Src Signaling in Breast Cancer

Institution: University of California, Berkeley
Investigator(s): Steven Martin, Ph.D. -
Award Cycle: 2003 (Cycle IX) Grant #: 9IB-0092 Award: $75,000
Award Type: IDEA
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (2003)
The v-Src (Rous sarcoma virus) tyrosine kinase was the first oncogenic gene discovered. The corresponding cellular gene, c-Src, is a non-receptor signaling kinase located at the cell membrane. The central hypothesis on which this research is based is that the Src tyrosine kinase and its effectors control not only cell proliferation, but also the pathways and genes that dictate cell polarity and tissue architecture. To explore this hypothesis we have teamed-up with colleagues, Dr. Mina Bissell’s group, at the Lawrence Berkeley National Laboratory. Breast cancer cells cultured in three-dimensional reconstituted basement membrane (3D-rBM) cultures revert to normal mammary ductal structures when signaling by the EGF-receptor (EGFR) or β1-integrins is inhibited. These findings have highlighted the importance of three-dimensional tissue architecture in the control of tumor cell proliferation. Both EGFR and integrin signaling pathways are known to be mediated by the non-receptor tyrosine kinase, Src. Furthermore, Src and some of its downstream effectors, such as the transcription factors Stat3 and Myc, are known to be increased in expression and/or activity in a variety of breast cancer types. Yet the detailed role of the Src-Stat3-Myc pathway in breast cancer and mammary cell biology remains uncertain.

We hypothesize that an increase in the activity of Src or its effectors is sufficient to convert normal mammary epithelium into “tumor-equivalent” cells that cannot form normal mammary acini (def. Terminal Ductal Lobular Units TDLU or acini). We also hypothesize that a decrease in the function of Src or its effectors may be sufficient to induce reversion of tumorigenic cells to regain the ability to form normal mammary structures. To test these possibilities, we will use a normal mammary epithelial line, HMT-3522-S1, and its tumorigenic derivative HMT-3522-T4-2. We will determine whether blocking the function of Src, Stat3 or Myc is sufficient to cause reversion of tumorigenic mammary epithelial cells to form normal mammary acini in three-dimensional matrix cultures. To examine the effect of malignant conversion and reversion on the function of Src, Stat3 and Myc we will use antibodies or DNA probes to determine their level of expression, degree of activation, extent of phosphorylation, or in vitro activity. In addition, to determine the effect of an increase in the activity of Src or its effectors, we will introduce into normal mammary epithelial cells wild-type or ”activated” Src, Stat3 and Myc constructs under the control of an inducible promoter.

Src, Stat3 and Myc are all under active investigation in both academic and commercial settings as possible targets for anti-cancer therapy. Yet, the effect of inhibiting or activating Src signaling in normal or tumorigenic cells is not well understood. In particular, it is not clear whether this pathway regulates the morphogenesis of normal breast acini or whether inhibiting this pathway in tumorigenic cells can result in reversion to the normal phenotype.

Final Report (2004)
Introduction: The differentiation and function of normal breast epithelial cells is dependent on cell polarity and tissue architecture. This tissue architecture, which can be replicated in a three-dimensional cell culture system, breaks down in mammary tumors. The signaling pathways that regulate cell polarity and tissue architecture are not well understood, although alterations in these pathways are of fundamental importance in cancer. The level and/or activity of the Src oncoprotein have been shown to be elevated in breast cancer, yet its role in breast cancer remains unclear.

Topic addressed: The central hypothesis on which this research is based is that Src signaling regulates not only cell proliferation but also the pathways and genes that dictate cell polarity and tissue architecture.

Progress towards specific aims: The major goal of these studies is to determine whether inhibition of Src and Src effector pathways is sufficient to induce reversion of tumorigenic mammary epithelial cells. In collaboration with Dr. Mina Bissell at Lawrence Berkeley National Laboratory, we used a number of early- and late-stage transformed breast cancer cell lines. The experiments involved treating the cells with Src family kinases, followed by morphology and motility studies using two-dimensional and three-dimensional tissue culture conditions. We found that Src inhibitors induce reversion of two distinct human mammary epithelial lines, as judged by the ability of the cells to polarize and form normal mammary epithelial structures, called acini. A third mammary cell line that is more aggressive and possesses the ability to invade the extracellular matrix did not undergo reversion under these conditions: instead inhibition of Src inhibited the ability of the cells to invade the extracellular matrix. We hypothesize that Src has a dual role in controlling mammary cell architecture. In cells that have not undergone an epithelium-to-mesenchyme transition (EMT), activation of Src is involved in disruption of acinar architecture, while in cells that have undergone this transition, Src is involved in the formation of structures involved in matrix invasion. These findings suggest that inhibitors of Src function may be of value both in preventing the progression of early lesions and in blocking invasion by tumors that are no longer capable of reversion.

Future directions and impact: We are currently using activated mutants of Src and Src effectors to study the mechanisms by which Src activity blocks acinus formation and promotes invasion. We are also extending these studies to a large panel of human mammary carcinoma cell lines, so that we can correlate the effects of these inhibitors with the genetic constitution of the tumors. Newer generations of Src inhibitors are being actively investigated as cancer therapeutics, and these studies should provide information that will guide use of these inhibitors.

Symposium Abstract (2005)
The primary goal of this project is to elucidate the function that the non-receptor tyrosine kinase, c-Src, plays in mammary carcinoma cells. c-Src regulates many signaling pathways that control cell processes such as proliferation, survival, polarity, migration, and invasion. However, although the levels and activity of Src have been shown to be elevated in numerous breast cancer cell lines and tissue samples from breast cancer patients, the specific role that Src plays in mammary cells remains undefined. We have investigated this question by examining mammary cells cultured in a three-dimensional reconstituted basement membrane (Matrigel). When cultured in Matrigel, normal mammary cells will polarize and form acinar structures similar to the terminal end buds formed in the mammary tissue during lactation and pregnancy. Tumorigenic cells fail to polarize in Matrigel and will form large, disorganized colonies. Treatment of the tumorigenic cells with compounds that inhibit certain signaling pathways can induce certain cells to revert and form acinar-like structures similar to normal mammary cells.

Using two Src-specific pharmacological inhibitors, we have found that Src inhibition induces reversion of two tumorigenic cell lines (T4-2 and MDA-MB-435S) and formation of acinar structures in 3-D cultures as assessed by the polarized localization of protein markers in these cells. A third, more aggressive and invasive cell line, (MDA-MB-231) failed to form acini in 3-D cultures upon Src inhibition. However, formation of cytoplasmic projections that may be involved in cell motility and invasion was prevented in this cell line when Src was inhibited. Further analysis of reverted T4-2 cells in 3-D cultures has shown that the activation of a kinase involved in tumorigenesis and cell survival, AKT, and expression of a component of a membrane receptor involved in increased proliferation, integrin beta-1, are downregulated upon Src inhibition. Previous studies have shown that direct inhibition of AKT activation or inhibition of beta-1 integrin in T4-2 cells can induce reversion in 3-D cultures. Thus, AKT and beta-1 may represent potential downstream targets of Src in T4-2 cells.

We are currently examining other potential downstream signaling targets of Src in order to better understand the mechanism of reversion induced by Src inhibition. In addition we are utilizing DNA recombinant technology to genetically inhibit Src activity in breast carcinoma cell lines to validate the pharmacological studies. Src plays a role in many biological processes that become deregulated during tumorigenesis and therefore may represent an attractive therapeutic target in the treatment of breast cancer disease. Indeed, Gleevec, a small molecule pharmacological inhibitor of another tyrosine kinase, Abl, that is in clinical use for the treatment of chronic myelogenous leukemia (CML), provides evidence that targeting tyrosine kinases may be a viable therapeutic route in cancer treatment. The results from this study have begun to address the role of Src in breast cancer in a more physiological 3-D culture system. Results from this study may help us better understand the role tyrosine kinases play in breast cancer pathogenesis and may yield clues in the development of more effective therapies.