Metastasis Suppressor Genes for Breast Cancer

Institution: Stanford University
Investigator(s): Stanley Cohen, M.D. -
Award Cycle: 2000 (Cycle VI) Grant #: 6JB-0008 Award: $312,937
Award Type: IDEAS II
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (2000)
Despite the advances in our understanding of the genetic causes of cancer, there are gaps in our ability to associate specific genes with key processes in the disease. The critical clinical threshold in breast cancer is metastasis. Diagnosis prior to metastasis is favorable and usually treatable. Unfortunately there are no effective therapeutic approaches aimed specifically at the genetic causes of breast tumor metastasis. We currently know a great deal about many cancer cell proteins associated with metastasis, such as adhesion receptors, proteases, and angiogenic factors. However, we still know little about the genes that could serve as the common regulatory elements.

We hypothesize that there exist discrete breast tumor metastasis suppressor genes (MSGs). In this project we plan to use a novel genetic strategy (RHKO: Random Homologous Knock-Out). This makes it possible to reproduce the genetic events that occur spontaneously during the acquisition of metastatic capabilities by cancer cells, and it also makes practical the isolation and characterization of MSGs. This procedure was developed in our laboratory to identify previously unknown genes. A critical feature of RHKO is that it enables random inactivation of both alleles of chromosomal genes. This is accomplished using a special ‘search’ function that includes a regulated antisense promoter. Two experimental advantages evolve from this method. First, we can readily reverse the RHKO process, so the role of candidate MSGs can be tested and validated. Second, MSGs inactivated by RHKO will contain a "tag," so they can be readily isolated and characterized. Finally, the MSGs of interest can be rapidly tested for their relevance to breast cancer by using microarrays of human cDNAs. This is accomplished by either raising or lowering the MSG of interest, and then using the microarray to see which metastasis proteins (e.g., proteases, adhesion receptors, etc.) are affected. We believe that the MSGs will represent the ‘master switches’ for metastasis processes.

The extraordinary translational potential of the work resides in its ability to provide validated targets for: 1) the development of new therapeutic measures aimed at altering the metastatic capabilities of human breast cancers, 2) the design of epidemiological approaches for assessing the genetic susceptibility of individuals to develop tumor metastases, 3) the creation of tests of prognostic value for managing patients with breast cancer, and 4) the evaluation of new approaches that may be proposed to limit the metastatic spread of cancer cells.

Final Report (2002)
Cancer metastasis—the spread of tumor cells from the primary neoplasm to different organs and their proliferation at distant sites—is the principal cause of death from cancer. Yet, other than the destruction and removal of cancer cells per se, there currently are no effective therapeutic approaches aimed specifically at controlling the processes that allow cancer metastasis to occur; this is due in large measure to our limited current understanding of basic biology of cancer metastasis. The goal of this research is to further an understanding of the genetics of cancer cell metastasis by identifying and isolating genes that have the ability to suppress the metastasis of human breast cancers [i.e., metastasis suppressor genes (MSGs)].

Our experimental strategy was aimed at the direct isolation of cancer cells defective in MSGs is Random Homozygous Knock Out (RHKO; Li and Cohen, 19961), a method for discovering genes whose homozygous functional inactivation results in biological effects of interest. This procedure allows the experimental reproduction of the spontaneous genetic events that create metastatic cancer cells. In addition, since we start from cells that initially lack metastatic capabilities, we can identify the specific genes that have a crucial role in preventing cancer metastases. We successfully tested the RHKO system and vectors, prepared libraries of cancer cells that acquired metastatic properties as a result of RHKO, and isolated and cloned these cells. We have identified genes whose inactivation has resulted in metastases and studied their actions. One of these genes encodes an internal signaling kinase that potentially may be a therapeutic target. Although this gene appears to be an “oncogene” rather than a MSG, it is clearly metastasis-associated. A second MSG-like gene has been tentatively identified, but the precise location has not yet been mapped. Finally, we have initiated a collaboration with a biotech company, called Xenogen, Inc. in Alameda, CA, to utilize a special mouse system that allow fluorescent detection of metastasis “externally.” Thus, the mice do not need to be killed and dissected at experimental time points, and we can detect tumors containing only a few thousand cells and in all body locations. This is an important advance, since many animal experiments examine only pre-selected metastatic sites, such as lung and liver.

Further study of MSGs may provide a tool of important prognostic value for the care of breast cancer patients. In addition, these genes may prove predictive of the eventual genetic susceptibility of individuals to develop tumor metastases. However, the more immediate application is the creation of a framework for evaluating new approaches aimed at limiting the metastatic capabilities of breast cancer cells.