Research interests:
Key Words:Insulin-like growth factors (IGF), IGF-I receptor, Mechanisms of transcription, Tumor suppressors, WT1, p53, BRCA1.
The insulin-like growth factors (IGF-I and IGF-II) are a family of mitogenic polypeptides with important roles in normal growth and development. The biological actions of the IGFs are mediated by the IGF-I receptor (IGF-IR), a transmembrane tyrosine kinase which is structurally related to the insulin receptor. In addition to its basic requirement for cell cycle progression, the IGF-IR plays a pivotal role in malignant transformation. The receptor is highly expressed in most tumors and cancer cell lines, where it functions as an antiapoptotic agent, conferring enhanced survival to malignant cells. On the other hand, a decrease in the number of receptors was shown to cause massive apoptosis, suggesting that a critical determinant of tumor behavior is the concentration of cell-surface receptors. Over the last several years our work focused on defining the molecular mechanisms that are responsible for the expression of the IGF-IR gene during normal development, on understanding the events and factors that govern its levels of expression (and therefore determine, to a large extent, the proliferative status of the cell) and, in particular, on analyzing the transcriptional mechanisms that underlie the pathological expression of the IGF-IR in malignancy.
Following the cloning and characterization of the regulatory region of the IGF-IR gene, we discovered that this promoter is a molecular target to WT1, a transcription factor with tumor suppressor activity whose deletion or mutation has been linked to the etiology of Wilms' tumor, a pediatric kidney cancer. Using a combination of methods, including transient and stable transfections, electrophoretic mobility shift assays, and DNase footprintings, we showed that the activity of the IGF-IR promoter is negatively regulated by WT1, whereas mutant forms of WT1 had no effect. Based on these results, we hypothesized that inhibition of the IGF-IR gene by WT1 in the terminally-differentiated kidney cell prevents it from progressing through the cell cycle. Disruption of the WT1 gene in Wilms' tumors and related cancers may result in a defective protein unable to suppress the IGF-IR promoter. The net consequence will be an increase in proliferation rate. A particular malignancy in which we showed that this is, in fact, the case, is desmoplastic small round cell tumor (DSRCT), an abdominal neoplasia of young males. DSRCT is characterized by a recurrent chromosomal translocation that "hits" the WT1 gene and fuses its C-terminal, DNA-binding domain, to the N-terminal, activation domain of the Ewings' sarcoma gene, EWS. As predicted, this rearrangement abrogates the tumor suppressor role of WT1 and generates a potential oncogene capable of binding and transactivating the IGF-IR promoter. Current studies are intended to elucidate the molecular interactions between WT1 and EWS-WT1 in controlling transcription of the IGF-IR gene. The rationale for these studies is the fact that tumor cells displaying the translocation retain one normal allele encoding WT1. Therefore, the expression of the IGF-IR gene, and the proliferative status of the cell, depend on the potential interplay between native WT1 and mutant EWS-WT1 proteins.
This paradigm of tumor suppressor regulation of the IGF-IR gene in pediatric solid tumors prompted us to investigate the potential transcriptional regulation of the IGF-IR gene by BRCA1, a gene whose mutation is linked to a large proportion of familial breast and ovarian cancers. Because the IGF-IR is highly overexpressed in most breast tumors, we postulated that its expression may be under inhibitory control by the BRCA1 gene product. Using transient transfection assays we demonstrated that expression of BRCA1 resulted in a dose-dependent suppression of cotransfected IGF-IR promoter constructs. Although the molecular targets of BRCA1 have not yet been identified, it is possible that part of the proapoptotic activity of BRCA1 is achieved via suppression of the strongly antiapoptotic IGF-IR gene. Mutant versions of BRCA1 lacking transactivation activity can potentially derepress the IGF-IR promoter, resulting in augmented levels of IGF-IR mRNA and IGF-I binding in breast cancer. Current studies are intended to understand the molecular mechanisms responsible for transcriptional suppression of the IGF-IR gene by BRCA1.
In addition to tumor-specific tumor suppressors (e.g., WT1, BRCA1) we have also explored the transcriptional regulation of the IGF-IR gene by p53, the most frequently mutated tumor suppressor in human cancer. Wild-type p53 displayed a potent suppressive effect towards the IGF-IR promoter whereas tumor-derived, mutant forms of p53 significantly stimulated its activity. Furthermore, wild-type p53 decreased the IGF-I-induced tyrosine phosphorylation of the IGF-IR and of IRS-1, whereas mutant p53 stimulated their phosphorylation. Taken together, these results suggest that, at least part of, the effects of wild-type p53 on apoptosis and cell cycle arrest are mediated via suppression of the IGF-IR promoter. Lack of inhibition by mutant p53 may accelerate tumor growth and inhibit apoptosis, thus providing an increased survival capacity to malignant cell populations.
In summary, our studies over the last years have significantly contributed to the understanding of the molecular mechanisms responsible for the expression of the IGF-IR gene in the malignant cell. These mechanisms are extremely complex and involve interactions between numerous DNA-binding and non DNA-binding transcription factors. In view of the central role of the IGF-IR in cell cycle progression and in the etiology of a number of malignancies, we feel that a clear understanding of the interplay between wild-type and mutant forms of tumor suppressors and oncogenes in transcriptional control of the IGF-IR gene will provide the foundation for future studies aimed at interrupting the IGF growth loop.
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