Retinoblastoma tumor suppressor gene/ cell cycle/ RNA Processing

The main goal of the laboratory is to understand how the retinoblastoma tumor suppressor protein (Rb1) functions to prevent the onset of cancer. Mutational inactivation of both Rb1 alleles is the cause of the childhood cancer retinoblastoma. As such, Rb1 is the only known human gene whose mutation is necessary and sufficient for a human cancer. Deregulation of the Rb1 pathway is also a major contributing factor to most adult cancers. The frequency of Rb1 deregulation in many clinically common adult cancers is greater than 80%, making it one of the most frequent molecular alterations detected in human cancer. Rb1 and the regulatory pathway in which it functions is clearly important for understanding how cancer arises and for designing therapeutic strategies for cancer treatment. Despite its importance, the identities of the molecular mechanisms that mediate Rb1 tumor suppression are currently unclear.

The Rb1 encoded protein (pRb) is an important regulator of the cell cycle. One way that pRb restrains cell cycle progression is through its ability to bind and negatively regulate the E2F family of transcription factors. E2Fs control the expression of genes required for the transition of cell cycle checkpoints. It is currently hypothesized that the ability of pRb to restrain cell cycle progression through inhibition of E2Fs is centrally important for pRb mediated tumor suppression. To test this hypothesis, we have genetically engineered mice to express mutant pRb that is specifically deficient in regulating E2Fs. By crossing this mutant allele into various mouse models of cancer, we can determine the relative contribution of the pRb/E2F mechanism to tumor suppression in a number of tissues. We are currently extending this approach by creation of additional Rb1 mutations that compromise the ability of pRb to interact with other proteins, but retain the ability to bind and regulate E2Fs.

 

Rb1 protein has been documented to bind over one hundred and fifty different cellular proteins, in addition to the E2F transcription factors. Even if many of these are not physiologically important, the number of possible molecular mechanisms mediating pRb tumor suppression may be large. A major gap in our knowledge, therefore, is the identity of the cellular proteins that pRb associates with in different tissues, and the functional consequences of these interactions. We have engineered an affinity tagged Rb1 allele in the mouse to help fill this gap. The tag placed at the end of the Rb1 gene facilitates purification of pRb containing protein complexes by affinity chromatography without affecting the function of the protein. Thus we can purify pRb complexes from any tissue accessible in the mouse, and identify cellular proteins that interact with pRb in those tissues. We are combining this approach with the mutational analysis described above. Thus we can compare the cellular proteins that interact with wild type and mutant pRb. By correlating the specific molecular defects of a series of such mutants with their ability to prevent tumors in mice, we will be able to identify the mechanisms that make important contributions to Rb mediated tumor suppression.

 

Through this type of analysis we have identified another protein that may be important for Rb1 tumor suppression. This protein, encoded by the Thoc1 gene, binds pRb at a position distinct from where E2Fs bind, suggesting multiple cellular proteins may simultaneously bind a single molecule of pRb. Another major area of interest in the lab is to determine the normal function of Thoc1, and to test whether it is important for Rb1 tumor suppression. We are using both molecular and genetic approaches to study this gene, including the creation and analysis of mice containing Thoc1 mutations.

We have found that the E2F binding deficient Rb1 allele has no detectable tumor suppressor activity in neuroendocrine tissues such as the pituitary and thyroid glands. These tissues expressing the pRb mutant display abnormal cell cycle regulation. Hence the pRb/E2F mechanism is likely critically important for tumor suppression in these tissues. In contrast, the mutant pRb has significant tumor suppressor activity in the prostate gland. Thus the pRb/E2F mechanism is not absolutely required for tumor suppression in the prostate. We are currently attempting to identify the molecular mechanisms that may account for mutant pRb mediated tumor suppression in the prostate, primarily using affinity tagged versions of the mutant Rb1 allele. These findings unexpectedly indicate that pRb may use different molecular mechanisms to suppress tumorigenesis in different tissues. This has important implications for therapies designed to target the Rb1 pathway, as the response to these therapies may vary from tissue to tissue.

 

We have also created several mutant alleles of the mouse Thoc1 gene in order to assess its normal physiological function. Mice homozygous for a Thoc1 null allele are embryonic lethal, with development ceasing at the early blastocyst stage. This demonstrates that Thoc1 is required for normal mouse development. We have also created a hypomorphic Thoc1 allele that expresses reduced levels of protein. Mice homozygous for this allele are viable. However, these mice are smaller than wild type mice, are infertile, and develop lymphoma at increased frequency. We are currently studying the defects in testes development that cause infertility in male mice. Finally, we have constructed a conditional null allele of mouse Thoc1. Thus, we can delete the Thoc1 gene in adult mice in a tissue specific manner. Surprisingly, loss of Thoc1 has little effect on a number of adult tissues, including the mammary gland and the prostate. Thus differentiated cells of the adult animal are more permissive of Thoc1 loss than cells of the developing embryo. Interestingly, we also find that cancer cells are more sensitive to Thoc1 loss than normal differentiated cells. We are currently attempting to verify this hypothesis in vivo using mouse models of cancer. If true, the hypothesis suggests Thoc1 may be a useful molecular target for cancer therapy. Antagonizing Thoc1 function is predicted to preferentially compromise the viability of cancer cells, while sparing normal cells.

 

Using molecular approaches, we have also determined that Thoc1 is the human functional orthologue of the yeast HPR1 gene. Both yeast and human gene products are involved in transcriptional elongation and RNA processing. Preliminary data suggest pRb can inhibit the activity of the Thoc1 protein, potentially providing an additional molecular mechanism by which pRb negatively regulates RNA expression. We are currently validating the physiological significance of the Rb1/Thoc1 interaction in vivo.