BERKELEY, CA – Researchers in the Life Sciences Division of the Department of Energy's Lawrence Berkeley National Laboratory have discovered a key molecular pathway by which an enzyme that normally helps remodel tissues initiates the pathway to breast cancer. The same molecular pathway, the researchers found, links both the loss of tissue organization in cancerous organs and the loss of genomic stability in individual cancer cells.

"This study demonstrates how structure and function in a tissue are intimately related, and how loss of structure could itself lead to cancer," says Mina Bissell, who pioneered the view that a cell's environment is as important as its genes in determining the formation and progression of tumors. "Thus the unit of function in organs — which are made of tissues — is the organ itself."

When treated with an enzyme that destroys the tissue scaffolding (MMP-3), the structures of normal cells (left) are distorted and tissues become disorganized (right).

Enzymes known as matrix metalloproteinases (MMPs) are important during an organism's development and during wound healing, but they can also promote carcinogenesis. The new study shows that one type, MMP-3, causes normal cells to express a protein, Rac1b, that has previously been found only in cancers. Rac1b stimulates the production of highly reactive oxygen molecules, which promote cancer in two ways — by leading to tissue disorganization and by damaging genomic DNA.

"What comes first in cancer, the mutations within the genome of the tumor cells or the loss of tissue organization?" asks Derek Radisky, a postdoctoral fellow in Bissell's laboratory who has focused on the molecular pathways mediating interactions between tumors and their surrounding tissues. "It's a chicken-and-egg problem. Our study shows that the relationship is reciprocal."

Lead author Radisky, with Bissell and their colleagues Dinah Levy, Hong Liu, Celeste Nelson, and Jimmie Fata of Berkeley Lab; Laurie Littlepage, Donna Albertson, and Zena Werb of the University of California at San Francisco; Devin Leake and Elizabeth Godden of Dharmacon, Inc.; and M. Angela Nieto of the Instituto de Neurociencias de Alicante, Spain, report their findings in the 7 July issue of Nature.

When tissue organization breaks down

Epithelial cells are the source of the majority of cancers; the investigators thus concentrated on factors controlling epithelial cell organization. Epithelial tissues are specialized for managing the flow of substances into and out of the body and for protecting underlying organs. To form these tissues, epithelial cells are tightly interconnected in sheets that line hollow organs and glands, including the breast, prostate, colon, and lung, as well as external surfaces of the body.

Epithelial cell organization is determined by a specialized structure known as the basement membrane, a form of the ubiquitous extracellular matrix (ECM) that acts as both a structural scaffold for cells in a tissue and a medium through which the cells communicate. Breakdown of the basement membrane is associated with the spread of tumors; in earlier studies with transgenic mice, Bissell and her colleagues, including UCSF's Werb, showed that loss of integrity of the basement membrane can itself cause tumors. But the mechanism was not understood.

Among the chief agents controlling the configuration of the basement membrane are the matrix metalloproteinases, digestive enzymes that normally act as bulldozers to clear the way for building new organ structures or repairing old ones. Breast tumors have an increased amount of MMPs, however, and this promotes the spread of tumors by degrading the basement membrane and digesting the contacts that bind the epithelial cells into sheets.

In cancers MMPs also wreak havoc in another way, as Bissell and her colleagues showed previously: MMPs induce the so-called epithelial-mesenchymal transition (EMT). This transition from one cell state to another causes epithelial cells to disassociate from their neighbors, break free, and acquire the ability to move through the body. In the embryo, EMT is essential for normal organ development. In breast cancer, however, the process renders tumor cells mobile and helps them penetrate barriers like the walls of lymph and blood vessels, facilitating metastasis.

Pathways of destruction

In the present study, the researchers demonstrate that the link between MMPs and the epithelial-mesenchymal transition lies in a family of proteins called Rho GTPases, which control the proteins that define the cell skeleton. They found that treating normal cells with a particular MMP, MMP-3, causes the cells to express an unusual form of Rho GTPase, previously found in cancers, called Rac1b. Rac1b dramatically alters the cell skeleton, making it easier for epithelial cells to separate and move away from surrounding cells.

Epithelial cells (left) are tightly interconnected in sheets by numerous cell-cell interactions; their organization is determined by the basement membrane (lower left). During epithelial-mesenchymal transition (EMT), epithelial cells become independent of their neighbors and can move freely (center). In this way, tumors derived from epithelial cells become motile and may invade lymph or blood vessels (right).

Changes in the cell skeleton induced by Rac1b trigger the formation of extremely reactive molecules known as reactive oxygen species, or ROS. In turn, the increased amount of ROS activates key genes that control the epithelial-mesenchymal transition — the first slippage in an avalanche of tissue disorganization.

The reactive oxygen species induced by Rac1b also stimulate the development of cancer by directly affecting genomic DNA.

"Reactive oxygen species can damage DNA directly," says Radisky, "and we found that this was the case in cells exposed to MMPs. Examining the genomes of these cells showed that huge regions of DNA were either duplicated or missing altogether. These sorts of changes are a key characteristic for development of cancer."

Much effort in cancer research has gone into identifying and investigating the key genes known as oncogenes; when mutated to high activity, oncogenes stimulate the cell to form cancers. But a number of investigators, including Bissell and her colleagues, have shown that genetic alterations of oncogenes are not, as once believed, sufficient in themselves to cause cancer.

"Even activated oncogenes require changes in the tissue structure to produce cancer," Bissell says. "By altering the tissue structure, MMPs accomplish both functions: activating oncogenes and compromising genomic integrity."

New avenues to therapy

The fact that cells exposed to MMP-3 express Rac1b "presents both a challenge and an opportunity," says Radisky. "Rac1b is produced through a process known as alternative gene splicing, through which a single gene can produce many different proteins with dramatically different activities. Alternative splicing is emerging as a key mechanism for controlling cellular function, and identification of MMPs as influencing these processes provides a unique tool for understanding their regulation."

Finding new mechanisms of cancer control can lead to specific opportunities for targeting cancer development. In the course of defining the pathways by which MMP-3 induces malignancy, the researchers developed a method for specifically blocking the formation of the highly active Rac1b, using a recently discovered process known as RNA interference, or RNAi. Reducing the levels of Rac1b by RNAi completely blocked the effects of MMP-3, they found — suggesting a possible method for intervening in this particular pathway of tumor development.

Targeting Rac1b in this fashion is now being investigated for therapeutic potential. Besides inhibiting Rac1b, the study suggests additional therapeutic possibilities, for example by suppressing alterations of the cell skeleton, or blocking the effects of reactive oxygen species, or targeting the process by which the reactive oxygen species activate genes that induce the epithelial-mesenchymal transition.

"Many people are discouraged by the extra complexity demonstrated by this study, but personally I find it encouraging," says Radisky. "It suggests many more points for intervention in the treatment of cancer."

"MMP-3-induced Rac1b stimulates formation of ROS, causing EMT and genomic instability," by Derek C. Radisky, Dinah D. Levy, Laurie E. Littlepage, Hong Liu, Celeste M. Nelson, Jimmie E. Fata, Devin Leake, Elizabeth L. Godden, Donna G. Albertson, M. Angela Nieto, Zena Werb, and Mina J. Bissell, appears in the 7 July 2005 issue of Nature.

Berkeley Lab is a U.S. Department of Energy national laboratory located in Berkeley, California. It conducts unclassified scientific research and is managed by the University of California. Visit our website at http://www.lbl.gov.