This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Grady, W. M. Articles by Markowitz, S. Search for Related Content PubMed PubMed Citation Articles by Grady, W. M. Articles by Markowitz, S.

Mutational Inactivation of Transforming Growth Factor ß Receptor Type II in Microsatellite Stable Colon Cancers¹

Department of Medicine [W. M. G., J. K. V. W., S. M.], Ireland Cancer Center [W. M. G., L. L. M., S. E. S., J. D. L., J. K. V. W., S. M.], Department of Surgery [A. R.], and BSTP Program [A. N.], Case Western Reserve University and University Hospitals of Cleveland, Cleveland, Ohio 44106; Department of Surgery, University of Texas Health Sciences Center, San Antonio, Texas 78284 [M. G. B.]; Laboratory of Cell Regulation and Carcinogenesis, National Cancer Institute, Bethesda, Maryland 20892 [J. C., S-J. K.]; Molecular Genetics Laboratory, The Johns Hopkins Oncology Center, Baltimore, Maryland 21231 [S. T., K. W. K., B. V.]; and Howard Hughes Medical Institute [J. D. L., B. V., S. M.]

	ABSTRACT

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

 
We previously demonstrated that mutational inactivation of transforming growth factor ß type II receptors (RIIs) is very common among the 13% of human colon cancers with microsatellite instability. These mutations principally cluster in the BAT-RII polyadenine sequence repeat. Among microsatellite stable (MSS) colon cancers, we now find that non-BAT-RII point mutations inactivate RII in another 15% of cases, thus doubling the known number of colon cancers in which RII mutations are pathogenetic. Functional analysis confirms that these mutations inactivate RII signaling. Moreover, another 55% of MSS colon cancers demonstrate a transforming growth factor ß signaling blockade distal to RII. The transforming growth factor ß pathway and RII in particular are major targets for inactivation in MSS colon cancers as well as in colon cancers with microsatellite instability.

	Introduction

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

 
TGF-ß⁴ inhibits the growth of epithelial cells in general (1 , 2) and can inhibit growth and/or induce apoptosis in nontransformed colon epithelial cells (3 , 4) . TGF-ß signaling is transduced by a heteromeric receptor complex composed of type I and type II components, both of which are serine/threonine-directed kinases (5) . A role for RII as a human colon cancer tumor suppressor gene was demonstrated by the discovery of inactivating RII mutations in colorectal cancers that show MSI due to defects in DNA mismatch repair (6, 7, 8) . These cancers with MSI account for 13% of all colon cancers (6 , 7 , 9) . Furthermore, the restoration of wild-type RII in cell lines from colon cancers with MSI abolishes their tumorigenicity in athymic mice (10) . RII mutations in colon cancers with MSI usually result in frameshifts clustered in a naturally occurring 10-bp microsatellite-like polyadenine tract in the 5' coding half of the gene (BAT-RII; Refs. 7 and 8 ). In a few colon cancers with MSI, inactivation of one of the RII alleles occurs via non-BAT-RII mutations that alter the RII kinase domain (7 , 9 , 11) , demonstrating an underlying selective advantage for RII inactivation, irrespective of whether this occurs via BAT-RII or non-BAT-RII mutational events. We hypothesized that the RII mutation should function similarly as a tumor suppressor gene in MSS colon cancers. Accordingly, we examined the RII sequence in 19 MSS colorectal cancer cell lines. These Vaco cell lines have been matched to antecedent tumor and normal tissues and have been extensively characterized as a model for human colon cancer (7 , 12, 13, 14) . Three of the 19 cell lines were shown to express mutant-only RII transcripts. Functional analysis showed that the RII mutations inactivated RII signaling in each case. Moreover, an additional 11 of these cell lines demonstrated a loss of TGF-ß responsiveness, apparently through post-RII signaling defects. Therefore, TGF-ß signaling in general and RII in particular are targets for inactivation in MSS colon cancers as well as in colon cancers with MSI.

	Materials and Methods

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

 
Cell Lines and Primary Tumors.
The establishment of the panel of the Vaco colon cancer cell lines has been described previously (7 , 12) . The Vaco8-2 cell line was established from a stage IV cecal colon adenocarcinoma from a 56-year-old male. Vaco400 was established from a liver metastasis from a 54-year-old man. Vaco410 was derived from a stage IV colon adenocarcinoma metastasis in the right lobe of the liver of a 35-year-old woman. These cell lines were maintained as described previously (12) . Genomic DNA from the original paraffin-embedded formalin-fixed tumor blocks was extracted as described previously (15) . DNA from matched normal tissue for each colon cancer was obtained when available.

TGF-ß Growth Inhibition Assay.
The colon cancer cell lines were plated at clonogenic density (100–500 cells/well in 24-well plates for adherent cell lines and 1000–5000 cells/well in 6-well plates for collagen-dependent adherent cell lines) and treated with TGF-ß1 (10 ng/ml) 3 h after plating. The cell lines were grown until discrete microscopic colonies appeared (usually for 7–10 days), and then the number of colonies in each well was manually counted. Mean and SEs of the means were calculated from experiments performed in triplicate wells and repeated in at least nine independent determinations.

RT-PCR Amplification and Sequencing of RII.
RNA from the cell lines was prepared by extraction with guanidine isothiocyanate (7) . RT-PCR and cloning of the reaction products and manual sequencing of the cloned DNA were performed as described previously (7) , or the reaction products were sequenced by automated sequencing using a ABI 377 DNA Sequencer. For amplification of RII from genomic DNA, sense primer 1871 (5'-GGTGTGTGAGACGTTGACTGAGTG-3') was paired with antisense primer 2001 (5'-AATCTTCTCCTCCGAGCAGCTC-3') for 30 cycles of 95°C for 30 s, 58°C for 1 min, and 70°C for 1 min. All RII mutations were confirmed to be present in at least two independently amplified PCR reactions compared with the reference sequence (GenBank accession number M85079).

TGF-ß Signaling Analysis.
The cell lines were transiently transfected following the manufacturers’ protocols using Lipofectin (Life Technologies, Inc., Gaithersburg, MD), Superfect (Qiagen), or FuGENE (Boehringer Mannheim) with a TGF-ß-responsive firefly luciferase reporter plasmid (p3TP-Lux; Ref. 16 ) and an internal control reporter plasmid containing cDNA encoding R. reniformis luciferase under a thymidine kinase promoter (pRL-TK) or a cytomegalovirus promoter (pRL-CMV; Promega, Madison, WI). The p3TP-Lux plasmid was kindly provided by Dr. Joan Massagué (Memorial Sloan-Kettering Cancer Center and Howard Hughes Medical Center, New York, NY). After transfection, the cell lines were exposed to TGF-ß1 (10 ng/ml) for 72 h and then assessed for reporter activity. The luciferase activity was measured using the Dual-Luciferase Reporter Assay System following the protocol included with this kit (Promega). The samples were assayed on a Turner Dual Injector Luminometer (Promega) or a MLX Multiplate Luminometer (Dynex Technologies).

Retroviral Transduction.
The cell lines were infected with the MFG-RII or MFG-CAT retroviruses (17) . Cells were plated in 6-well plates at a density of 200,000 or 400,000 cells/well; on the following day, they were incubated in MFG-RII or MFG-CAT viral supernatant produced by a virus producer cell line and polybrene (4 µg/ml) for 4 h. The cells then were rinsed three times in PBS. G418 (600 µg/ml) was added 2 days after the viral infection. TGF-ß1 (10 ng/ml) was added to designated subsets of the infected cell lines 3 h after infection to assess for reconstitution of TGF-ß-induced growth inhibition. The growth inhibition assays were performed as described above. All of these experiments were performed in triplicate and repeated at least three times.

	Results and Discussion

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

 
MSS Colon Cancers Are Commonly TGF-ß Resistant.
We initially determined whether TGF-ß growth-inhibitory responses were intact in 19 colon cancer cell lines that had been previously determined to be MSS (7) . TGF-ß growth inhibition was assayed by determining the ability of TGF-ß1 to inhibit the colony formation of cells plated at clonogenic density. Cell lines in which TGF-ß1 (10 ng/ml) reduced colony formation at 7–10 days after plating by >25% were considered to be responsive to TGF-ß-mediated growth inhibition. Five cell lines (26%) were determined to demonstrate sensitivity to TGF-ß-mediated growth inhibition, with TGF-ß-mediated suppression of colony formation ranging from 42–79%. However, 74% (14 of 19) of the MSS cell lines were found to be resistant to the growth-inhibitory effects of TGF-ß.

RII Mutations Detected in MSS Colon Cancers.
Ninety percent of colon carcinoma cell lines with MSI demonstrate both TGF-ß resistance and BAT-RII mutations that inactivate the RII receptor (7 , 9) . Accordingly, we initially examined the MSS colon cancers for BAT-RII mutations using our previously described assay (8 , 9) . Consistent with our prior results, no BAT RII mutations were detected in any of these cell lines (7) . Therefore, we determined the complete RII cDNA sequences in RT-PCR cDNA pools amplified from each of the 14 MSS cell lines that demonstrated TGF-ß resistance. In three cell lines (15%), only mutant RII sequences were obtained, and no wild-type RII was expressed. In the remaining cell lines, RII was expressed and was wild-type in sequence. RII mutations were previously demonstrated to be ubiquitous among the approximately 13% of colon cancers that show MSI (7 , 9 , 18) . These new findings therefore double the number of colon cancers in which RII mutations are pathogenetic to approximately 28% of all colon adenocarcinomas and show that such RII mutant cases occur in both the MSI and MSS subtypes.

The mutations detected in MSS cell lines Vaco8-2, Vaco400, and Vaco410 are located within conserved regions of the kinase domain of the RII gene (Vaco8-2, Vaco410, and Vaco400 cells) or in the 5' extracytoplasmic portion of the receptor (Vaco400 cells). In Vaco8-2 cells, a missense mutation (GACAAC) changed an aspartic acid to an asparagine at codon 522 (Fig. 1 ; Ref. 19 ), altering the charge of a residue within subdomain XI of the serine/threonine kinase domain of the receptor. In Vaco410 cells, a missense mutation (CGTCAT) changed an arginine to a histidine at codon 528 (Fig. 1 ; Ref. 19 ). This arginine, which is also within kinase subdomain XI, is strictly conserved among all serine/threonine protein kinases (20) . In Vaco400 cells, two mutations were observed in the pooled cDNA products; both were apparently heterozygous (Fig. 1) . Sequencing individual RT-PCR cDNA clones revealed that this was due to separate mutations present on each of the two expressed Vaco400 alleles. One Vaco400 allele carried a missense mutation (TATGAT) at codon 470 that changed a tyrosine to an aspartic acid (19) and altered the charge of an amino acid in subdomain IX of the receptor kinase domain. The second Vaco400 allele carried a missense mutation (AAAACA) at codon 52 that changed a lysine to a threonine in the extracytoplasmic region of the receptor.

View larger version (31K): [in this window] [in a new window]   Fig. 1. Point mutations of TGF-ß RII in colon cancer cell lines Vaco410, Vaco8-2, and Vaco400. RII cDNA was PCR-amplified in two overlapping pieces, cloned into the pCRII vector (Invitrogen), and then sequenced manually or by ABI automated sequencing. Vaco410 has a homozygous missense mutation in exon 7 at bp 1918 (GA) that changes codon 528 from Arg to His. Vaco8-2 has a homozygous missense mutation at bp 1899 (GA) that changes codon 522 from Asp to Asn. Vaco400 has two heterozygous missense mutations that are on opposite alleles. One mutation is located at bp 490 and changes codon 52 from Lys to Thr, and the other mutation is at bp 1743 and changes codon 470 from Tyr to Asp. The mutations in Vaco400 (at codon 470), Vaco410, and Vaco8-2 all affect the kinase domain of the receptor. The mutation at codon 52 in Vaco400 affects the extracytoplasmic portion of RII.

Kinase domain mutations in RII have previously been observed in occasional colon cancers with MSI both by us and by other investigators (9 , 11) and have also been noted in two squamous cell carcinomas of the oropharynx (21) and in one T-cell lymphoma (22) . The selection of these Vaco colon cancer mutations during tumorigenesis, their location within RII, and the nature of the amino acid changes produced suggested that these mutations likely inactivated the TGF-ß receptor and induced the TGF-ß resistance observed in the Vaco cell lines. Of note, RII mutations were not observed in an investigation of a different set of colon cancers studied by single-strand conformational polymorphism; this contrasting result is most likely a consequence of the lower sensitivity of single-strand conformational polymorphism compared to sequencing (11) .

RII Mutations Inactivate TGF-ß Receptor Signaling.
To confirm that the RII mutations detected in the MSS colon cancers inactivated the receptor, we determined the ability of wild-type RII to restore TGF-ß-mediated responses in these cell lines. We initially assayed the ability of wild-type RII to restore TGF-ß-mediated transcriptional responses as assayed by the TGF-ß-responsive firefly luciferase activity encoded by the reporter construct p3TP-Lux (16) . p3TP-Lux was transiently transfected into these cell lines accompanied by either RII expression vector, pRC/CMV-TGF-ßRII, or control DNA, and p3TP-Lux-mediated luminescence was determined in the presence and absence of exogenous TGF-ß1. Each determination was corrected for transfection efficiency by assaying for the luminescence from a cotransfected control construct, pRL-TK or pRL-CMV (Promega), a plasmid that expresses R. reniformis luciferase activity that is easily discriminated from firefly luciferase activity.

Consistent with the findings that Vaco8-2, Vaco400, and Vaco410 were resistant to TGF-ß-mediated growth inhibition, each of these cell lines demonstrated low basal activity of p3TP-Lux and no further response of the reporter to added TGF-ß1 (Fig. 2) . In Vaco410 and Vaco400 cells, cotransfection of wild-type RII augmented the basal p3TP-Lux activity. p3TP-Lux activity increased further after the addition of exogenous TGF-ß1, such that total the 3TP-Lux output reached 5–10-fold over baseline (Fig. 2) . The increase in p3TP-Lux activity due to the introduction of wild-type RII likely reflects the activation of the restored TGF-ß signaling pathway by the autocrine TGF-ß produced by these cell lines.⁵ The responsiveness of p3TP-Lux to exogenous TGF-ß1 clearly establishes that introducing wild-type RII restores TGF-ß-mediated signaling in these cell lines. Thus, the RII mutations in Vaco410 and Vaco400 directly account for the TGF-ß resistance of these cell lines. As discussed below, we hypothesized that the Vaco8-2 RII mutation also inactivated receptor signaling, but that additional progression events in this cell line interfered with the ability to reconstitute TGF-ß signaling by a single gene alone.

View larger version (23K): [in this window] [in a new window]   Fig. 2. Restoration by RII of TGF-ß-stimulated transcriptional responses. Relative luciferase activity from TGF-ß-responsive reporter plasmid 3TPLux in cell lines Vaco410, Vaco400, and Vaco8-2 after transient transfection with pRC/CMV RII is shown. The cells were grown for 2 days after transfection with or without TGF-ß1 (10 ng/ml). Vaco410 and Vaco400 demonstrate both a basal and TGF-ß-inducible increase in 3TPLux activity after transfection with RII. The basal induction in Vaco410 and Vaco400 is presumably secondary to autocrine TGF-ß produced by these cell lines. Vaco8-2 shows no increase in 3TPLux activity after RII transfection with or without the addition of TGF-ß1. The luciferase activity was normalized to the activity of a cotransfected control vector, pRL-TK or pRL-CMV, that contains the distinguishable R. reniformis luciferase.

View larger version (27K): [in this window] [in a new window]   Fig. 3. Suppression of colony-forming activity by wild-type RII. The cell lines were plated at a concentration to yield clonogenic density after G418 selection, infected with a RII retrovirus, and then assessed for colony formation 7–10 days after being grown in the presence or absence of exogenous TGF-ß1 (10 ng/ml). Reconstitution of RII suppresses the number of colonies formed in Vaco400 and Vaco410. This suppression is significantly augmented by exogenous TGF-ß. Vaco8-2 shows no suppression of colony formation after RII reconstitution. MFG-CAT, a MFG retrovirus encoding CAT and a neomycin resistance gene, was used to control for nonspecific effects of the retrovirus.

View larger version (14K): [in this window] [in a new window]   Fig. 4. A, transcriptional activity of wild-type and mutant RII transfected into Hct116. The relative luciferase activity from the 3TPLux reporter plasmid in the RII-null cell line Hct116 is shown after transfection with wild-type RII cDNA in pRC/CMV (wild-type RII), Vaco 8-2 mutant RII cDNA in pRC/CMV (Vaco8-2), or an empty pRC/CMV control vector (empty vector). Transfections were done with () and without () treatment with TGF-ß1 (10 ng/ml) for 48 h. Equal amounts of expression vectors for wild-type RII and Vaco8-2 mutant RII were also mixed and cotransfected (wild-type + Vaco8-2 RII). Transfections were normalized relative to the activity of a cotransfected pRL-TK or pRL-CMV vector. B, TGF-ß induced transcriptional responses in Vaco8–2. The relative luciferase activity from 3TPLux in Vaco8-2 is shown after transfection with wild-type RII, wild-type Smad4, or a combination of both wild-type RII and Smad4. Induction of 3TPLux activity is observed only after the transfection of RII and Smad4.

The binding of TGF-ß to its receptor activates a heteromeric complex of Smad2-Smad3-Smad4 transcription factors to translocate to the nucleus (23 , 24) . Previously, we and others have reported that mutations of Smad2 and Smad4 are present at a low frequency in some human colon cancers (13 , 14 , 25 , 26) . Accordingly, we examined the possibility of Smad inactivation as a separate event in the Vaco8-2 TGF-ß signaling pathway. Whereas neither Smad4 nor RII transfected singly into Vaco8-2 triggered the 3TP-Lux reporter, cotransfection of RII and Smad4 triggered a 5–10-fold p3TP-Lux response, thus suggesting that the inactivation of RII and Smad4 accounted for two separate defects in the Vaco8-2 TGF-ß signaling pathway (Fig. 4B) . Examination of the Vaco8-2 Smad4 gene revealed that neither Smad4 exon 1 nor exon 2 could be amplified from Vaco8-2 genomic DNA. Thus, biallelic deletion of Smad4 was indeed a second genetic event in the Vaco8-2 cell line TGF-ß pathway. Due to the small amounts of residual normal tissue remaining in even the microdissected tumor antecedent to the Vaco8-2 cell line, we could not establish whether Smad4 loss occurred in the predecessor Vaco8-2 tumor or only after establishment of the Vaco8-2 cell line. Nonetheless, it is intriguing to speculate that the presence of mutations in both the RII and Smad4 genes in the Vaco 8-2 cell line, although rare, seems to suggest that RII and Smad4 may have some functions that are distinct from one another.

In summary, multiple lines of evidence now suggest that the TGF-ß pathway is a potent tumor suppressor of human colorectal carcinogenesis. We have previously determined that among the 13% of human colon cancers of the MSI subtype, nearly all have inactivating RII frameshift mutations clustering at the BAT-RII tract (7 , 9) . Data from the Vaco colon cancer cell lines now suggest that among MSS human colon cancers, an additional 15% of tumors also bear inactivating RII mutations, and that restoring wild-type RII to these tumors is potently growth suppressive. Hence, RII mutations play a pathophysiological role in approximately 30% of human colon cancers, and RII gene therapy may have a future role in the treatment of these tumors. Analysis of TGF-ß sensitivity in the Vaco colon cancer cell line panel further suggests that an additional 55% of human colon cancers demonstrate functional inactivation of TGF-ß-mediated growth inhibition. Furthermore, these cancer lines have lost TGF-ß-mediated transcriptional responses as well.⁶ Presumptively, these additional cancers bear defects in the TGF-ß signaling pathway at points distal to RII. We have previously shown that Smad2 and Smad4 mutations are present in only a small percentage of human colon cancers (13 , 14 , 27) . However, the tumor-promoting activity of such downstream inactivation of TGF-ß signaling is suggested by recent studies in murine models showing that heterozygous Smad4 mutation promotes colon adenoma to carcinoma progression (28) , and homozygous Smad3 knockout leads directly to colon cancer (29) . Study of additional mechanisms leading to the inactivation of TGF-ß signaling in the remaining set of wild-type RII MSS colon cancers is currently ongoing.

	ACKNOWLEDGMENTS

 
We thank Dr. Joseph Willis for assistance in microdissecting clinical material for this study and Kim Yonkof for technical assistance in sequencing the TGF-ß RII gene.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by NIH Grants RO1 CA67409 and RO1 CA 72160 (to S. M.), by an Advanced Research Training Award from the American Digestive Health Foundation and NIH Grant KO8 CA77676-01 (to W. M. G.), and by NIH Grants P30 CA43703 and T32 CA 59366 (to Case Western Reserve University). S. M. is an investigator in the Howard Hughes Medical Institute.

² These authors contributed equally to this study.

³ To whom requests for reprints should be addressed, at UCRC #2, Room 200, Ireland Cancer Center, 11001 Cedar Road, Cleveland, OH 44106.

⁴ The abbreviations used are: TGF-ß, transforming growth factor ß; RII, TGF-ß receptor type II; MSI, microsatellite instability; MSS, microsatellite stable; RT-PCR, reverse transcription-PCR.

⁵ S. Markowitz and M. G. Brattain, unpublished data.

⁶ W. M. Grady, unpublished data.

Received 11/ 6/98. Accepted 12/ 8/98.

	REFERENCES

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

 

1. Markowitz S., Roberts A. Tumor supressor activity of the TGF-ß pathway in human cancers. Cytokine Growth Factor Rev., 7: 93-102, 1996.[Medline]
2. Moses H., Yang E., Pietonpol J. TGF-ß stimulation and inhibition of cell proliferation: new mechanistic insights. Cell, 63: 245-247, 1990.[Medline]
3. Markowitz S., Myeroff L., Cooper M., Traicoff J., Kochera M., Lutterbaugh J., Swiriduk M., Willson J. A benign cultured colon adenoma bears three genetically altered colon cancer oncogenes but progresses to tumorigenicity and transforming growth factor-ß independence without inactivating the p53 tumor suppressor gene. J. Clin. Investig., 93: 1005-1013, 1994.
4. Wang C. Y., Eshleman J., Willson J., Markowitz S. TGF-ß and substrate release are both inducers of apoptosis in a human colon adenoma cell line. Cancer Res., 55: 5101-5105, 1995.[Abstract/Free Full Text]
5. Massagué J., Attisano L., Wrana J. The TGF-ß family and its composite receptors. Trends Cell Biol., 6: 172-178, 1985.
6. Eshleman J., Markowitz S. Mismatch repair defects in human carcinogenesis. Hum. Mol. Genet., 5: 1489-1494, 1996.[Abstract]
7. Markowitz S., Wang J., Myeroff L., Parsons R., Sun L., Lutterbaugh J., Fan R., Zborowska E., Kinzler K., Vogelstein B., Brattain M., Willson J. Inactivation of the type II TGF-ß receptor in colon cancer cells with microsatellite instability. Science (Washington DC), 268: 1336-1338, 1995.[Abstract/Free Full Text]
8. Myeroff L., Parsons R., Kim S-J., Hedrick L., Cho K., Orth K., Mathis M., Kinzler K., Lutterbaugh J., Park K., Bang Y-J., Lee H., Park J-G., Lynch H., Roberts A., Vogelstein B., Markowitz S. A transforming growth factor ß receptor type II gene mutation common in colon and gastric but rare in endometrial cancers with microsatellite instability. Cancer Res., 55: 5545-5547, 1995.[Abstract/Free Full Text]
9. Parsons R., Myeroff L., Liu B., Willson J., Markowitz S., Kinzler K., Vogelstein B. Microsatellite instability and mutations of the transforming growth factor ß type II receptor gene in colorectal cancer. Cancer Res., 55: 5548-5550, 1995.[Abstract/Free Full Text]
10. Wang J., Sun L., Myeroff L., Wang X., Gentry L., Yang J., Liang J., Zborowska E., Markowitz S., Willson J., Brattain M. Demonstration that mutation of the type II transforming growth factor ß receptor inactivates its tumor supressor activity in replication error-positive colon carcinoma cells. J. Biol. Chem., 270: 22044-22049, 1995.[Abstract/Free Full Text]
11. Takenoshita S., Tani M., Nagashima M., Hagiwara K., Bennet W., Yokota J., Harris C. Mutation analysis of coding sequences of the entire transforming growth factor ß type II receptor gene in sporadic human colon cancer using genomic DNA and intron primers. Oncogene, 14: 1255-1258, 1997.[Medline]
12. Willson J., Bittner G., Oberley T., Meisner G., Weese J. Cell culture of human colon adenomas and carcinomas. Cancer Res., 47: 2704-2713, 1987.[Abstract/Free Full Text]
13. Thiagalingam S., Lengauer C., Leach F., Schutte M., Hahn S., Overhauser J., Willson J., Markowitz S., Hamilton S., Kern S., Kinzler K., Vogelstein B. Evaluation of candidate tumour suppressor genes on chromosome 18 in colorectal cancers. Nat. Genet., 13: 343-346, 1996.[Medline]
14. Riggins G., Thiagalingam S., Rozenblum E., Weinstein C., Kern S., Hamilton S., Willson J., Markowitz S., Kinzler K., Vogelstein B. Mad-related genes in the human. Nat. Genet., 13: 347-349, 1996.[Medline]
15. Grady W., Rajput A., Myeroff L., Liu D., Kwon K-H., Willis J., Markowitz S. Mutation of the type II transforming growth factor-ß receptor is coincident with the transformation of human colon adenomas to malignant carcinomas. Cancer Res., 58: 3101-3104, 1998.[Abstract/Free Full Text]
16. Wrana J., Attisano L., Carcamo J., Zentella A., Doody J., Laiho M., Wang X-F., Massagué J. TGFß signals through a heteromeric protein kinase receptor complex. Cell, 71: 1003-1014, 1992.[Medline]
17. Chang J., Park K., Bang Y-J., Kim W., Kim D., Kim S-J. Expression of transforming growth factor ß type II receptor reduces tumorigenicity in human gastric cancer cells. Cancer Res., 57: 2856-2859, 1997.[Abstract/Free Full Text]
18. Eshleman J., Markowitz S. Microsatellite instability in inherited and sporadic neoplasms. Curr. Opin. Oncol., 7: 83-89, 1995.[Medline]
19. Lin H. Y., Wang X-F., Ng-Eaton E., Weinberg R. A., Lodish H. F. Expression cloning of the TGF-ß type II receptor, a functional transmembrane serine/threonine kinase. Cell, 68: 775-785, 1992.[Medline]
20. Derynck R., Feng X-H. TGF-ß receptor signaling. Biochim. Biophys. Acta, 1333: F105-F150, 1997.[Medline]
21. Garrigue-Antar L., Munoz-Antonia T., Antonia S., Gesmonde J., Vellucci V., Reiss M. Missense mutations of the transforming growth factor ß type II receptor in human head and neck squamous carcinoma cells. Cancer Res., 55: 3982-3987, 1995.[Abstract/Free Full Text]
22. Knaus P., Lindemann D., DeCoteau J., Perman R., Yankelev H., Hille M., Kandin M., Lodish H. A dominant inhibitory mutant of the type II transforming growth factor ß receptor in the malignant progression of a cutaneous T-cell lymphoma. Mol. Cell. Biol., 16: 3480-3489, 1996.[Abstract]
23. Macias-Silva M., Abdollah S., Hoodless P., Pirone R., Attisano L., Wrana J. MADR2 is a substrate of the TGFß receptor, and its phosphorylation is required for nuclear accumulation and signaling. Cell, 87: 1215-1224, 1996.[Medline]
24. Massagué J. TGF-ß signaling: receptors, transducers, and mad proteins. Cell, 85: 947-950, 1996.[Medline]
25. Riggins G., Kinzler K., Vogelstein B., Thiagalingam S. Frequency of smad gene mutations in human cancers. Cancer Res., 57: 2578-2580, 1997.[Abstract/Free Full Text]
26. Eppert K., Scherer S., Ozcelik H., Pirone R., Hoodless P., Kim H., Tsui L-C., Bapat B., Gallinger S., Andrulis I., Thomsen G., Wrana J., Attisano L. MADR2 maps to 18q21 and encodes a TGFß-regulated MAD-related protein that is functionally mutated in colorectal cancer. Cell, 86: 543-552, 1996.[Medline]
27. Schutte M., Hruban R., Hedrick L., Cho K., Nadasdy G., Weinstein C., Bova G., Isaacs W., Cairns P., Nawroz H., Sidransky D., Casero R., Meltzer P., Hahn S., Kern S. DPC4 gene in various tumor types. Cancer Res., 56: 2527-2530, 1996.[Abstract/Free Full Text]
28. Takaku K., Oshima M., Miyoshi H., Matsui M., Seldin M., Taketo M. Intestinal tumorigenesis in compound mutant mice of both Dpc4 (Smad4) and Apc genes. Cell, 92: 645-656, 1998.[Medline]
29. Zhu Y., Richardson J., Parada L., Graff J. Smad3 mutant mice develop metastatic colorectal cancer. Cell, 94: 703-714, 1998.[Medline]

This article has been cited by other articles:

				K. Imai and H. Yamamoto Carcinogenesis and microsatellite instability: the interrelationship between genetics and epigenetics Carcinogenesis, April 1, 2008; 29(4): 673 - 680. [Abstract] [Full Text] [PDF]

				S. Bharathy, W. Xie, J. M. Yingling, and M. Reiss Cancer-Associated Transforming Growth Factor {beta} Type II Receptor Gene Mutant Causes Activation of Bone Morphogenic Protein-Smads and Invasive Phenotype Cancer Res., March 15, 2008; 68(6): 1656 - 1666. [Abstract] [Full Text] [PDF]

				N. Sharifi, R. J Lechleider, and W. L Farrar Transforming growth factor-{beta} receptor III downregulation in prostate cancer: is inhibin B a tumor suppressor in prostate? J. Mol. Endocrinol., November 1, 2007; 39(5): 329 - 332. [Abstract] [Full Text] [PDF]

				S. H. Wrzesinski, Y. Y. Wan, and R. A. Flavell Transforming Growth Factor-{beta} and the Immune Response: Implications for Anticancer Therapy Clin. Cancer Res., September 15, 2007; 13(18): 5262 - 5270. [Abstract] [Full Text] [PDF]

				S. I. Berndt, W.-Y. Huang, N. Chatterjee, M. Yeager, R. Welch, S. J. Chanock, J. L. Weissfeld, R. E. Schoen, and R. B. Hayes Transforming growth factor beta 1 (TGFB1) gene polymorphisms and risk of advanced colorectal adenoma Carcinogenesis, September 1, 2007; 28(9): 1965 - 1970. [Abstract] [Full Text] [PDF]

				G. Chen, P. Ghosh, H. Osawa, C. Y. Sasaki, L. Rezanka, J. Yang, T. J. O'Farrell, and D. L. Longo Resistance to TGF-{beta}1 correlates with aberrant expression of TGF-{beta} receptor II in human B-cell lymphoma cell lines Blood, June 15, 2007; 109(12): 5301 - 5307. [Abstract] [Full Text] [PDF]

				Y. Xu and B. Pasche TGF-{beta} signaling alterations and susceptibility to colorectal cancer Hum. Mol. Genet., April 15, 2007; 16(R1): R14 - R20. [Abstract] [Full Text] [PDF]

				N. M. Munoz, M. Upton, A. Rojas, M. K. Washington, L. Lin, A. Chytil, E. G. Sozmen, B. B. Madison, A. Pozzi, R. T. Moon, et al. Transforming Growth Factor {beta} Receptor Type II Inactivation Induces the Malignant Transformation of Intestinal Neoplasms Initiated by Apc Mutation. Cancer Res., October 15, 2006; 66(20): 9837 - 9844. [Abstract] [Full Text] [PDF]

				J. S. Sunde, H. Donninger, K. Wu, M. E. Johnson, R. G. Pestell, G. S. Rose, S. C. Mok, J. Brady, T. Bonome, and M. J. Birrer Expression Profiling Identifies Altered Expression of Genes That Contribute to the Inhibition of Transforming Growth Factor-{beta} Signaling in Ovarian Cancer. Cancer Res., September 1, 2006; 66(17): 8404 - 8412. [Abstract] [Full Text] [PDF]

				S. E. Beck, B. H. Jung, A. Fiorino, J. Gomez, E. D. Rosario, B. L. Cabrera, S. C. Huang, J. Y. C. Chow, and J. M. Carethers Bone morphogenetic protein signaling and growth suppression in colon cancer. Am J Physiol Gastrointest Liver Physiol, July 1, 2006; 291(1): G135 - G145. [Abstract] [Full Text] [PDF]

				S. K. Halder, G. Anumanthan, R. Maddula, J. Mann, A. Chytil, A. L. Gonzalez, M. K. Washington, H. L. Moses, R. D. Beauchamp, and P. K. Datta Oncogenic Function of a Novel WD-Domain Protein, STRAP, in Human Carcinogenesis. Cancer Res., June 15, 2006; 66(12): 6156 - 6166. [Abstract] [Full Text] [PDF]

				L. Maggio-Price, P. Treuting, W. Zeng, M. Tsang, H. Bielefeldt-Ohmann, and B. M. Iritani Helicobacter Infection Is Required for Inflammation and Colon Cancer in Smad3-Deficient Mice Cancer Res., January 15, 2006; 66(2): 828 - 838. [Abstract] [Full Text] [PDF]

				P. Alhopuro, H. Alazzouzi, H. Sammalkorpi, V. Davalos, R. Salovaara, A. Hemminki, H. Jarvinen, J.-P. Mecklin, S. Schwartz Jr., L. A. Aaltonen, et al. SMAD4 Levels and Response to 5-Fluorouracil in Colorectal Cancer Clin. Cancer Res., September 1, 2005; 11(17): 6311 - 6316. [Abstract] [Full Text] [PDF]

				M. Macarthur, L. Sharp, G. L. Hold, J. Little, and E. M. El-Omar The Role of Cytokine Gene Polymorphisms in Colorectal Cancer and Their Interaction with Aspirin Use in the Northeast of Scotland Cancer Epidemiol. Biomarkers Prev., July 1, 2005; 14(7): 1613 - 1618. [Abstract] [Full Text] [PDF]

				J. S. Edmiston, W. A. Yeudall, T. D. Chung, and D. A. Lebman Inability of Transforming Growth Factor-{beta} to Cause SnoN Degradation Leads to Resistance to Transforming Growth Factor-{beta}-Induced Growth Arrest in Esophageal Cancer Cells Cancer Res., June 1, 2005; 65(11): 4782 - 4788. [Abstract] [Full Text] [PDF]

				Y. Tang, V. Katuri, R. Srinivasan, F. Fogt, R. Redman, G. Anand, A. Said, T. Fishbein, M. Zasloff, E. P. Reddy, et al. Transforming Growth Factor-{beta} Suppresses Nonmetastatic Colon Cancer through Smad4 and Adaptor Protein ELF at an Early Stage of Tumorigenesis Cancer Res., May 15, 2005; 65(10): 4228 - 4237. [Abstract] [Full Text] [PDF]

				W. M. Grady Transforming Growth Factor-{beta}, Smads, and Cancer Clin. Cancer Res., May 1, 2005; 11(9): 3151 - 3154. [Full Text] [PDF]

				R. L. Elliott and G. C. Blobe Role of Transforming Growth Factor Beta in Human Cancer J. Clin. Oncol., March 20, 2005; 23(9): 2078 - 2093. [Abstract] [Full Text] [PDF]

				T. Seo, J. Park, and J. Choe Kaposi's Sarcoma-Associated Herpesvirus Viral IFN Regulatory Factor 1 Inhibits Transforming Growth Factor-{beta} Signaling Cancer Res., March 1, 2005; 65(5): 1738 - 1747. [Abstract] [Full Text] [PDF]

				H. Yamagata, K. Matsuzaki, S. Mori, K. Yoshida, Y. Tahashi, F. Furukawa, G. Sekimoto, T. Watanabe, Y. Uemura, N. Sakaida, et al. Acceleration of Smad2 and Smad3 Phosphorylation via c-Jun NH2-Terminal Kinase during Human Colorectal Carcinogenesis Cancer Res., January 1, 2005; 65(1): 157 - 165. [Abstract] [Full Text] [PDF]

				M. Yan, R. M. Rerko, P. Platzer, D. Dawson, J. Willis, M. Tong, E. Lawrence, J. Lutterbaugh, S. Lu, J. K. V. Willson, et al. From the Cover: 15-Hydroxyprostaglandin dehydrogenase, a COX-2 oncogene antagonist, is a TGF-{beta}-induced suppressor of human gastrointestinal cancers PNAS, December 14, 2004; 101(50): 17468 - 17473. [Abstract] [Full Text] [PDF]

				S.S. Prime, M. Davies, M. Pring, and I.C. Paterson THE ROLE OF TGF-{beta} IN EPITHELIAL MALIGNANCY AND ITS RELEVANCE TO THE PATHOGENESIS OF ORAL CANCER (PART II) Crit. Rev. Oral. Biol. Med., November 1, 2004; 15(6): 337 - 347. [Abstract] [Full Text] [PDF]

				S. Biswas, A. Chytil, K. Washington, J. Romero-Gallo, A. E. Gorska, P. S. Wirth, S. Gautam, H. L. Moses, and W. M. Grady Transforming Growth Factor {beta} Receptor Type II Inactivation Promotes the Establishment and Progression of Colon Cancer Cancer Res., July 15, 2004; 64(14): 4687 - 4692. [Abstract] [Full Text] [PDF]

				A. M. Dunning, P. D. Ellis, S. McBride, H. L Kirschenlohr, C. S. Healey, P. R. Kemp, R. N. Luben, J. Chang-Claude, A. Mannermaa, V. Kataja, et al. A Transforming Growth Factor{beta}1 Signal Peptide Variant Increases Secretion in Vitro and Is Associated with Increased Incidence of Invasive Breast Cancer Cancer Res., May 15, 2003; 63(10): 2610 - 2615. [Abstract] [Full Text] [PDF]

				E. B. Brunschwig, K. Wilson, D. Mack, D. Dawson, E. Lawrence, J. K.V. Willson, S. Lu, A. Nosrati, R. M. Rerko, S. Swinler, et al. PMEPA1, a Transforming Growth Factor-{beta}-induced Marker of Terminal Colonocyte Differentiation Whose Expression Is Maintained in Primary and Metastatic Colon Cancer Cancer Res., April 1, 2003; 63(7): 1568 - 1575. [Abstract] [Full Text] [PDF]

				P. M. Hempen, L. Zhang, R. K. Bansal, C. A. Iacobuzio-Donahue, K. M. Murphy, A. Maitra, B. Vogelstein, R. H. Whitehead, S. D. Markowitz, J. K. V. Willson, et al. Evidence of Selection for Clones Having Genetic Inactivation of the Activin A Type II Receptor (ACVR2) Gene in Gastrointestinal Cancers Cancer Res., March 1, 2003; 63(5): 994 - 999. [Abstract] [Full Text] [PDF]

				C. Benckert, S. Jonas, T. Cramer, Z. von Marschall, G. Schafer, M. Peters, K. Wagner, C. Radke, B. Wiedenmann, P. Neuhaus, et al. Transforming Growth Factor {beta}1 Stimulates Vascular Endothelial Growth Factor Gene Transcription in Human Cholangiocellular Carcinoma Cells Cancer Res., March 1, 2003; 63(5): 1083 - 1092. [Abstract] [Full Text] [PDF]

R. A. Gupta, P. Sarraf, J. A. Brockman, S. B. Shappell, L. A. Raftery, T. M. Willson, and R. N. DuBois Peroxisome Proliferator-activated Receptor gamma and Transforming Growth Factor-beta Pathways Inhibit Intestinal Epithelial Cell Growth by Regulating Levels of TSC-22 J. Biol. Chem., Feb