Research Grants Awarded
Dbc2/Rhobtb2: An Atypical Rho Gtpase And Tumor Suppressor In Breast Cancer Development?
Background: The Ras oncogenes are the founding members of a large superfamily of Ras-related genes, with Rho (Ras homologous) family genes comprising a major branch of this family. While the role and importance of aberrant Ras function in cancer is well-established, only recently have studies begun to implicate Rho family proteins in oncogenesis. Rho family small GTPases are signaling proteins that modulate diverse normal cellular processes, including cell migration, proliferation, and survival. There is considerable evidence that the aberrant expression and function of multiple members of the Rho family proteins promote breast cancer development and growth. In particular, recent studies implicated an atypical Rho GTPase, DBC2 (for deleted in breast cancer 2) in breast cancer. DBC2 gene expression is found in normal breast, but lost in more than 50% of breast cancers. Since restoration of DBC2 expression in DBC2-deficient breast carcinoma cells caused growth inhibition, DBC2 loss may promote the aberrant growth of a majority of breast cancers.
Objective/Hypothesis: Like Ras, DBC2 is a GTPase (also called RhoBTB2), and hence, likely to serve a function in signal transduction. Additionally, DBC2 is a substrate for the Cullin 3 ubiquitin ligase complex, and hence, may regulate protein degradation. Since aberrant signaling and protein degradation processes contribute to cancer cell growth, we hypothesize that the loss of DBC2 function is likely to contribute significantly to the transformed and tumorigenic growth of breast cancer cells. Thus, establishing a critical role for aberrant DBC2 function in breast cancer growth, and determining its mechanism of tumor suppression, may advance our understanding of breast cancer development and may establish DBC2 as a diagnostic marker as well as a novel target for drug discovery for breast cancer treatment. Currently, the precise contribution of DBC2 loss to the aberrant growth properties of breast cancer cells, and the roles of the GTPase and Cullin 3 ubiquitin ligase function in DBC2 growth suppression, remain largely unknown. Our goals are to establish the biological ramifications of DBC2 loss and to determine how DBC2 may function as a tumor suppressor, and thus, validate DBC2 as a potential diagnostic marker or target in the therapeutic fight against breast cancer.
Specific Aims and Study Design: First, we will evaluate the contribution of DBC2 loss to different facets of the aberrant breast cancer phenotype (e.g., anchorage-independent growth, invasion, tumorigenicity). To address this question, human breast tumor cell lines in which DBC2 levels will be either reintroduced by ectopic expression or silenced by interfering RNA will be used. Second, in addition to a Rho GTPase domain, DBC2 contains a tandem pair of BTB domains, domains that promote protein-protein interactions, and the first BTB domain has been shown to interact with the Cullin 3 protein ubiquitination complex, promoting DBC2 degradation. By generating mutants of the Rho GTPase domain or the BTB domains, we will determine if these biochemical functions of DBC2 are important for its function as a tumor suppressor in breast cancer and in promoting protein ubiquitination. Using proteomic analyses, we have identified DBC2-interacting proteins that may represent candidate GTPase effectors and/or Cullin 3 substrates. Lastly, using confocal microscopy analysis and protein targeting, we will determine if the endomembrane localization of DBC2 is important for its biological activity.
Potential outcomes and benefits of the research: Based on the validated functional roles of other Rho family GTPases in breast and other cancers, we anticipate that our studies will establish an important consequence of DBC2 loss for breast cancer growth. The overall impact of our research on breast cancer treatment may be the establishment of DBC2 as a diagnostic marker as well as a potential target for anti-cancer drug discovery. The prevailing bias is that protein kinases are the most attractive and ?druggable? targets for anti-cancer drug development. However, despite the recent success in the FDA approval of protein kinase inhibitors for cancer treatment, it is also clear that protein kinase-based therapies will not be effective for the treatment of many cancers. Therefore, there is a need to identify and validate new classes of proteins for anti-cancer drug discovery. Multiple members of the Rho family of small GTPases have been implicated in breast cancer, with the apparent loss of DBC2 expression seen in more than 50% of breast cancers. Hence, if our studies can further validate DBC2 loss in breast cancer growth, and elucidate mechanisms by which DBC2 regulate cell growth, our findings may impact the treatment of a majority of breast cancers. We believe that our studies are innovative and novel in evaluating an unconventional protein target for breast cancer treatment. Our studies may also stimulate greater consideration of Rho family GTPases as an important new class of signaling proteins for target-based treatment of breast cancer.
The importance of aberrant cell signal transduction in breast cancer development and malignant growth transformation is well established. For example, overexpression of the HER2 receptor tyrosine kinase is associated with advanced and invasive breast cancers, and inhibition of HER2 signaling by Herceptin is an important treatment for breast cancer. Recently, a new class of signaling proteins, Rho (Ras homologous) family GTPases (20 human members), has been implicated in breast cancer. Like the related Ras GTPases, which are the most frequently mutated oncogenes in human cancers, Rho proteins control many normal cellular processes, including cell growth and survival, cell movement, and gene expression. Therefore, it is not surprising that there is considerable evidence for the involvement of aberrant Rho GTPase function in breast cancer development. Recent studies have implicated an atypical member of this family, DBC2 (for deleted in breast cancer 2), in sporadic breast cancer growth. Also called RhoBTB2, DBC2 was identified as a gene missing or inactivated in human breast cancers. Whereas DBC2 expression is detected in 100% of normal breast tissue, it is lost in more than 50% of breast cancer cells. Missense mutations in the DBC2 gene have also been isolated from primary breast tumors. Furthermore, when DBC2 expression was transiently restored in DBC2-deficient breast cancer cells, growth inhibition was seen. Based on these observations, it is hypothesized that DBC2 acts as a tumor suppressor gene in breast cancer. However, the precise contribution of DBC2 loss to breast cancer growth, and how DBC2 functions as a tumor suppressor, are issues that remain unresolved. DBC2 is expected to have two functions in normal cells: (1) regulation of signal transduction and (2) regulation of protein degradation. Both of these processes are important factors in the conversion of normal cells to cancerous cells. Since recent target-based therapies that block these cancer-associated perturbations have met with clinical success, we propose that DBC2 may be an important candidate for target-based anti-breast cancer drug discovery. The broad goals of our studies are to validate DBC2 as an important diagnostic marker or drug target for breast cancer treatment. First, we want to determine what aspect of breast cancer growth and invasion the loss of DBC2 may contribute to. Second, we want to determine how DBC2 works as a tumor suppressor, which may then provide clues for designing therapeutic approaches to ?reactivate? DBC2 function in breast cancers. This will be important to do, since the simple approach of gene therapy to restore DBC2 expression is currently not feasible. Our general approaches will use human breast cancer cells and we will study how DBC2 protein expression regulates breast cancer cell growth in vitro or in immunodeficient mice. In summary, our studies may critically determine the usefulness of DBC2 as a novel diagnostic and therapeutic target for breast cancer. Although protein kinases have traditionally been the most attractive targets for anti-cancer drug development, with the growing importance of Rho GTPases in breast cancer, we believe that more effort is needed to critically determine if GTPases will also be useful drug targets. Since DBC2 gene expression is lost in more than 50% of breast cancer cells, the results of our studies may establish new directions for the treatment of a majority of breast cancer patients. We believe that our studies are innovative and important, and are novel since we are pursuing a less conventional target for drug discovery. Thus, we anticipate that our studies will expand the repertoire of targets for rational drug discovery for breast cancer treatment.