Genetic alterations of the transforming growth factor beta receptor genes in pancreatic and biliary adenocarcinomas

Transforming growth factor beta (TGF-beta) is an extracellular ligand that binds to a heterodimeric receptor, initiating signals that regulate growth, differentiation, and apoptosis. Many cancers, including pancreatic cancer, harbor defects in TGF-beta signaling and are resistant to TGF-beta-mediated growth suppression. Genetic alterations of DPC4, which encodes a DNA binding protein that is a downstream component of the pathway, most frequently occur in pancreatic and biliary carcinomas. We searched for other targets of mutation of the TGF-beta pathway in these cancers. We report somatic alterations of the TGF-beta type I receptor gene ALK-5. Homozygous deletions of ALK-5 were identified in 1 of 97 pancreatic and 1 of 12 biliary adenocarcinomas. A germ-line variant of ALK-5, presumably a polymorphism, was identified, but no somatic intragenic mutations were identified upon sequencing of all coding regions of ALK-5. Somatic alterations of the TGF-beta type II receptor gene (TGFBR2) were identified in 4 of 97 (4.1%) pancreas cancers, including a homozygous deletion in a replication error-negative cancer and three homozygous frameshift mutations of the poly(A) tract of the TGF-beta type II receptor in replication error-positive cancers. We also studied other related type I receptors of the TGF-beta superfamily. In a panel of pancreas cancers preselected for loss of heterozygosity at the ALK-1 locus, sequencing of all coding exons of the ALK-1 gene revealed no alterations. No homozygous deletions were detected in the ALK-1, ALK-2, ALK-3, or ALK-6 genes in a panel of 86 pancreatic cancer xenografts and 11 pancreatic cancer and 22 breast cancer cell lines. The rate of genetic inactivation of TGF-beta pathway members was determined in 45 pancreatic cancers. Eighty-two % of these pancreatic cancers had genetic inactivation of the DPC4, p15, ALK-5, or TGFBR2 genes. Our results indicate that the TGF-beta type I and type II receptor genes are selective targets of genetic inactivation in pancreatic and biliary cancers.     

Functional characterization of transforming growth factor beta type II receptor mutants in human cancer

We recently identified missense mutations at amino acid residues 526 and 537 located within the highly conserved subdomain XI of the transforming growth factor beta type II receptor (TbetaR-II) serine-threonine kinase in two human squamous carcinoma cell lines. These cell lines are resistant to transforming growth factor beta-mediated inhibition of growth. Moreover, treatment with transforming growth factor beta fails to increase the levels of type 1 plasminogen activator inhibitor and fibronectin synthesis. To test the effects of the mutations on receptor function, mutant TbetaR-II cDNAs were expressed in TbetaR-II-deficient T47D cells. Cyclin A promoter activity was reduced by 50% in cells expressing wild-type TbetaR-II but increased 2-fold in cells transfected with either of the two mutant receptors. Conversely, plasminogen activator inhibitor type 1 promoter activity was increased 6-fold in cells transfected with wild-type receptor but not with either of the two mutant receptors. Moreover, the activity of both mutant serine-threonine kinases was strongly reduced compared to that of the wild-type receptor. Thus, the amino acid residues at positions 526 and 537 seem to be essential for kinase function and signaling activity of the TbetaR-II.     

Aberrant expression of type I fibroblast growth factor receptor in human pancreatic adenocarcinomas

Acidic and basic fibroblast growth factors are mitogenic polypeptides that are overexpressed in pancreatic cancer. To determine whether fibroblast growth factors may exert direct effects on pancreatic cancer cells in vivo, we compared the expression of the high-affinity type I fibroblast growth factor receptor (FGFR-1) in human pancreatic tissues. In the normal pancreas, FGFR-1 immunostaining was seen mainly in acinar cells. In pancreatic cancers, FGFR-1 was abundant in ductal-like cancer cells which also exhibited many FGFR-1 mRNA in situ hybridization grains. Analysis by the polymerase chain reaction and RNase protection revealed that the 2-immunoglobulin-like and the 3-immunoglobulin-like forms of FGFR-1 were expressed in all tissue samples, and that the 2-immunoglobulin-like form was overexpressed in the cancer tissues by comparison with the normal tissues. These findings suggest that the 2-immunoglobulin-like form of FGFR-1 may contribute to aberrant autocrine and paracrine pathways in pancreatic cancer.     

Reduced levels of transforming growth factor beta receptor type II in human prostate cancer: an immunohistochemical study

In previous studies we demonstrated that the growth of human prostatic adenocarcinoma is associated with aberrant accumulation of transforming growth factor (TGF) beta1, a growth factor that has been shown to be a potent inhibitor of epithelial cell proliferation. We investigated the expression of TGF-beta receptor II (TGFbetaR-II) in benign prostate tissue and in prostate cancer using standard immunohistochemical techniques. Quantitation of immunopositivity for TGFbetaR-II was assessed on a visual analogue scale ranging from 0 (absence of staining) to 4+ (intensely positive staining). All of the benign glandular epithelia stained intensely, either 3+ or 4+, representative of the ubiquitous nature of TGFbetaR-II in normal tissue. Overall, staining was reduced in prostate cancer sections, and there was progressively diminished staining as the histological grade of the cancer increased (P < 0.01, Kruskal-Wallis test). This immunohistochemical study indicates that a decline in the levels of TGFbetaR-II is correlated with advancing histological aggressiveness of the cancer and suggests that aberrant TGFbetaR-II function may play a role in human prostate carcinogenesis.     

Expression of transforming growth factor beta receptors in normal human colon and sporadic adenocarcinomas

BACKGROUND & AIMS: An absence or a presence of mutated transforming growth factor (TGF)-beta receptors is a possible hypothesis explaining the resistance of cancer cells to the growth-inhibitory effect of TGF-beta. Mutations involving microsatellite-like regions of the type II TGF-beta receptor have been described in subgroups of colorectal cancers. The aim of this study was to investigate the expression and distribution of TGF-beta receptors in sporadic colorectal cancers and normal tissues. METHODS: Thirty-three sporadic colorectal cancers and 20 normal colonic tissues were explored by immunohistochemistry for the expression of type I and type II TGF-beta receptors. Eighteen tumor and 20 normal samples were used for radioactive thermocycling and sequencing of the two microsatellite-like regions of the type II receptor. RESULTS: Both receptors were overexpressed in tumors compared with normal samples. There was a relationship between the abundance of type II receptor expression and the degree of differentiation of the tumors but not the Dukes' staging or the localization of the neoplasias. No mutation was observed in the microsatellite-like regions of receptor II in any of the samples. CONCLUSIONS: Sporadic colorectal cancers do not show an absence or a presence of mutated TGF-beta receptors that could explain a resistance to TGF-beta-mediated growth inhibition. The pathways to tumorigenesis of sporadic colorectal cancers may be different from those of some hereditary ones.     

Function of the type V transforming growth factor beta receptor in transforming growth factor beta-induced growth inhibition of mink lung epithelial cells

The type V transforming growth factor beta (TGF-beta) is a 400-kDa nonproteoglycan membrane protein that co-expresses with the type I, type II, and type III TGF-beta receptors in most cell types. The type V TGF-beta receptor exhibits a Ser/Thr-specific protein kinase activity with distinct substrate specificity (Liu, Q., Huang, S. S., and Huang, J. (1994) J. Biol. Chem. 269, 9221-9226). In mink lung epithelial cells, the type V TGF-beta receptor was found to form heterocomplexes with the type I TGF-beta receptor by immunoprecipitation with antiserum to the type V TGF-beta receptor after 125I-TGF-beta affinity labeling or Trans35S-label metabolic labeling of the cells. The kinase activity of the type V TGF-beta receptor was stimulated after treatment of mink lung epithelial cells with TGF-beta. TGF-beta stimulation resulted in the growth inhibition of wild-type mink lung epithelial cells and to a lesser extent of the type I and type II TGF-beta receptor-defective mutants, although higher concentrations of TGF-beta were required for the growth inhibition of these mutants. TGF-beta was unable to induce growth inhibition in human colorectal carcinoma cells lacking the type V TGF-beta receptor but expressing the type I and type II TGF-beta receptors. These results suggest that the type V TGF-beta receptor can mediate the TGF-beta-induced growth inhibitory response in the absence of the type I or type II TGF-beta receptor. These results also support the hypothesis that loss of the type V TGF-beta receptor may contribute to the malignancy of certain carcinoma cells.     

Analyses of mutation and loss of heterozygosity of coding sequences of the entire transforming growth factor beta type II receptor gene in sporadic human gastric cancer

Mutations in the transforming growth factor beta type II receptor (TGFbetaRII) gene have been detected in several human cancer types exhibiting microsatellite instability. Using intron primers previously reported for examination of the entire coding region of the TGFbetaRII gene, 29 sporadic gastric cancers were screened with non-radioactive single strand conformation polymorphism and subsequent DNA sequencing analysis. Mutations of the TGFbetaRII gene were detected in three out of 29 tumors (10%). Two cases showed deletions in a polyadenine tract in both alleles and was positively associated with replication error. One case had an insertion of GA dinucleotide sequence in one allele. Mutations of the TGFbetaRII gene were restricted to exon 3 and other coding regions were not affected. Loss of heterozygosity was detected by analyzing a polymorphic site in intron 2. Three out of nine (33%) informative cases, which were all of intestinal type and advanced cases, showed loss of heterozygosity but neither TGFbetaRII mutation nor replication error was found in these cases. Immunoreactivity of TGFbetaRII in tumor tissues was reduced to a different extent in the gastric cancer with genetically abnormal transforming growth factor. Although the numbers studied are small, homozygous (A)10 deletion or loss of heterozygosity of TGFbetaRII is involved in tumorigenesis and progression of at least some part of sporadic gastric cancer.     

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.     

Mutation analysis of coding sequences of the entire transforming growth factor beta type II receptor gene in sporadic human colon cancer using genomic DNA and intron primers

Mutations in the transforming growth factor beta type II receptor (TGFbeta RII) gene have been detected in several human cancers exhibiting microsatellite instability. To extend analyses of this gene, we previously investigated the exon-intron organization of the TGFbeta RII gene and defined seven exons and flanking intron sequences. In this study, we further determined the nucleotide sequences surrounding these seven exons and designed eight sets of intron-based primers to examine the entire coding region of the TGFbeta RII gene. Using these primers, we screened genomic DNA sequences from 30 sporadic colorectal cancers for mutations of the TGFbeta RII gene. TGFbeta RII mutations were detected in two of 30 tumors and both displayed microsatellite instability. One had a deletion in a polyadenine tract in exon 3 and the other had a point mutation in the kinase domain located in exon 7. There were no mutations in exons 1, 2, 4, 5 and 6. These results further implicate the polyadenine tract and kinase domain as mutational hotspots in the TGFbeta RII gene in cells with genomic instability and suggest that TGFbeta RII gene mutations occur rarely in cells lacking genomic instability.     

Absence of mutations in the analysis of coding sequences of the entire transforming growth factor-beta type II receptor gene in sporadic human breast cancers

The transforming growth factor-beta (TGFbeta) binds the type II TGFbeta growth factor receptor (TGFbetaRII) to inhibit the growth of most epithelial tissues. Most human colon and gastric cancers with microsatellite instability (MI) have frameshift mutations in polynucleotide repeats within the TGFbetaRII coding region; these mutations truncate the receptor protein and disable the serine/threonine kinase to produce TGF-beta resistance. To further investigate the type, frequency and tissue distribution of TGFbetaRII gene mutations, in this study, we examined 36 sporadic breast cancers. We previously produced eight intron based primer pairs for mutational analysis of the entire coding region of the TGFbetaRII gene. Using these primers, we developed protocols for polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis of PCR products from genomic DNA samples of 36 breast cancer patients and we tested them for microsatellite instability (MI) at eight microsatellite loci. One case demonstrated MI (2.8%) and we found no mutations. These and other recent data indicate that TGFbetaRII mutations are essentially confined to colon and gastric cancers with MI. The narrow spectrum of tissues containing RII mutations illustrates the complexity of genetic checkpoints in human carcinogenesis.     

Mutation of the transforming growth factor-beta type II receptor gene is a rare event in human sporadic gastric carcinomas

Mutations of the transforming growth factor-beta type II receptor (TGF-beta RII) gene have been detected in several human cancers. However, mutation analysis of coding sequences of TGF-beta RII in gastric carcinomas has not yet been fully elucidated. We performed PCR-SSCP analysis and direct DNA sequencing of the entire coding region of TGF- RII in 38 human sporadic gastric cancers and 8 gastric cancer cell lines. Mutations of the TGF-beta RII were detected in two tumors and three cell lines. Two tumors had one base deletion in the polyadenine tract in exon 3, the cystein-rich extracellular domain. Three cell lines had a silent mutation in the kinase domain located in exon 4. Polymorphisms were detected in introns 2 and 3. An a/g polymorphism was observed at the seventh base in intron 2 and an a/t polymorphism was observed at the fourth to last base in intron 3. There were no mutations in exons 1, 2, 5, 6 and 7. These results indicate that the polyadenine tract in the TGF-beta RII is a mutational hot spot in human gastric cancer. However, these results also suggest that mutations of the gene are rare events in human sporadic gastric cancer.     

Expression of a dominant-negative type II transforming growth factor beta (TGF-beta) receptor in the epidermis of transgenic mice blocks TGF-beta-mediated growth inhibition

To determine whether a functional type II receptor of transforming growth factor beta (TGF-beta) is required to mediate the growth inhibitory effect of TGF-beta on the skin in vivo, we have generated transgenic mice that overexpress a dominant negative-type II TGF-beta receptor (delta beta RII) in the epidermis. The delta beta RII mice exhibited a thickened and wrinkled skin, and histologically the epidermis was markedly hyperplastic and hyperkeratotic. In vivo labeling with BrdUrd showed a 2.5-fold increase in the labeling index over controls, with labeled nuclei occurring in both basal and suprabasal cells of transgenic epidermis. In heterozygotes, this skin phenotype gradually diminished, and by 10-14 days after birth the transgenic mice were indistinguishable from their normal siblings. However, when F1 mice were mated to homozygosity, perinatal lethality occurred due to the severe hyperkeratotic phenotype, which restricted movement. Cultured primary keratinocytes from delta beta RII mice also exhibited an increased rate of growth in comparison with nontransgenic controls, and were resistant to TGF-beta-induced growth inhibition. These data document the role of the type II TGF-beta receptor in mediating TGF-beta-induced growth inhibition of the epidermis in vivo and in maintenance of epidermal homeostasis.     

Human malignant thyroid tumors displayed reduced levels of transforming growth factor beta receptor type II messenger RNA and protein

Transforming growth factor beta (TGF-beta) is a physiological regulator of thyroid epithelial cell growth and differentiation. This factor signals through a heteromeric complex composed of type I (TGF-beta receptor type I) and type II [TGF-beta receptor type II (TbetaRII)] receptors. Loss of TbetaRII expression has been related to resistance to TGF-beta inhibition of cell proliferation. In the present work, we analyzed the TbetaRII expression in a series of human thyroid tumors, from benign lesions (adenomas) to neoplastic lesions of increasing aggressiveness (papillary and follicular carcinomas) up to the extremely aggressive anaplastic tumors. Results obtained indicated a clear reduced expression of TbetaRII mRNA only in the group of thyroid carcinomas when compared with their relative normal tissues. Immunohistochemical analyses with specific anti-TbetaRII antibodies confirm these observations. These data indicate that loss of expression of TbetaRII can contribute to thyroid cancer progression, inducing cancer cells to escape the growth-inhibitory effect of TGF-beta.     

Purification of transforming growth factor beta 1 from human platelets

Transforming growth factor-beta (TGF beta) is a growth and differentiation factor which can be released from many cell types. In previous studies, platelets were identified as a rich source of TGF beta. Here we present a rapid and convenient method for TGF beta purification from human platelets which includes acid-ethanol extraction and gelfiltration, cation exchange and reversed phase chromatography. All purification steps are performed under acidic conditions to prevent adsorption of TGF beta to the vial walls. In addition, volatile solvents and buffers were used which allowed easy removal of solvent and salt by lyophilization. Using this method pure TGF beta can be easily obtained in high yield (370 micrograms) from 20 units of platelet concentrate.     

Mutations of the transforming growth factor-beta type II receptor gene are strongly related to sporadic proximal colon carcinomas with microsatellite instability

BACKGROUND: Mutations of the transforming growth factor-beta type II receptor gene (TGF-beta RII) have been found in several replication error-positive sporadic colorectal carcinomas and hereditary nonpolyposis colorectal carcinoma cell lines. The aim of this study was to clarify the role of TGF-beta RII in sporadic colorectal carcinogenesis. METHODS: The authors screened for mutations at simple repeated sequences in the TGF-beta RII gene by polymerase chain reaction-single strand conformation polymorphism. They also examined genomic instability, using five microsatellite DNA markers in 69 sporadic colorectal carcinomas. When the carcinomas exhibited the TGF-beta RII mutations, the authors screened further for mutations in two DNA mismatch repair genes, hMSH2 and hMLH1. RESULTS: Seven of the 69 cancers (10%) showed one or two A deletions in TGF-beta RII and resultant frameshift mutations in nucleotide positions 709-718 containing a (A) 10 repeated sequence; but none of these appeared in the corresponding normal DNA, indicating a somatic mutation. All of the seven cancers were located in the proximal colon; there were none in the distal colon (P < 0.01). On the other hand, 22 of the 69 carcinomas (32%) showed the replication error-positive phenotype. The frequency of replication errors in proximal colon carcinomas was higher than that in distal colon carcinomas (P < 0.05). All 7 cancers with TGF-beta RII mutations showed replication errors. One of them revealed a nonsense mutation at codon 413, and 1 revealed a loss of heterozygosity in hMSH2. CONCLUSIONS: These data indicate that mutations of TGF-beta RII are strongly related to proximal colon carcinomas with microsatellite instability and that the mechanism of carcinogenesis in some proximal colon carcinomas is similar to that in hereditary nonpolyposis colorectal carcinoma.     

Cloning of a cDNA encoding the human transforming growth factor-beta type II receptor: heterogeneity of the mRNA

A combined study of anterograde axonal degeneration and Golgi electron microscopic technique was designed to examine the distribution and density of axon terminals from the mediodorsal thalamic nucleus (MD) over layer III pyramidal cells in the prelimbic cortex of the rat. The reconstructive analysis of serial ultrathin sections of gold-toned apical and basal dendrites of layer III pyramidal cells showed that degenerating thalamocortical axon terminals from MD formed asymmetrical synaptic contacts predominantly with dendritic spines of the identified basal dendrites as well as apical dendrites. There was little difference in the numerical density of thalamocortical synapses from MD per unit length of both apical and basal dendrites.     

A novel missense mutation and frameshift mutations in the type II receptor of transforming growth factor-beta gene in sporadic colon cancer with microsatellite instability

Microsatellite instability of DNA samples of 79 sporadic colon cancer patients were analyzed. These samples were also screened to search mutations in the repeat sequences in the gene for the type II receptor of transforming growth factor-beta (TGF-beta RII) using polymerase chain reaction (PCR), electrophoresis with urea gel, and PCR-single strand conformation polymorphism (PCR-SSCP) method. The incidence of microsatellite instability, defined as severe replication error phenotype (RER) with microsatellite alterations in more than three loci, was 6%. Deletion and insertion of an A residue in the (A)10 region, which cause frameshift mutation, were found in four samples and their incidence in the samples with microsatellite instability was 80%. A novel nucleotide substitution of T for G at 1918, which causes missense mutation of arginine to leucine at codon 528, was found in a sample with microsatellite instability. The mutation at 1918 was in highly conservative amino acid residue.     

Overexpression of transforming growth factor beta type I receptor abolishes malignant phenotype of a rat bladder carcinoma cell line

The disposition of ISIS 2922, a phosphorothioate oligonucleotide for treatment of cytomegalovirus associated retinitis, was evaluated in rabbits. Vitreous humor and retina samples were collected from rabbits that received a single intravitreal injection of 66 microg [14C]-labeled ISIS 2922 and were analyzed using anion exchange HPLC. Four hr postdosing, the concentration of ISIS 2922 in vitreous humor was 3.3 microM. The elimination of ISIS 2922 from the vitreous humor exhibited first-order kinetics with a t1/2 of 62 hr. By 10 days postdosing, the mean concentration of ISIS 2922 in rabbit vitreous humor had decreased to 0.17 microM, which represented 22% of the total radioactivity remaining in the vitreous. The remaining 78% coeluted on anion exchange HPLC with shorter oligonucleotides. In retina, ISIS 2922 accumulated over the first 5 days postdosing, reaching a maximum concentration of 3.5 microM, and then declined thereafter with an estimated t1/2 of 79 hr. By 10 days postdosing when only 24% of the total radioactivity in the retina was parent compound, the concentration of ISIS 2922 remained at 1.6 microM, which was 10 times higher than the concentration in the vitreous humor. Whereas the elimination of full-length ISIS 2922 and total radioactivity from the vitreous humor occurred at nearly equal rates, ISIS 2922 disappeared more rapidly than did total radioactivity from the retina, suggesting a greater role for metabolism in the clearance process from retina than the vitreous. Alternatively, the results are consistent with metabolites being cleared from the vitreous at approximately the same rate as parent compound while in the retina metabolites may be cleared more slowly. The data were analyzed with a user-defined pharmacokinetic model, which was then used to predict the potential for accumulation of ISIS 2922 during clinical dosing.