More than 200,000 women in the United States will be diagnosed with ‘invasive’ breast cancer this year, and more than 40,000 women will lose their lives. Despite significant advances made in understanding the behavior of breast cancer cells, we are still failing to interfere with the mechanical process of metastasis, the primary cause of death for breast cancer patients. Metastasis is the spreading of cancerous cells from the breast tissue into the blood stream or lymphatic system where they survive within the immune system and then exit at a distant organ where secondary tumors form. The mechanics of how a cancer cell achieves the ability to do this is poorly understood, and is the motivation for this investigation.
In 2008, the Cunliffe laboratory at TGen were the first to report the presence of a protein called Fn14 that is produced on the cell surface of invasive breast cancer cells. Importantly, this protein alone, was shown to be sufficient to control the cancer cells’ invasive behavior. This was accomplished by deliberatively blocking the function of the Fn14 protein in breast cancer cells and measuring their ability to invade substances mimicking normal breast tissue. Conversely, adding an artificial copy of the Fn14 gene into to early-stage breast cancer cells made them highly invasive. These preliminary studies highlight Fn14 as a novel therapeutic target in breast cancer. An extension of these preliminary studies is required in a preclinical animal model to verify the role of the Fn14 protein in the metastatic spread of breast cancer cells. If blockade of Fn14 in a mouse model of breast cancer invasion and metastasis can halt the spread of cancerous cells, this evidence will propel efforts to develop anti-Fn14 drugs for clinical use.Goal
The goal of this project is to determine whether the incidence of distant metastases in an established laboratory model of breast cancer is decreased following blockade of the Fn14 protein.Current Status
If our hypothesis is correct, our breast cancer cell line engineered to no longer produce the Fn14 protein will have reduced capability to metastasize to distant organs of the mouse. Our first objective was to consider each of the immortalized breast cancer cell lines that overproduce Fn14 and behave in an invasive manner in the laboratory, and select one such model to move into a laboratory mouse model. We selected the first breast cancer cell line called MDA-MB-231. We have two strains of this particular cell line, which differ in ‘where’ they selectively metastasize to in mice. The first cell strain, when injected into the mouse breast or into the mouse blood stream, will metastasize primarily to the lungs. The second strain we selected, MDA-MB-231-BR, is known to selectively metastasize to the brain and occasionally to the lung. We chose to genetically modify both strains to no longer make the Fn14 protein for this study.
We have successfully achieved blockade of the Fn14 protein in the first cell strain and confirmed they no longer behave in an invasive manner. Interestingly, after two attempts, we have failed to block the production of Fn14 protein in the second cell strain. To determine why this might be the case, we examined the genetic differences between the two strains, and identified the second strain to have an extra copy of the Fn14 gene from chromosome 16, which appears to be resulting in overproduction of the Fn14 protein so that it cannot be effectively blocked by the technology we are employing. It is interesting to speculate that this may be a contributing factor in the ability of this strain of cancer cells to spread to the brain rather than lung. We have not yet tested this hypothesis.
We moved forward with studies to inject the first strain and our engineered which no longer make no longer makes Fn14 protein into SCID (Severe Combined Immuno-Deficient) mice. We use these mice as they have been bred to have a depleted immune system, thereby will not reject the injected human cancer cells in experimental models. A requirement of the mouse facility we are collaborating with at the Van Andel Research Institute (Grand Rapids, MI), is to certify that all incoming immortalized cancer cell lines are certified pathogen free. This testing was performed, and all cell lines were certified pathogen free. The first strain of cells has been injected into a small series of mice and was able to form tumors in the fat pad and formed lung metastases, as expected. Our current focus is to set up a larger experiment to determine whether our cell strain missing the Fn14 protein no longer has the ability to form lungFinancial Update
We are currently meeting our budget projections based on workflow timelines.What’s Next.
We are in the process of conducting our final experiment to determine whether the breast cancer cells which no longer make the Fn14 protein have reduced ability to metastasize to the mouse lung.Milestones.
1) We have successfully engineered an invasive human breast cancer cell line to no longer produce the Fn14 protein. 2) All cell strains were certified pathogen-free to enter the mouse vivarium at the Van Andel Research Institute. 3) Received approval from the Institutional Animal And Use Committee (IACUC) to perform the proposed mouse xenograft studies. 4) Mouse xenograft studies with breast cancer cells producing the Fn14 protein were successfully injected into SCID mice and formed metastatic tumors. Current studies are to determine whether the strain missing the Fn14 protein has reduced capacity to metastasize in the SCID mouse model.