-C, the weights of resected mammary tumors were significantly lower and serum total iodine concentration was significantly higher than in I-A. BrdU indices were significantly lower in groups I-B and I-C, compared with I-A. TGF-β and apoptotic index were inversely related to BrdU. These results suggest that iodine is transported from the serum into mammary tissues and induces apoptosis through the expression of TGF-β. In conclusion, wakame suppressed the proliferation of DMBA-induced mammary tumors.     

Progressive tumor features accompany epithelial-mesenchymal transition induced in mitochondrial DNA-depleted cells

The growth of LNCaP, a human prostate adenocarcinoma cell line, and MCF-7, a human breast adenocarcinoma cell line, is initially hormone dependent. We previously demonstrated that LNρ0-8 and MCFρ0, derived from LNCaP and MCF-7 by depleting mitochondrial DNA (mtDNA), exhibited hormone-independent growth that led to progressed phenotypes. Here, we demonstrate that LNρ0-8 and MCFρ0 have invasive characters as evaluated by the ability of invasion through the extracellular matrix (ECM) in vitro . In addition, the induction of vimentin and the repression of E-cadherin expression in ρ0 cells indicate that they are mesenchymal cells. Since LNρ0-8 and MCFρ0 were derived from epithelial cancer cell lines, LNCaP and MCF-7 must have lost epithelial features and gained the mesenchymal phenotype by epithelial-mesenchymal transition (EMT) during the mtDNA depletion. In the ρ0 cell lines, the Raf/MAPK signaling cascade was highly activated together with the expressions of transforming growth factor-beta (TGF-β) and type I TGF-β receptor (TGF-βRI). EMT requires cooperation of TGF-β signaling with activation of the Raf/MAPK cascade, suggesting that EMT was induced in mtDNA depleted cells resulting in the acquisition of progressive tumor features, such as higher invasiveness and loss of hormone dependent growth. Our results indicate that decreasing mtDNA content induces EMT, enabling the progressive phenotypes observed in cancer. ( Cancer Sci 2008; 99: 1584–1588)     

Clonal Heterogeneity in Human Esophageal Squamous Cell Carcinomas on DNA Analysis

Cancers are thought to arise through multistep accumulation of somatic mutations in the progeny of a single cell. Multiple mutations may induce molecular intratumor heterogeneity. Therefore, we examined molecular clonal heterogeneity in esophageal squamous cell carcinomas. Twenty-four esophageal squamous cell carcinomas and associated lymph node metastases were examined for microsatellite alterations, and abnormalities of the p53 and transforming growth factor-β type II receptor ( TGF-β RII ) genes. There were eight cases (33%) showing different patterns of loss of heterozygosity in primary tumors and metastatic lymph nodes with microsatellite markers. On the other hand, the abnormalities of p53 were identical in all these cases. No mutation was detected in the simple repeated sequences of the TGF-β RII gene. These results indicate that molecular clonal heterogeneity exists in esophageal squamous cell carcinomas. Therefore, care is necessary in preoperative genetic diagnosis using biopsy samples.     

The use of cystatin C to inhibit epithelial–mesenchymal transition and morphological transformation stimulated by transforming growth factor-β

Introduction  

Transforming growth factor-β (TGF-β) is a potent suppressor of mammary epithelial cell (MEC) proliferation and is thus an inhibitor of mammary tumor formation. Malignant MECs typically evolve resistance to TGF-β-mediated growth arrest, enhancing their proliferation, invasion, and metastasis when stimulated by TGF-β. Recent findings suggest that therapeutics designed to antagonize TGF-β signaling may alleviate breast cancer progression, thereby improving the prognosis and treatment of breast cancer patients. We identified the cysteine protease inhibitor cystatin C (CystC) as a novel TGF-β type II receptor antagonist that inhibits TGF-β binding and signaling in normal and cancer cells. We hypothesized that the oncogenic activities of TGF-β, particularly its stimulation of mammary epithelial–mesenchymal transition (EMT), can be prevented by CystC.     

Frequent Microsatellite Instabilities and Analyses of the Related Genes in Familial Gastric Cancers

Microsatellite instability or replication error seems to be related to defective DNA mismatch repair genes, such as hMSH2 , hMLH1 , hPMS1 and hPMS2 , which have been identified as causative genes of hereditary nonpolyposis colorectal cancers (HNPCC). Recently, it was reported that mutations at the simple repeated sequences in the transforming growth factor-β type II receptor ( TGF-β RII ) gene occurred in replication error-positive colorectal cancers. To determine genetic alterations in familial gastric cancers (FGC), we examined replication error using eight microsatellite DNA markers, and screened mutations in the hMSH2 , hMLH1 and TGF-β RII genes in six cases from four FGC kindreds. Moreover, hMTH1 , a human homolog of the bacterial mutT gene, was also screened. Four of six (67%) cancers showed the replication error-positive phenotype, indicating that microsatellite instability is highly associated with not only HNPCC, but also FGC. No germline mutation was found in the whole coding sequences of hMSH2 and hMTH1 , or in the conservative regions of hMLH1 in any patient, while one cancer DNA showed a somatic mutation at codon 682 (threonine to alanine) in hMSH2 . No alteration was found at the small repeated sequences in TGF-β RII in FGC tumor DNA. These results indicate that the carcinogenetic process of FGC may be different from that of HNPCC.     

Potentiation of Invasive Capacity of Rat Ascites Hepatoma Cells by Transforming Growth Factor-β

The in vitro invasive capacity of poorly invasive cells (W₁), which were cloned from rat ascites hepatoma cells (AH 130), was potentiated dose- and time-dependently by pretreating the cells with transforming growth factor-β (TGF-β). This potentiation of invasive capacity was completely abolished by anti-TGF-β antibody. When the treated cells were ip inoculated into rats, the cells extensively invaded the peritoneum, and formed penetrating tumor nodules. The effect of TGF-β was reversed by subculturing the treated cells without TGF-β. The potentiation of invasive capacity by TGF-β might participate in platelet-associated enhancement of tumor cell metastasis.