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.     

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.     

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.     

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.     

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.      

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.     

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.     

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.     

转化生长因子β1基因多态性与结直肠癌的相关性分析

目的分析转化生长因子β1(TGF-β1)基因多态性与结直肠癌的相关性。方法选取2008年1月至2013年1月间诊治的109例结直肠癌患者为观察组,120例健康体检者作为对照组。分析对比两组TGF-β1基因多态性及其与结直肠癌的相关性。结果两组TGF-β1 869C及869T等位基因频率差异无统计学意义(P>0.05)。观察组患者TGF-β1的509T等位基因频率显著高于对照组(χ~2=6.109,P=0.013)。Logistic回归分析显示,携带TGF-β1(509T)基因是发生结直肠癌的独立危险因素(OR=3.512,95%CI为2.136~5.641)。结论 TGF-β1 509基因与结直肠癌发病相关,其T等位基因可能是结直肠癌发病的危险因素。     

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.     

Coordinated functions of E-cadherin and transforming growth factor beta receptor II in vitro and in vivo

In epithelial cells, E-cadherin plays a key role in cell-cell adhesion, and loss of E-cadherin is a hallmark of tumor progression fostering cancer cell invasion and metastasis. To examine E-cadherin loss in squamous cell cancers, we used primary human esophageal epithelial cells (keratinocytes) as a platform and retrovirally transduced wild-type and dominant-negative forms of E-cadherin into these cells. We found decreased cell adhesion in the cells expressing dominant-negative E-cadherin, thereby resulting in enhanced migration and invasion. To analyze which molecular pathway(s) may modulate these changes, we conducted microarray analysis and found up-regulation of transforming growth factor beta receptor II (TbetaRII) in the wild-type E-cadherin-overexpressing cells, which was confirmed by real-time PCR and Western blot analyses. To investigate the in vivo relevance of this finding, we analyzed tissue microarrays of paired esophageal squamous cell carcinomas and adjacent normal esophagus, and we could show a coordinated loss of E-cadherin and TbetaRII in approximately 80% of tumors. To determine if there may be an E-cadherin-dependent regulation of TbetaRII, we show the physical interaction of E-cadherin with TbetaRII and that this is mediated through the extracellular domains of E-cadherin and TbetaRII, respectively. In addition, TbetaRI is recruited to this complex. When placed in the context of three-dimensional cell culture, which reflects the physiologic microenvironment, TbetaRII-mediated cell signaling is dependent upon intact E-cadherin function. Our results, which suggest that E-cadherin regulates TbetaRII function, have important implications for epithelial carcinogenesis characterized through the frequent occurrence of E-cadherin and TbetaRII loss.     

Forkhead家族转录因子FoxM1与肿瘤

目的 随着分子生物学和分子肿瘤学的深入研究,运用肿瘤发生发展中的关键分子做为诊断标志和治疗靶点已成为肿瘤诊治的新趋势.本研究旨在分析Forkhead家族转录因子FoxM1介导肿瘤发生发展的机制及其在分子诊断和靶向干预中的价值.方法 应用PubMed及CNKI期刊全文数据库检索系统,以“FoxM1和肿瘤”为关键词,检索2011-03-2016-02的所有相关文献,共检索到英文文献383篇,中文文献97篇.纳入标准:(1)FoxM1介导肿瘤发生发展的机制;(2)FoxM1与肿瘤分子诊断及靶向干预.根据纳入标准,符合分析的文献44篇.结果 FoxM1通过调控细胞周期G1/S和G2/M期促转化分子Skp2、Cksl、C myc和细胞周期调节分子Cdc25A、Cdc25B、Cyclin D1、p27kip1和p21cip1等表达,影响细胞有丝分裂、细胞周期、DNA损伤修复、细胞增殖与分化等生物学过程.FoxM1过表达导致细胞异常增殖、新生血管形成、上皮间质转化、维持干细胞特性、代谢异常和细胞逃逸衰老等生物学表征,促进细胞恶性转化,参与肿瘤发生发展.FoxM1还可调节多种肿瘤细胞对药物敏感性与耐药性.基于FoxM1的恶性肿瘤诊断系统对多种早期癌症的诊断具有良好价值.以FoxM1为靶点的抗肿瘤化合物通过抑制肿瘤细胞中过度表达的FoxM1成为临床治疗的潜在的新策略.结论 FoxM1过表达促进肿瘤发生发展,检测FoxM1分子有助于肿瘤早期识别,抑制FoxM1可以靶向干预肿瘤的发生发展,FoxM1是肿瘤诊疗的潜在靶标.     

Mutation analysis of transforming growth factor beta type II receptor, Smad2, Smad3 and Smad4 in esophageal squamous cell carcinoma

Mutations of the transforming growth factor beta type II receptor (TGFbetaRII) gene and Smad family genes have been observed in several human cancers. However, there has been no report on mutation analysis of the entire coding regions of these genes in esophageal cancer, and the role of these genes in the development of esophageal cancer remains unknown. We performed polymerase chain reaction-single strand conformation polymorphism analysis of TGFbetaRII, Smad2, Smad3 and Smad4 genes and microsatellite assay in 20 esophageal squamous cell carcinomas (ESCC). We detected polymorphisms at exon 2 of Smad3 and intron 6 of Smad4. No mutation was found in TGFbetaRII, Smad2, Smad3 and Smad4 genes. These results suggest that mutation of TGFbetaRII, Smad2, Smad3 and Smad4 genes is a rare event in ESCC.