胃癌组织Ⅱ型β转化生长因子受体基因突变的研究

目的　 β转化生长因子 ( transforming growth factorβ,TGF- β) 型受体 TGF- βR 基因突变是某些肿瘤细胞逃逸 TGF- β生长抑制调控的机制之一。有报道表明 TGF- βR 基因的两个突变热点 ( c DNA70 9- 718,1931- 1936)在人类结肠癌细胞系有高的突变率。据此我们检测了 TGF- βR 基因这两个热点在 4 4例人胃癌组织的改变。方法　组织 DNA提取后进行 PCR- SSCP银染分析 ,将阳性标本进行荧光测序。结果　在 TGF- β R 的 c DNA70 9- 718位点突变率为 6.8% ( 3/ 4 4 ) ,而另一位点 c DNA1931- 1936未发现突变 ( 0 / 4 4 )。结论　TGF- β R 基因在人胃癌组织较之结肠癌具有低的突变率。     

Gene mutation of transforming growth factor beta1 type II receptor in hepatocellular carcinoma.

Alteration of transforming growth factor beta1 (TGF-beta1) type II receptor (RII) appears to cause unresponsiveness to TGF-beta1 in tumorigenic cells. Defect in the mononucleotide repeat sequence, i.e., poly A region of TGF-beta1RII gene has been reported to be related to replication error-positive cancer cells. We examined if there is any TGF-beta1RII mutation in a coding microsatellite in hepatocellular carcinoma (HCC). Genomic DNAs were extracted from formalin-fixed, paraffin-embedded liver tissues obtained at surgery or autopsy in 3 normal individuals and 96 patients with hepatitis C virus-induced chronic liver disease; 3 with chronic hepatitis, 20 with liver cirrhosis and 73 with HCC. The DNA was PCR-amplified at 2 segments of TGF-beta1RII: poly A region which includes the (A)10 microsatellite sequence, and poly GT region. PCR products were directly sequenced. DNA from normal and patients with chronic liver disease contained the 10 wild-type adenines but 3 cases with liver cirrhosis in whom there were only 9 adenines within poly A tract. This microdeletion of one A resulted in a frameshift and truncated a predicted length of amino acids. In HCC lesions, the same deletion was noted in 4 cases (25%) of well-differentiated type, 10 (40%) of moderately differentiated type, 18 (53%) of poorly differentiated type. None of the lesions had mutations within the GT region. Our findings indicate that one adenine deletion of poly A microsatellite tract within TGF-beta1RII is frequently detected in patients with HCC, and the mutation may cause the abrogation of the function of TGF-beta1RII gene.     

The progression of invasiveness regarding the role of transforming growth factor beta receptor type II in gastric cancer.

AIMS: Transforming growth factor beta (TGF beta) is a potent growth inhibitor of epithelial cells. The expression of TGF beta receptors is required for the effect of TGF beta. In this study, we used immunohistochemistry to demonstrate the roles of the expression of TGF beta type I (T beta R-I) and type II (T beta R-II) receptors in the progression of gastric carcinoma. METHODS: To evaluate the potential prognostic value of T beta R-I and T beta R-II, 158 consecutive gastric cancer tissues specimens obtained over a 3-year period were examined. RESULTS: A total of 50 (32%) and 28 (18%) patients had T beta R-I(+) and T beta R-II(+), respectively. The 5-year survival rates of the patients with T beta R-I(+) and those with T beta R-I(-) were 74% and 71%, respectively. In contrast, the 5-year survival rates of the patients with T beta R-II(+) and those with T beta R-II(-) were 57% and 75%, respectively, and the difference was statistically significant (P<0.05). The extent of T beta R-II was closely correlated to the macroscopic types based on the Borrmann classification (P<0.01), and curability (P<0.05). However, a significant difference between the 5-year survival rates of the patients with T beta R-II(+) and those with T beta R-II(-) was only obtained in advanced cases (P<0.05) not in either curative cases, non-curative cases, or early cases. CONCLUSIONS: Our data suggest that when T beta R-II expression correlates with the progression of invasiveness in gastric cancer, it may lead to a non-curative resection and a poor prognosis.     

Inhibiting mutations in the transforming growth factor beta type 2 receptor in recurrent human breast cancer

Members of the transforming growth factor beta (TGF-beta) family are potent inhibitors of the growth of many epithelial cell types. Transmembrane signaling by TGF-beta occurs via a complex of the serine/threonine kinases TGF-beta type 1 receptor and TGF-beta type 2 receptor (TGFBR2), and inactivating mutations in the latter have recently been detected in some primary tumors and in several types of tumor-derived cell lines. The most common mutations that have been identified in TGFBR2 are frameshifts in a repetitive polyadenine region in replication error-positive colorectal carcinomas that result in a truncated protein and absence of receptor expression at the cell surface. A number of point mutations in the highly conserved serine/threonine kinase domain of TGFBR2 have also been reported, some of which have been correlated with either loss of trans-phosphorylation of TGF-beta type 1 receptor or constitutive activation of trans-phosphorylation. No TGFBR2 mutations have been reported in human breast tumors, but anomalous expression of TGF-beta in breast carcinomas suggests that TGF-beta signaling may be defective. We have therefore systematically examined unmatched sets of 17 primary and 17 recurrent breast tumor samples for mutations in TGFBR2, restricted to those regions of the gene in which mutations have previously been reported. None of the previously reported mutations was detected, but four novel mutations (V387M, N435S, V447A, and L452M) were found in the kinase domain in recurrent tumors. No mutations were detected in primary tumors. TGF-beta signaling was significantly inhibited by each of the N435S, V447A, and L452M mutations.     

Mutational analysis of the transforming growth factor beta receptor type II gene in hereditary nonpolyposis colorectal cancer and early-onset colorectal cancer patients.

Somatic mutations in the transforming growth factor beta receptor type II (TGF-beta RII) gene have been observed in various human cancers showing microsatellite instability. Most of the mutations observed were additions or deletions of the mononucleotide repeat sequence present in TGF-beta RII coding region, suggesting that the TGF-beta RII may be a target gene of genomic instability in tumorigenesis. Recently, we reported germ-line frameshift mutations in the mononucleotide repeat sequence of the hMSH6 gene, which is believed to be one of the target genes of genomic instability in tumorigenesis, suggesting the possibility of germ-line mutation in mononucleotide repeat sequences. Moreover, one case of germ-line mutation in the TGF-beta RII gene was identified in a hereditary nonpolyposis colorectal cancer (HNPCC) kindred, indicating the involvement of TGF-beta RII inactivation in tumorigenesis of HNPCC. However, germ-line mutation analysis of all of the coding sequences and the mononucleotide repeat sequence of the TGF-beta RII in HNPCC patients has not yet been fully elucidated. Therefore, to further investigate the presence of germ-line mutations, we screened all of the coding region sequences and mononucleotide repeat sequence of TGF-beta RII from 35 HNPCC, 44 suspected HNPCC, and 45 sporadic early-onset colorectal cancer patients. However, no pathogenic mutations other than silent mutations, introgenic mutation, and polymorphisms were identified. Two silent mutations at codons 309 (ACG to ACA) and 340 (CAT to CAC) in the kinase domain located in exon 4 were detected. A 1-bp cytidine deletion was observed 6 bases from the 3' end of intron. Two polymorphisms were identified at codon 389 (AAC to AAT) and at the fourth-to-last base in intron 3. The polymorphism at codon 389 was more frequent in HNPCC (20%; 7 of 35) and suspected HNPCC patients (18%; 8 of 44) than in nonmalignant control group (10%; 5 of 50). Moreover, the frequency was significantly higher in early-onset colorectal cancer patients (31%; 14 of 45). This is the first report of a different frequency of polymorphism in HNPCC, suspected HNPCC, early-onset colorectal cancer patients, and healthy normal individuals. This result suggests that: (a) germ-line mutation of the TGF-beta RII gene may be a rare event during tumorigenesis in HNPCC and sporadic early-onset colorectal cancer; (b) the mononucleotide repeat sequence of the TGF-beta RII gene is an apparent target of genomic instability but not of germ-line mutation; and (c) the polymorphism of codon 389 (AAC to AAT) is frequent, especially in early-onset colorectal cancer patients, in which it is more frequent than in control group.     

Mutations of the transforming growth factor β type II receptor gene and microsatellite instability in gastric cancer

Forty-three sporadic gastric cancers were analyzed with regard to whether mutations of simple repeated sequences in the transforming growth factor β type II receptor (TβR-II) gene are associated with microsatellite instability (MSI) and gastric carcinogenesis. In 12 of the 43 cancers (28%), MSI was observed at least at 1 of the 2 microsatellite loci. Frameshift mutations of the TβR-II gene, all of which were 1 base deletion of 10 adenine repeats, were detected in 3 of 6 cancers, with MSI at 2 loci. However, mutations were not detected in 6 cancers, with MSI only at 1 locus and 31 cancers without MSI. Moreover, micro-analysis in these cases revealed that the mutant-type alleles of TβR-II were invariably common in different areas within the tumor, in contrast to the markedly variable alleles of microsatellite loci. Our results suggest that frameshift mutation of the TβR-II gene may be a critical event associated with MSI and may contribute to carcinogenesis of the stomach. One of the possible mechanisms of escape from growth control by TGFβ during gastric carcinogenesis could involve frameshift mutations of the TβR-II gene caused by DNA replication errors. © 1996 Wiley-Liss, Inc.     

Microsatellite instability, Epstein-Barr virus, mutation of type II transforming growth factor beta receptor and BAX in gastric carcinomas in Hong Kong Chinese

Microsatellite instability (MI), the phenotypic manifestation of mismatch repair failure, is found in a proportion of gastric carcinomas. Little is known of the links between MI and Epstein-Barr virus (EBV) status and clinicopathological elements. Examination of genes mutated through the MI mechanism could also be expected to reveal important information on the carcinogenic pathway. Seventy-nine gastric carcinomas (61 EBV negative, 18 EBV positive) from local Hong Kong Chinese population, an intermediate-incidence area, were examined. Eight microsatellite loci, inclusive of the A10 tract of type II transforming growth factor beta receptor (TbetaR-II), were used to evaluate the MI status. MI in the BAX and insulin-like growth factor II receptor (IGF-IIR) genes were also examined. High-level MI (>40% unstable loci) was detected in ten cases (12.7%) and low-level MI (1-40% unstable loci) in three (3.8%). High-level MI was detected in two EBV-associated cases (11%) and the incidence was similar for the EBV-negative cases (13%). The high-level MIs were significantly associated with intestinal-type tumours (P = 0.03) and a more prominent lymphoid infiltrate (P = 0.04). Similar associations were noted in the EBV-positive carcinomas. The high-level MIs were more commonly located in the antrum, whereas the EBV-associated carcinomas were mostly located in body. Thirteen cardia cases were negative for both high-level MI and EBV. All patients aged below 55 were MI negative (P = 0.049). Of the high-level MIs, 80% had mutation in TbetaR-II, 40% in BAX and 0% in IGF-IIR. Of low-level MIs, 33% also had TbetaR-II mutation. These mutations were absent in the MI-negative cases. Of three lymphoepithelioma-like carcinomas, two cases were EBV positive and MI negative, one case was EBV negative but with high-level MI. In conclusion, high-level MIs were present regardless of the EBV status, and were found in a particular clinicopathological subset of gastric carcinoma patient. Inactivation of important growth regulatory genes observed in these carcinomas confirms the importance of MI in carcinogenesis.     

Mutational analysis of the transforming growth factor beta receptor type I gene in primary non-small cell lung cancer

Transforming growth factor-β receptor-dependent signals are critical for cell growth and differentiation and are often disrupted during tumorigenesis. The entire coding region of TGFβRI and flanking intron sequences from 53 primary non-small cell lung cancer (NSCLC) tissues were examined for alterations using SSCP and direct sequencing. No somatic point mutations other than two silent mutations and a polymorphism were found in the TGFβRI gene. The two silent mutations located at codon 344 (AAT to AAC) and codon 406 (TTA to CTA), respectively, and the polymorphism was at the 24th base of intron 7 (G to A). To investigate whether the presence of this polymorphism is associated with NSCLC, we determined its allele distribution in all the 53 carcinomas and 89 normal controls. Interestingly, we found that the subjects with homozygous genotype A/A displayed more than 3-fold increased risk of developing NSCLC than the common wild genotype G/G. As the first report, the present study showed that TGFβRI gene is not a frequent site of spontaneous mutational inactivation while the detected polymorphism is frequent in the pathogenesis of NSCLC.     

Mutational analysis of transforming growth factor beta receptor type II and DNA mismatch repair genes in sporadic endometrial carcinomas with microsatellite instability

To clarify how microsatellite instability (MI) is involved in carcinogenesis of sporadic endometrial carcinoma, we examined mutations of the transforming growth factor beta receptor type II (TGF beta RII) gene in 32 patients with MI-positive sporadic endometrial carcinoma. Moreover, mutations of 4 DNA mismatch repair (MMR) genes (hPMS1, hPMS2, hMLH1, hMSH2), which are considered to cause MI, were investigated as well. With respect to the TGF beta RII gene, mutations in the 10-bp polyadenine repeat sequence were observed in 7 of 29 informative cases (24%). Concerning MMR genes, a T to C point mutation at the -6 intronic splice acceptor site of exon 13 of hMSH2 was detected in 43% (6/14). However, there was no mutation in any exon of these 4 MMR genes. These results suggest that there is a carcinogenic mechanism via mutation of the TGF beta RII gene in some cases of MI-positive sporadic endometrial carcinoma. It seems unlikely that the unknown MMR genes are responsible for MI. The implication of the mutation at the intronic splice acceptor site in hMSH2 remains to be clarified.     

Alteration of cell growth and morphology by overexpression of transforming growth factor beta type II receptor in human lung adenocarcinoma cells

TGF-beta is a potent inhibitory regulator of cell growth, which is transduced through interaction between type I (RI) and type II (RII) receptors that form heteromeric kinase complexes. Abnormal expression of these receptors has been identified in several human epithelial cancers and has been shown to be highly associated with resistance to TGF-beta. In this study, we investigated the expression of RI and RII in 13 human non-small cell lung cancer cell lines (NSCLCs) and demonstrated decreased or loss of RII expression in five lung cancer cell lines, but not of RI. Of these cell lines, the role of RII in NCI-H358 adenocarcinoma, which lacks RII and is insensitive to TGF-beta, was investigated by transducing this cell line with a recombinant retrovirus expressing full-length TGF-beta RII. Stably transfected cells showed significant increase in RII mRNA and protein expression. These cells responded to exogenous TGF-beta1 with suppressed proliferation in a dose-dependent manner and G1 arrest accompanied by morphological change distinct from control cells. We also investigated whether overexpression of dominant-negative RII (dnRII) in NCI-H441 adenocarcinoma, which is sensitive but expresses low levels of RII, could block signaling through the receptor complex. The overexpression of this kinase-domain-truncated RII by expressing the retroviral dnRII construct led to loss of the ability to respond to TGF-beta1 and an exhibition of uncontrolled growth. These results suggest a close association between the loss of the expression of wild-type TGF-beta RII and carcinogenesis in human lung cancer cells.     

Cancer-associated transforming growth factor beta type II receptor gene mutant causes activation of bone morphogenic protein-Smads and invasive phenotype

Transforming growth factor beta (TGFbeta) plays a key role in maintaining tissue homeostasis by inducing cell cycle arrest, differentiation and apoptosis, and ensuring genomic integrity. Furthermore, TGFbeta orchestrates the response to tissue injury and mediates repair by inducing epithelial to mesenchymal transition and by stimulating cell motility and invasiveness. Although loss of the homeostatic activity of TGFbeta occurs early on in tumor development, many advanced cancers have coopted the tissue repair function to enhance their metastatic phenotype. How these two functions of TGFbeta become uncoupled during cancer development remains poorly understood. Here, we show that, in human keratinocytes, TGFbeta induces phosphorylation of Smad2 and Smad3 as well as Smad1 and Smad5 and that both pathways are dependent on the kinase activities of the type I and II TGFbeta receptors (T beta R). Moreover, cancer-associated missense mutations of the T beta RII gene (TGFBR2) are associated with at least two different phenotypes. One type of mutant (TGFBR2(E526Q)) is associated with loss of kinase activity and all signaling functions. In contrast, a second mutant (TGFBR2(R537P)) is associated with high intrinsic kinase activity, loss of Smad2/3 activation, and constitutive activation of Smad1/5. Furthermore, this TGFBR2 mutant endows the carcinoma cells with a highly motile and invasive fibroblastoid phenotype. This activated phenotype is T beta RI (Alk-5) independent and can be reversed by the action of a dual T beta RI and T beta RII kinase inhibitor. Thus, identification of such activated T beta RII receptor mutations in tumors may have direct implications for appropriately targeting these cancers with selective therapeutic agents.     

Transforming growth factor beta type II receptor expression in gastric cancer: evidence for two independent subgroups

Transforming growth factor beta type II receptor (TGFbeta-IIR) has been found to be altered in primary gastrointestinal carcinomas. So far relatively few facts are known about the expression of TGFbeta-IIR in primary gastric cancer. Therefore, in the present study, TGFbeta-IIR expression was analyzed in 130 primary gastric carcinomas and correlated with clinicopathological findings, the presence of a mutator phenotype, the mutational status of the TGFbeta-IIR polyadenine tract and survival. TGFbeta-IIR expression was analyzed immunohistochemically. Microsatellite instability was evaluated using a PCR-based assay and the polyadenine run inside the TGFbeta-IIR gene was sequenced. A complete loss of TGFbeta-IIR expression could be found in 55 (42.3%) of these carcinomas. Loss of TGFbeta-IIR expression was significantly correlated with diffuse-type carcinomas according to the Lauren classification as well as with signet ring cell carcinomas and a lower grade of differentiation. No correlation was found with the overall prognosis, the presence of a mutator phenotype, or a mutated TGFbeta-IIR. Thus, our data suggest the existence of a further definite subgroup of diffuse-type gastric carcinomas with altered TGFbeta-IIR expression, independent from a mutator phenotype with TGFbeta-IIR gene mutations. However, according to our results, in gastric cancer neither loss of TGFbeta-IIR expression nor mutations of the TGFbeta-IIR are of prognostic value.     

Mutation and downregulation of the transforming growth factor beta type II receptor gene in primary squamous cell carcinomas of the head and neck

In the present study, we analyzed 28 squamous cell carcinomas of the head and neck (SCCHN) for mutations in the coding region of TbetaR-II using 'Cold' SSCP and automatic DNA sequencing analyses. Twenty-one percent (6/28) of the SCCHN examined contained TbetaR-II mutations compared with patient-matched normal tissues. These alterations included five missense mutations (A:T-->G:C transitions in codons 250, 401, 448 and 488, and a G:C-->T:A transversion in codon 373), and a 38- bp deletion between nucleotides 1825 to 1862. In addition to these code- altering mutations, one case exhibited a silent mutation (A:T-->G:C transition in codon 451) and three cases contained one of two potential population polymorphisms (codons 354 and 389). In contrast to colon and gastric cancers exhibiting microsatellite instability (MI) or replication errors (RER+), no 'indirect' frameshift mutations were identified within a 10-bp polyadenine repeat present in the TbetaR-II coding sequence. All of the mutations in the present study occurred within the highly conserved serine/threonine kinase domain and represent the first report of such 'direct' TbetaR-II mutations in primary human tumors. In addition, we analyzed a subset of SCCHN and corresponding normal samples for TbetaR-II mRNA expression using semi- quantitative multiplex RT-PCR. Expression of TbetaR-II was decreased by 24% to 74% in 20 of 23 SCCHN (87%) compared with patient-matched normal tissues. Taken together, the results from this study suggest that alterations in the nucleic acid sequence and mRNA expression of TbetaR- II are prevalent events in the development of SCCHN, which may deregulate cell cycle control.      

Alterations of transforming growth factor beta receptor II, insulin growth factor receptor II genes in microsatellite unstable prostate carcinomas

DNA from 45 primary prostate tumors and corresponding normal tissues were analyzed to detect whether the alterations of transforming growth factor beta receptor II (TGFbetaRII) and insulin growth factor receptor II (IGFRII) are associated with microsatellite instability (MSI). We identified that 25 tumors were microsatellite unstable (55%). The remaining 20 tumors are found to be microsatellite stable. Loss of heterozygosity (LOH) was also tested at various loci. Results indicate that in case of TGFbetaRII, the rate of frame-shift mutation depends on the number of polyadenine [poly(A)] tracts. Twelve percent of the tumors had frame-shift alteration at BAT-RII locus which has 10 poly(A) repeats. Twenty percent of the tumors had frame-shift at BAT-25 locus which has 25 poly(A) repeats. In addition, IGFRII gene was examined for the presence of mutation in the repetitive sequences. Seven of the 25 tumors showed deletion of a G within eight poly(G) repeats. Besides these changes there were two tumors which showed a novel insertion of A within this poly(G) repeat making a change in 9 samples (R4, 36%). On the other hand, 4 tumors showed changes within the 5CT repeats. In addition, 3 tumors showed another novel insertion of C within the CT repeats.     

Prognostic significance of transforming growth factor beta receptor II in estrogen receptor-negative breast cancer patients.

PURPOSE: The role of transforming growth factor beta (TGF-beta) in breast cancer is ambiguous; it can display both tumor suppressing and enhancing effects. Activation of the TGF-beta signal transduction system is subject to hormonal regulation. This study was conducted to further analyze the role of TGF-beta receptors in breast cancer and to evaluate their significance as prognostic markers. EXPERIMENTAL DESIGN: Expression of TGF-beta receptor I (TbetaRI) and TGFbeta receptor II (TbetaRII) was retrospectively analyzed by immunohistochemistry in 246 breast cancer patients. RESULTS: Expression of TbetaRI was strongly correlated with tumor size (P < 0.001) and nodal status (P = 0.012) but only weakly with overall survival (P = 0.056). In contrast, TbetaRII was prognostic for overall survival in univariate analysis (P = 0.0370). In estrogen receptor (ER) -negative patients TbetaRII expression was correlated with highly reduced overall survival (P = 0.0083). In multivariate analysis TbetaRII proved to be an independent and highly significant prognostic marker with a hazard ratio of 6.8. Simultaneous loss of both ER and TbetaRII was associated with longer overall survival times comparable with those of ER-positive patients. CONCLUSIONS: The results of this exploratory study show that TbetaRII is an independent, highly significant prognostic indicator for overall survival in ER-negative patients. In addition our results are supportive of a mechanism of breast cancer progression in which a selective loss of the tumor inhibitory action of TGFbeta takes place, whereas tumor- promoting aspects remain intact.     

Tumorigenicity of mouse thymoma is suppressed by soluble type II transforming growth factor beta receptor therapy

Many types of tumor cells overexpress transforming growth factor beta (TGF-beta), which is believed to promote tumor progression. We hypothesized that overexpression of the extracellular region of the type II TGF-beta receptor (soluble TbetaRII) would compete for or block TGF-beta binding to TbetaRs on immune cells, preventing TGF-beta-mediated immunosuppression and consequently resulting in the eradication of tumor cells. We tested this in the mouse thymoma cell line EL4, which has been reported to suppress cellular immunity by secreting a large amount of TGF-beta. Transduction of EL4 with recombinant retrovirus encoding soluble TbetaRII resulted in the secretion of heterogeneously glycosylated, 25 to 35 kDa truncated TbetaRII. Inoculation of 1 x 10(4) to 5 x 10(4) soluble TbetaRII-modified EL4 cells (EL4/Ts, EL4 cells transduced with recombinant retrovirus encoding soluble TbetaRII and neomycin resistance gene) s.c. to mice showed reduced tumorigenicity, as indicated by lower overall tumor incidence (7%, 1 of 14; P < 0.001) compared with unmodified EL4 (100%, 9 of 9) or vector-modified EL4 cells (EL4/neo, EL4 cells transduced with recombinant retrovirus encoding neomycin resistance gene; 100%, 4 of 4). Administration of mitomycin C-treated EL4/Ts cells (1 x 10(6)) after EL4 inoculation (1 x 10(4)) reduced tumor incidence from 100% (5 of 5 in mice inoculated with mitomycin C-treated EL4/neo) to 40% (4 of 10, P < 0.05), indicating that supply of soluble TbetaRII could actually block TGF-beta-mediated tumorigenesis. In vitro tumor cytotoxicity assays revealed 3-5-fold higher cytotoxic activity with lymphocytes from EL4/Ts-bearing mice compared with those from EL4- or EL4/neo-bearing mice, indicating that the observed tumor rejection was mediated by restoration of the tumor-specific cellular immunity. These data suggest that expression of soluble TbetaRII is an effective strategy for treating highly progressive tumors secreting TGF-beta.