TGF-B3 and small GTPases in Invasive Breast Cancer

Institution: Childrens Hospital, Los Angeles
Investigator(s): Vesa Kaartinen, Ph.D. -
Award Cycle: 2000 (Cycle VI) Grant #: 6KB-0015 Award: $323,925
Award Type: New Investigator Awards
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (2000)
Breast cancer is deadly because the cancerous cells are capable of leaving the original tumor site and entering the circulation. This allows cancer cells to move to distant locations and form new colonies (metastasize). Despite the recent progress in understanding mechanisms leading to the development of breast cancer, many basic questions remain unanswered.

In this research proposal, we will study the role of two types of molecules in the processes that allow tumor cells to invade the surrounding tissue, penetrate through blood vessel walls and form metastases. One of the molecules is a transforming growth factor (TGF-beta 3). The other is a GTPase molecule acts as a molecular switch that transmits the signal from an activated receptor to the inside of the cell.

We postulate that these molecules are key players in introducing changes in cancerous cells that give them the ability to form metastases.

This hypothesis will be tested by the following experiments: The results of these experiments will provide a well-defined starting point for designing ways to block invasive characteristics generated by these molecules, with the ultimate objective of therapeutic intervention in metastatic breast cancer.

Final Report (2003)
The overall objective of this project was to define molecular mechanisms by which malignant cancerous cells turn invasive and cause the deadly metastatic disease of the mammary gland. It has been shown earlier that many epithelial cells undergo a phenotypic change to motile “fibroblastoid” cells when exposed to transforming growth factor-? (TGF??). This process is called epithelial-to-mesenchymal transdifferentiation (EMT). Based on these observations we hypothesized that during breast cancer progression TGF-? induces EMT (i.e., change from stationary cells to mobile cells), and that this process requires activity of the small Rho-related GTPases (Rac and Rho), and involves changes in cell-matrix interactions. We proposed to test this hypothesis by i) determining changes in expression patterns, localization and function of several cell surface receptors (integrins) in a mammary epithelial cells during TGF-?3-induced EMT, ii) by determining the role of Rho/Rac signaling in TGF-?3-induced mammary epithelial cell transdifferentiation to mesenchymal cells using a cell culture model and iii) by defining the role of Rac3 in mammary gland tumorigenesis in vivo using a transgenic approach, in which constitutively active V12Rac3 gene expression is targeted to mammary epithelium.

Using a tissue culture model we have shown that migration of mammary epithelial cells is inhibited by inactivation of ?1-integrin and that ?1-integrin expression is dramatically reduced after TGF? induction. Moreover, we have shown that TGF? stimulation leads to a rapid activation of the small GTPase RhoA, followed by a gradual downregulation of the related small GTPases (Cdc42 and Rac). We have also shown that inactivation of the Rho signaling pathway interferes with TGF?3-induced EMT. Therefore, we generated transgenic mice, which express the activated small GTPase, Rac3, in mammary epithelium. These transgenic mice are not capable of nursing their offspring despite of the normal expression of main milk proteins, casein and whey acidic protein. Moreover, the sustained Rac3 activation leads to the delayed post-lactational mammary gland involution. The older transgenic mice display benign mammary gland lesions with signs of micro-glandular adenomas and a fibrocystic disease combined with inflammation. These findings suggest that Rac3 is a factor influencing proliferation of mammary epithelium and formation of premalignant lesions. To determine, whether Rac3 activation influences the mammary tumor formation and/or invasiveness, we crossed MMTV-V12Rac3 mice with the MMTV-erbB2 mice, which develop mammary tumors with high frequency. In the Rac3 transgenic and control backgrounds tumors appeared at the average age of 226 days (n=2) and 253 days (n=3), respectively. Currently we are testing the statistical significance of this phenomenon by using a larger number of samples. In addition, we are pursuing the biological significance of TGF-beta/BMP type I receptor Alk2 in mammary gland biology and disease by utilizing the tissue-specific knockout mouse model we recently generated.

Crkl enhances leukemogenesis in BCR/ABL P190 trangenic mice
Periodical:Cancer Research
Index Medicus: Cancer Res
Authors: Hemmeryckx B, van Wojk A, Reichert A, Kaartinen V, de Jong R, Pattengale, et al.
Yr: 2001 Vol: 61 Nbr: 4 Abs: Pg:1398-1405

The small GTPase Rac3 interacts with the integrin-binding protein CIB and promotes integrin alpha(IIb)beta(3)-mediated adhesion and spreading
Periodical:Journal of Biological Chemistry
Index Medicus: J Biol Chem
Authors: Haataja, L., Kaartinen, V., Groffen, J., Heisterkamp, N.
Yr: 2002 Vol: 277 Nbr: 10 Abs: Pg:8321-8

TGFbeta3-induced activation of RhoA/Rho-kinase pathway is necessary but not sufficient for epithelio-mesenchymal transdifferentiation: implications for palatogenesis.
Periodical:International Journal of Molecular Medicine
Index Medicus: Int. J. Mol. Med.
Authors: Kaartinen V, Haataja L, Nagy A, Heisterkamp N, Groffen J.
Yr: 2002 Vol: 9 Nbr: 6 Abs: Pg:563-70