Demonstration That Mutation of the Type II Transforming Growth Factor β Receptor Inactivates Its Tumor Suppressor Activity in Replication Error-positive Colon Carcinoma Cells

Abstract

Escape from negative growth regulation by transforming growth factor β (TGF-β) as a result of the loss of TGF-β type II receptor (RII) expression has been found to be associated with the replication error (RER) colorectal cancer genotype, which is characteristic of hereditary nonpolyposis colorectal cancers. The RER-positive HCT 116 colon carcinoma cell line was examined for RII mutations. A 1-base deletion was found within a sequence of 10 repeating adenines (nucleotides 709-718), which resulted in a frameshift mutation. Although it is reasonable to predict that the loss of RII function would be an important determinant of malignancy, the large number of potential mutations in cells of this phenotype raises the possibility that an RII mutation may not be a key event in the tumorigenic phenotype of these cells. One way to test directly the importance of RII mutations in determining the malignant phenotype would be to restore its expression. If restoration of expression leads to diminished tumorigenicity, it would indicate that RII mutation is an important determinant of malignancy in the RER phenotype. To determine whether restoration of RII would lead to reversal of malignancy in RER colon cancers, an RII expression vector was transfected into the HCT 116 cell line. RII stable clones showed mRNA and protein expression of transfected RII. The fibronectin mRNA level was increased by exogenous TGF-β 1 treatment in a dose-dependent manner in RII-positive clones, whereas the control cells remained insensitive. The RII transfectants showed reduced clonogenicity in both monolayer culture and soft agarose. They were growth arrested at a lower saturation density than control cells. TGF-β 1 -neutralizing antibody stimulated the proliferation of RII-transfected but not control cells, indicating that the alterations in the growth parameters of the transfected cells were due to the acquisition of autocrine-negative activity. Tumorigenicity in athymic mice was reduced and delayed in RII transfectants. These results indicate that reconstitution of TGF-β autocrine activity by reexpression of RII can reverse malignancy in RER colon cancers, thus verifying that the malignancy of hereditary nonpolyposis colorectal cancer can be directly associated with the loss of RII expression.

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

“…These and other observations are consistent with the hypothesis that TGFb RII is a tumor suppressor gene whose mutational inactivation is important in human carcinogenesis (Sun et al, 1994;Wang et al, 1995).…”

Transcriptional repression of the transforming growth factor-β type I receptor gene by DNA methylation results in the development of TGF-β resistance in human gastric cancer

“…However, a strong correlation between TGF-b RII mutation and microsatellite instability has been demonstrated, only for colon and gastric cancers (Eshleman and Markowitz, 1995;Kolodner, 1996;Markowitz et al, 1995;Modrich and Lahue, 1996;Myero et al, 1995;Parsons et al, 1995). Since then, defective expression of TGF-b RII has been demonstrated in gastric, colon, small-cell lung, breast, endometrial, hepatocellular, bladder, and squamous cell cancers as well as retinoblastoma, pheochromocytoma, osteosarcoma, and lymphomas (Chang et al, 1997;Garrigue-Antar et al, 1995;Inagaki et al, 1993;Kadin et al, 1994;Kim and Kim, 1996;Kimchi et al, 1988;Park et al, 1994;Markowitz et al, 1995;Markowitz and Roberts, 1996;Sun et al, 1994;Wang et al, 1995).…”

Repression of transforming growth factor-β receptor type I promoter expression by Sp1 deficiency

“…Since RI and RII are vital for TGF-b mediated growth suppression, loss of either receptor may contribute to TGF-b resistance and subsequent tumor progression. Previous work has indicated TGF-b resistance either due to a mutation of the RII gene in gastric and colon carcinoma cells Park et al, 1994;Wang et al, 1995) or transcriptional repression of RII due to decreased binding of nuclear proteins to the RII promoter in keratinocytes and breast cancer cells (Ammanamanchi et al, 1998;Kim et al, 1997). A signi®cant percentage of gastric and colon cancer cells show loss or reduced expression of RI and/or RII transcript and protein levels without mutation, indicating abnormalities in transcriptional or post-transcriptional regulation of TGF-b receptors may be involved in the absence of TGF-b growth control and its tumor suppressor activities.…”