TßR-I(6A): A New Cancer Susceptibility Gene
By Boris Pasche, MD, PhD
Transforming growth factor beta (tgf-b) is one of the most potent naturally occurring inhibitors of cell growth. TGF-b exerts its action by binding to a type I (TbR-I) and a type II (TbR-II) transmembrane receptor located on the cell membrane. Downstream signaling is mediated by TbR-I once the ligand has complexed with both receptors. Three proteins, Smad2, Smad3, and Smad4/DPC4 have been found to be essential downstream components of the TGF-b signaling pathway in mammalian cells.1
The growth inhibitory effects of TGF-b on target cells include induction of G1 arrest, promotion of terminal differentiation or activation of cell death mechanisms.2 Unrestricted cell growth due to a lack of growth inhibitory activity appears as the most important of the possible consequences of a defect in TGF-b function. This hypothesis was confirmed by the discovery that mice with decreased TGF-b levels have an increased susceptibility to tumor development.3 The relevance of TGF-b pathway alterations in human cancer is confirmed by the finding that a TGF-b type II receptor (TbR-II) is inactivated by mutations in gastrointestinal cancer with microsatellite instability.4 Targeting of the TGF-b pathway in cancer is additionally demonstrated by the identification of inactivating mutations in Smad4/DPC4 in colon, breast, ovary, lung, and head and neck cancer and Smad2 in colon cancer.5-8 Homozygous deletion of TbR-I is observed in pancreatic and biliary carcinomas and a tumor-specific mutation has recently been reported in breast cancer.9,10 Additionally, in several human cancer cell lines that lack active TGF-b receptors, restoration of functional receptors reverses the transformed phenotype of the cell lines.11,12
The search for tumor specific mutations led to the discovery of a polymorphic allele of the type I receptor TbR-I(6A) that has a deletion of three alanines within a 9-alanine stretch in the coding sequence of exon 1.13 (See Figure 1.) Intriguingly, the frequency of TbR-I(6A) homozygotes in both tumor and non-tumor samples from patients with a diagnosis of cancer was higher than expected, suggesting that TbR-I(6A) might contribute to tumor development.13
Epidemiological Assessment of TbR-I(6A)
To test the hypothesis of an association of TbR-I(6A) with cancer, a case control study was performed to determine TbR-I(6A) frequency among patients with a diagnosis of cancer and controls. The control group consisted of healthy volunteers of identical gender, ethnic, and geographical background. Peripheral blood was obtained from both patients and volunteers and genomic DNA was extracted. Polymerase chain reaction (PCR) amplification was followed by gel electrophoresis that allowed for the identification of the various genotypes. Three rare additional variants were discovered during the study, each with a different number of alanine residues: TbR-I(5A), TbR-I(8A), and TbR-I(10A).
Analysis of the data shows that the frequency of TbR-I(6A) heterozygotes is significantly higher among patients with a diagnosis of cancer (14.6%) than among healthy volunteers of identical gender, ethnic, and geographical status (10.6%) (P = 0.02, Fisher’s exact test). Furthermore, there are nine TbR-I(6A) homozygotes among patients with a diagnosis of cancer and none among controls (P < 0.01, Fisher’s exact test). A subset analysis reveals that four of 112 patients with colon cancer were TbR-I(6A) homozygotes (P < 0.01).14 TbR-I(6A) homozygotes are noted in single cases of lymphoma, non-small cell lung cancer, and ovarian cancer, and in two cases of germ cell cancer.
The over-representation of TbR-I(6A) homozygotes in patients with colon cancer is further confirmed in an analysis of a subset of the study population presumed to be in Hardy-Weinberg equilibrium. Indeed, there were two TbR-I(6A) heterozygotes and two TbR-I(6A) homozygotes among 25 colon cancer cases of Ashkenazi background. Compared to a TbR-I(6A) heterozygote frequency of 12% observed in Ashkenazi controls, the finding of two homozygotes is highly significant (P < 0.01).14
The finding of TbR-I(6A) overrepresentation among patients with a diagnosis of cancer is corroborated by two other studies.14,15 One study conducted on U.S. and Jamaican residents assessed TbR-I(6A) frequency among 66 patients with a diagnosis of cancer of the cervix and 64 matched controls.15 The other study evaluated TbR-I(6A) among 347 Northern Italian patients with a diagnosis of either breast, colon, or bladder cancer and 50 random controls from the same region without other identifiers.14 A meta-analysis of the three studies shows a remarkably similar TbR-I(6A) heterozygote frequency among patients with a diagnosis of cancer, which is significantly higher than that of the control group. (See Table I.) There are 11 TbR-I(6A) homozygotes among patients with a diagnosis of cancer and none among 853 controls, a highly significant difference. (See Table I.)
| Table I-TßR-I genotypes in cases from New York (NY), United States/Jamaica (USJ), and Italy (ITA)14,15 | ||||||
| Study | N | wt/wt | wt/6A | 6A/6A | wt/10A | 6A/10A |
| NY | 851 | 716 | 123(14.6%) | 9 | 2 | 1 |
| USJ | 66 | 56 | 10(15.2%) | 1 | 0 | 0 |
| ITA | 347 | 294 | 51(14.7%) | 1 | 1 | 0 |
| Total | 1,264 | 1,066 | 184(14.6%)* | 11** | 3 | 1 |
| * P = 0.037 when compared with 11.2% TbR-I(6A) heterozygotes among controls | ||||||
| ** P = 0.004 when compared with no TbR-I(6A) homozygotes among controls | ||||||
Functional Assessment of TbR-I(6A)
The epidemiological findings of an association between TbR-I(6A) and cancer and the major growth-inhibitory effects of TGF-b mandated thorough assessment of TbR-I(6A) signaling capabilities.
TbR-I(6A) was transfected into mink lung epithelial cells devoid of type I receptor, and TGF-b growth inhibition was compared to cells transfected with the wild-type receptor. Growth inhibition was measured by assessing the rate of DNA synthesis following exposure of the transfected cells to TGF-b for 18-24 hours. TbR-I(6A) cells were significantly less growth-inhibited upon exposure to TGF-b than their wild-type counterpart (P < 0.01).14 Similar results were obtained using the firefly luciferase reporter gene, pSBE4, a surrogate marker of TGF-b-mediated growth inhibition.15 TGF-b binding was investigated in cells stably transfected with either TbR-I(6A) or TbR-I cells and no significant differences were observed.14 Similarly, there was no difference in receptor turnover as assessed by metabolic labeling.14
Summary
Hence, TbR-I(6A) is a hypofunctional type I TGF-b receptor and TbR-I(6A) carriers have an increased risk of developing cancer. Additional studies are needed to determine the magnitude of cancer risk and the contribution of other genetic and environmental factors. TbR-I(6A) contribution to cancer development is potentially attributable to various reasons. TbR-I(6A) encodes for a receptor with decreased TGF-b growth inhibition, and cells carrying such a receptor may be more prone to uncontrolled growth and tumor development. Also, TbR-I(6A) is a dominant-negative receptor that contributes to tumor development through a mechanism different from decreased TGF-b growth inhibition. Finally, TbR-I(6A) is a marker for a tumor susceptibility gene located within the same chromosome. The functional data gathered so far suggest that TbR-I(6A) decreased ability to mediate TGF-b growth inhibition contributes to tumor development. Nonetheless, the intriguing finding of a higher proportion of TbR-I(6A) heterozygotes among patients with a diagnosis of cancer evokes the possibility of another, yet unknown, mechanism by which TbR-I(6A) may contribute to cancer development.
Recent reports indicate that germline mutations of TbR-II and Smad4/DPC4 may predispose to the development of hereditary nonpolyposis colon cancer and juvenile polyposis, respectively.16,17 (See Figure 2.) Taken together these results unveil the potential contributions of TGF-b signaling alterations in tumor development. (Dr. Pasche is a Fellow in the Department of Medicine and Cell Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY. Dr. Pasche is also the recipient of a K08 MCSD award from the National Cancer Institute.)
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