Inhibition of Breast Cancer Cell Invasion by Natural Indoles

Institution: University of California, Berkeley
Investigator(s): Christine Brew, Ph.D. -
Award Cycle: 2003 (Cycle IX) Grant #: 9FB-0149 Award: $80,000
Award Type: Postdoctoral Fellowship
Research Priorities
Detection, Prognosis and Treatment>Innovative Treatment Modalities: search for a cure



Initial Award Abstract (2003)
The metastatic spread of breast cancers accounts for the lethality of the disease. Metastatic cancers are frequently resistant to therapy and therefore there is a great need to develop new therapeutics that target the spread of both estrogen-dependent and estrogen-independent breast tumors. A promising new anti-breast cancer strategy is to develop the use of phytochemicals as natural chemotherapeutic or preventative agents. One potential source to uncover new types of therapeutics is from dietary plants because many phytochemicals, biologically active compounds in plants, have anti-carcinogenic properties. Indole-3-carbinol (I3C) and 3,3'-diindolylmethane (DIM), naturally occurring in Brassica vegetables such as broccoli and cabbage, have striking growth arrest properties in estrogen-dependent and independent breast tumors. Most importantly, these indoles prevent the metastases of breast cancer cells injected into mice, and inhibit invasion through synthetic membranes.

I will investigate the molecular mechanism of indole-regulated inhibition of invasion, since this process is critical to metastasis. I hypothesize that I3C and DIM exert their anti-invasive actions by inhibiting the production of matrix metalloproteinases (MMPs), enzymes that degrade the extracellular matrix. MMP-2 and MMP-9, which are commonly present at higher levels in patients with breast cancer, break down the main constituent of the extracellular membrane, and have been directly implicated in tumor metastasis. My preliminary data shows that I3C decreases the expression of the MMP-2 and MMP-9 genes. Thus, the objective of this proposal is to investigate the indole-regulated cellular pathways that control MMP-2 and MMP-9 and breast cancer cell invasion.

The first aim of these studies is to establish that the extracellular secretion of MMP-2 and MMP-9 decreases in both poorly and highly invasive breast cancer cells. Then, I will functionally test that the MMPs are within the indole-regulated pathways, by reversing their decreased levels. This will be accomplished by the introducing of additional copies of MMP-2 and MMP-9 genes into breast cancer cells and determining whether indoles can no longer prevent invasion. I will then determine the precise DNA sequences and DNA-binding factors that cause MMP-2 and MMP-9 gene synthesis to be down-regulated by indoles.

The results of these experiments will provide key insights into the molecular mechanism by which natural indoles prevent cancer cell metastasis. This study represents a novel application of phytochemical nutrients in the control of tumor dissemination.


Final Report (2005)
The metastatic spread of breast tumors accounts for the majority of cancer-related deaths, and these tumor colonies typically fail to respond to conventional therapies. The phytochemical indole-3-carbinol (I3C), from cruciferous vegetables such as broccoli and Brussels sprouts, has been shown to reduce breast cancer growth and metastatic spread. I3C inhibits metastasis by decreasing cellular motility and invasion, yet the molecular mechanism is relatively unknown.

We have found that I3C markedly decreases directional migration of an aggressive breast cancer cell line called MDA-MB-231. By staining the cells with an immunofluorescent dye, we observed that I3C dramatically alters actin and vinculin, components of the cytoskeleton that provide cellular shape, and the ability to move. Untreated cells have a polarized structure, consisting of growing actin filaments that provide the force for forward protrusions known as lamellipodia. Within the cell, contractile actin filament bundles, or stress fibers, translocate the cell body forward. Stress fibers anchor to a substratum through small, transient adhesions consisting of vinculin and other focal adhesion components, at the leading edge of the cell. In contrast, I3C-treated MDA-MB-231 cells no longer exhibited a polarized shape. There was an absence of lamellopodia, increase in cell size, formation of numerous stress fibers, and an enhancement in size and number of focal adhesions around the whole cell periphery. We confirmed the I3C-mediated increase in focal adhesion formation by examining protein-protein interactions between the focal adhesion components FAK, paxillin, and p130 Cas. These co-immunoprecipitation studies demonstrated that I3C treatment induced protein complex assembly, and that the protein components were activated by phosphorylation at their tyrosine amino acid residues. The RhoA/Rho kinase signaling pathway regulates the actin cytoskeleton, and leads to the formation of stress fibers and focal adhesions. We found that I3C increased the enzymatic activity of Rho kinase proteins, and increased phosphorylation of downstream substrate cofilin, which inhibits its actin filament severing activity. A Rho kinase inhibitor, Y-27632, reversed the I3C-mediated formation of stress fibers, and reduced focal adhesion size. In addition, I3C did not induce any cytoskeletal changes in MDA-MB-231 cells that contained an inactivated form of RhoA, establishing a functional requirement for RhoA. Thus, I3C activates the RhoA/Rho kinase pathway to induce the formation of stress fibers and focal adhesions at the cell periphery, disrupting cell polarity and preventing cell migration. Because migration is an integral component of cancer cell metastasis, inhibition of this process by I3C should limit metastatic capability of breast cancer cells.

This study represents a novel application of phytochemical nutrients to the control of tumor dissemination, providing a specific approach to regulate metastatic capability.


Symposium Abstract (2005)
The phytochemical indole-3-carbinol (I3C), from cruciferous vegetables such as broccoli and Brussels sprouts, has been shown to reduce breast cancer growth and metastatic spread. I3C inhibits metastasis by decreasing cellular motility and invasion, yet the molecular mechanism is relatively unknown. We have found that I3C markedly decreases directional migration of an aggressive breast cancer cell line called MDA-MB-231. By staining the cells with an immunofluorescent dye, we observed that I3C dramatically alters actin and vinculin, components of the cytoskeleton that provide cellular shape, and the ability to move. Untreated cells have a polarized structure, consisting of growing actin filaments that provide the force for forward protrusions known as lamellipodia. Within the cell, contractile actin filament bundles, or stress fibers, translocate the cell body forward. Stress fibers anchor to a substratum through small, transient adhesions consisting of vinculin and other focal adhesion components, at the leading edge of the cell. In contrast, I3C-treated MDA-MB-231 cells no longer exhibited a polarized shape. There was an absence of lamellipodia, increase in cell size, formation of numerous stress fibers, and an enhancement in size and number of focal adhesions around the whole cell periphery. We confirmed the I3C-mediated increase in focal adhesion formation by examining protein-protein interactions between the focal adhesion components FAK, paxillin, and p130 Cas. These co-immunoprecipitation studies demonstrated that I3C treatment induced protein complex assembly, and that the protein components were activated by phosphorylation at their tyrosine amino acid residues. The RhoA/Rho kinase signaling pathway regulates the actin cytoskeleton, and leads to the formation of stress fibers and focal adhesions. We found that I3C increased the enzymatic activity of Rho kinase proteins, and increased phosphorylation of downstream substrate cofilin, which inhibits its actin filament severing activity. A Rho kinase inhibitor, Y-27632, reversed the I3C-mediated formation of stress fibers, and reduced focal adhesion size. Thus, I3C activates the RhoA/Rho kinase pathway to induce the formation of stress fibers and focal adhesions at the cell periphery, disrupting cell polarity and preventing cell migration. Because migration is an integral component of cancer cell metastasis, inhibition of this process by I3C should limit metastatic capability of breast cancer cells.

Indole-3-carbinol activates the ATM signaling pathway independent of DNA damage to stabilize p53 and induce G1 arrest of human mammary epithelial cells.
Periodical:International Journal of Cancer
Index Medicus: Int J Cancer
Authors: Brew CT, Aronchik I, Hsu JC, Sheen JH, Dickson RB, Bjeldanes LF, Firestone GL.
Yr: 2006 Vol: 118 Nbr: 4 Abs: Pg:857 - 868