(Philadelphia, PA) - The relationship between tissue rigidity and tumor formation is fairly well established; however, what is not so well understood is what happens on a molecular level that contributes to such stiffness. Now, for the first time, researchers at the University of Pennsylvania School of Medicine have shown that tumor formation is generated by a complex interaction of both mechanical as well as chemical signals, and the resulting tissue stiffening induces molecular signals that promote the cancerous behavior of cells. Penn’s interdisciplinary research team – drawn from the fields of Biomedical Engineering and Cell and Developmental Biology – has demonstrated clearly that force, growth, and tumor behavior are inextricably linked and this enhanced understanding of the necessary fusion of these factors may lead to the development of new tumor therapies or targets.

“This study identifies the connection between oncogenes and the mechanics of the cell and its microenvironment in animal and culture experimental models,” explains senior author Valerie Weaver, PhD, Assistant Professor of Pathology and Laboratory Medicine. “Specifically, we have defined the vitality of mechanical force as an integral factor in tumor development.” Weaver and colleagues published their findings as the cover-story in the September issue of Cancer Cell.

Weaver and first authors, Matthew J. Pascek and Nastaran Zahir – both graduate students in Bioengineering – used a three-dimensional gel on which they grew breast cancer cells and could precisely control and measure the stiffness of the surrounding microenvironment. “We found that tissue rigidity enhances cell growth and destroys tissue organization to promote tumor-like behavior in normal cells,” says Pascek. “This happens because the stiffness helps to activate key growth signaling pathways and increase cell tension.”

Cells use surface receptors called integrins to communicate with the outside tissue environment, which consists primarily of connective tissue. Integrins regulate cell growth, death, and movement, as well as tissue organization. Integrins also play a role in cell division and proliferation through ERK, an extracellular signal-regulating molecule. Despite the fact that integrins were discovered and their activity found to be aberrant in tumors decades ago, how integrins could become altered and their importance to cancer has remained contentious among researchers.

Weaver and colleagues found that tissue stiffness induces tumor-like behavior in cells through ERK and Rho, another regulatory molecule. Although researchers have long appreciated that oncogenes such as Ras and Erb2 drive cell growth via the ERK pathway, this study showed how high levels of ERK also prime a cell to contract more via integrins.
Integrin activity also regulates the Rho molecular pathway, which in tumors regulates the stiffness of the cytoskeleton, a collection of protein filaments within a cell that give shape and the capacity for directed movement. When the researchers increased the stiffness of the gel in which experimental cells were grown, Rho activity increased, as well as the number and size of focal adhesions, clusters of integrins that create a connection between integrins and the cytoskeleton.

Overall, the researchers found that a self-perpetuating program of tissue destruction is set up – through changed integrin signaling – to create a double-pronged drive toward aberrant cell behavior. Both the stiffness of connective tissue surrounding developing tumors and the increased activity or expression of oncogenes can promote cells to become cancerous. For example, the researchers found that as stiffness increased in connective tissue, the cells of a normal breast duct started to grow atypically, causing the structure of the duct to degrade, as the uncontrolled cell growth of duct-lining cells invaded the duct tube.

The researchers also discovered that when cell tension becomes great enough, it overrides normal tissue behavior, but is reversible. “We showed that some breast tumors with elevated signaling for the growth factor ERK also have high tension and that their behavior would return to normal by inhibiting cell tension," says Zahir. With this knowledge, Weaver's group is now looking to see whether drugs that inhibit cell contractility could help prevent early metastasis. They are also fine-tuning how different cell types react to different levels of stiffness and how this is important for normal cell behavior, as well as aberrant activity and structure.

This research was funded by the Department of Defense and the National Institutes of Health. Co-authors are Kandice R. Johnson, Johnathon N. Lakins, Gabriela I. Rozenberg, Amit Gefen, Cynthia A, Reinhart-King, Susan S. Margulies, David Boettinger, and Daniel A. Hammer, all from Penn; as well as Mica Dembo from Boston University.

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