Connexin Expression and Breast Cancer Metastasis to Bone
Open Access
- Author:
- Schenk, Rachelle Lois
- Graduate Program:
- Anatomy
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- April 08, 2010
- Committee Members:
- Dr Patricia Mc Laughlin, Thesis Advisor/Co-Advisor
Henry Joseph Donahue, Thesis Advisor/Co-Advisor - Keywords:
- breast cancer
metastasis
bone
4T1.2
NMuMG
cadherins
connexins
connexin 43
connexin 32
connexin 26
E-cadherin
N-cadherin
OB-cadherin - Abstract:
- Each year, breast cancer causes over 500,000 deaths worldwide. If caught at early stages, metastasis can often be localized, confined, and treated. However, if time for progression of metastasis has occurred, it spreads to bone and surrounding tissues, and quickly metastasizes throughout the body. Breast cancer, in particular is among the most common to preferentially metastasize to bone, with about 73% of those affected developing bone metastasis as a result of breast cancer. However, at this point we do not fully understand why breast cancer cells preferentially metastasize to bone, and through what exact mechanism this occurs. Studies have been previously conducted utilizing human breast cancer cells extracted from metastatic mammary tumors as a model. These human studies investigated the connexin and cadherin expression profiles, as well as the metastatic characteristics of migration and adhesion. Studies have also demonstrated that it is possible to inject human cells into a mouse model if the model is in an immunocompromised state. However, the utilization of transgenic models, such as Cx43 deficient mice, presents a problem to this scenario because these models are not from an immunodeficient background. Therefore, it is important to develop an in vivo murine model of breast cancer metastasis to bone. However, in order to employ a murine model, it is critical to know the connexin and cadherin expression profiles in mice. The results of the murine studies are very different from those found in human models and, in many ways, actually present opposite results. It is important to consider this when working with mice as a model for breast cancer. At the initiation of this experiment, it was hypothesized that breast cancer cell lines express different connexin and cadherin expression profiles relative to normal breast epithelial cells. To test this hypothesis the connexin and cadherin expression profiles of murine breast cancer cells (4T1.2 cells) and normal breast epithelial cells (NMuMg cells) were characterized through Western blot and RT-PCR analyses. The results of these experiments supported the hypothesis that murine breast cancer cells (4T1.2) possess a different connexin expression profile than normal murine breast epithelial cells (NMuMG). Regarding connexin 43, though both 4T1.2 and NMuMG cells show slight expression of this gene in comparison to the positive control (MLO-Y4), the 4T1.2 cell line expressed it by almost a 50% greater amount than the NMuMG cell line. Regarding connexin 32, NMuMG cells moderately expressed Cx32 in comparison to the positive control (Mouse Liver cells), while the 4T1.2 cell line does not express this protein. Regarding, connexin 26, 4T1.2 cells display a greater expression than the NMuMG cell line, but neither cell line displays statistically significant expression of Cx26. The results of these experiments revealed that murine breast cancer cells displayed a different cadherin expression profile relative to normal breast epithelial cells. Regarding, E-Cadherin, the 4T1.2 cell line had an abundance of this protein, but little was detected in the NMuMG cell line. In fact, the 4T1.2 cell line expressed E-Cadherin 2.5 times more than the NMuMG cell line. Regarding, N-Cadherin, the 4T1.2 cell line exhibited great expression of N-Cadherin, while NMuMG showed only trace amounts of expression. Regarding OB-Cadherin, neither the 4T1.2 cell line nor the NMuMG cell line showed expression of this cadherin. It was also hypothesized that murine breast cancer cells would display greater levels of migration and adhesion than normal murine breast epithelial cells. To test this hypothesis, the migration and adhesion in both of these cell types were quantified. The results of these experiments showed murine breast cancer cells displayed increased levels of adhesion in comparison to normal murine epithelial cells. Regarding adhesion, the NMuMG cell line displayed statistically significant higher levels of adhesion and over three times more adhesion than the 4T1.2 cell line. The results from the migration assays revealed that murine breast cancer cells exhibit greater levels of migration than normal mouse epithelial cells. In conclusion, murine breast cancer cells display decreased levels of adhesion and significantly increased levels of migration relative to normal murine breast epithelial cells. These findings support previous human and murine studies on the metastatic properties of adhesion and migration. Over the last fifteen years, survival rates among breast cancer patients have steadily increased due to major gains in our understanding of this disease. However, future advances will require a better understanding of the genetics and biology of human breast cancer, which will assist with development of new preventative and therapeutic strategies. Scientific findings must be tested in a setting in which potential clinical responsiveness can be assessed. This requires better in vitro and in vivo models as well as an understanding of the similarities and differences in expression and metastatic behavior between animal models and human models. This study focused on understanding the characteristics of an established murine breast cancer cell line and a normal murine epithelial cell line through which a highly effective in vivo model can be developed.