The discovery of cellular oncogenes in the early 1970s launched a generation of research on the molecular basis of cancer. The resulting explosion of information about the molecular pathways of cell transformation has led to several highly successful targeted approaches to cancer treatment, such as the recent success of imatinib (Gleevec), a tyrosine kinase inhibitor, for treating chronic myelogenous leukemia. But many glaring gaps remain in understanding the genesis and progression of cancer.
The frustrating lack of progress in treating many solid tumors has led some researchers to propose a radical shift in thinking about the nature of cancer. Several investigators are beginning to suggest that cancer may arise from disruptions in tissue organization rather than from accumulated mutations in individual cells. And their ideas are now being heard.
The National Cancer Institute has made understanding the tumor microenvironment one of its top priorities for 2004 by allocating $40 million to understanding the role of the cells and extracellular components that surround and interact with cancer cells and how this interaction can control or promote tumor growth.
"I argue that there is a language and alphabet of tissue structure and organ structure that we have not yet discovered," said Lawrence Berkeley National Laboratory scientist Mina Bissell, Ph.D., a pioneer in the study of the tissue microenvironment and cancer.
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Bissell has argued since the early 1980s that it is the interaction between cancer cells and the surrounding tissue and extracellular matrix proteinswhat she calls dynamic reciprocitythat directs gene expression and determines whether cancer is contained or spreads. She argues that, to study cancer, it is crucial to develop cell culture systems that mimic as closely as possible the complex structures and organization of tissues in the body.
To do this she developed a three-dimensional culture system in which breast epithelial cells grow on an extracellular matrix that closely mimics what is found in normal breast tissue. When grown on this environment, the cells differentiate into structures, called acini, that look like normal breast ducts and even produce milk proteins.
In a series of experiments, most recently reported at the April Experimental Biology 2004 meeting in Orlando, Fla., Bissell has shown that, by manipulating the tissue microenvironment, cells termed malignant are capable of reverting to normal cell behavior and that normal cells can turn malignant in a malfunctioning microenvironment.
Experiments in Bissell's laboratory revealed that a malignant population of
the popular breast tumor cell line HMT-3522 made increased amounts of
1-integrin, a receptor for the basement membrane component laminin 1.
When the scientists blocked
1-integrin with an antibody, the cells
stopped growing in a disorganized mass and differentiated into acini.
Bissell and her colleagues reported in the Oct. 2, 2002, issue of the Journal of the National Cancer Institute similar experiments in which breast cancer cell lines reverted to normal by inhibiting epidermal growth factor receptor (EGFR), mitogen-activated protein kinase (MAPK), or phosphatidylinositol 3-kinase (PI3K), which are all implicated in tumorigenicity. Since then, she has begun using microarrays to examine gene expression patterns of the reverted cells and has shown that they use different molecular pathways to revert to normal.
"Even though they all end up with the same structure and they all revert correctly, they didn't use the same pathway," said Bissell. "There are biochemical rules we have yet to work out that all have to come together to make a tissue."
Now experiments from other laboratories have begun to bear out her ideas.
Kornelia Polyak, M.D., Ph.D., and her colleagues at Dana-Farber Cancer Institute and Harvard Medical School, Boston, reported in the July 2004 issue of Cancer Cell that by studying the gene expression profiles of each cell type in normal breast and in situ and invasive breast cancers, they could identify specific gene expression changes in the cells surrounding the tumors that correlate to invasiveness of the tumor. Specifically, they discovered that a pair of chemokines, CXCL14 and CXCL12, molecules that bind to specific receptors on the breast epithelial cells and enhance their ability to proliferate and migrate, are made in large quantities in the myoepithelial cells surrounding breast tumors. The authors also found genetic changes only in the tumor epithelial cells and not in any of the surrounding cells. The results provide specific evidence that cross-talk between the tumor cells and the surrounding genetically normal cells can influence tumor invasion and proliferation.
Results such as this have some researchers suggesting a new way to control the spread of cancer.
"If you can obtain different phenotypes with the same genotype, why not consider that cancer is simply a problem of tissue organization and forget about mutations?" said Ana Soto, M.D., professor of anatomy and cellular biology at Tufts-New England Medical Center, Boston.
In the June 2004 issue of the Journal of Cell Science, Soto, Maricel Maffini, Ph.D., Carlos Sonnenschein, M.D., and their colleagues reported that the known chemical carcinogen, N-nitrosomethylurea (NMU), initiates neoplastic transformation by targeting the tissue stroma, not the epithelial cells that typically develop into tumors.
The scientists wanted to test the generally accepted idea that NMU causes cancer by creating mutations in epithelial cells. To do that they treated mammary epithelial cells with NMU in vitro and then transplanted them into rat mammary gland fat pads. The mutated epithelial cells were unable to form tumors in the untreated stroma. But when the researchers performed the reverse experiment, treating stroma in which the epithelial cells had been removed with NMU and then transplanting untreated epithelial cells into the ductal fat pads, the animals developed tumors.
Similar results are coming from other laboratories as well. A Vanderbilt
University research team led by Harold L. Moses, M.D., director of the
Vanderbilt-Ingram Cancer Center, and Eric G. Neilson, M.D., chair of medicine,
reported in the February 6 issue of Science that transgenic mice bred
to selectively eliminate production of transforming growth factor
(TGF-
) receptor in fibroblasts, key components of the tissue stroma,
developed precancerous prostate lesions and invasive cancers in their
forestomachs.
The findings suggest that when the TGF- signaling pathway is active,
the growth of adjacent epithelial cells is held in check, but when those
signals are interrupted, regardless of the mutational status of the epithelial
cells, growth is unchecked and tumors form.
Soto suggests that results such as these ought to make people rethink how tumors form. She and her collaborators have proposed a new theory, the tissue organization field theory, which hypothesizes that carcinogens alter stromalepithelial interactions and that proliferation is the default state of cells.
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The implications for cancer treatment are profound, Bissell says, and understanding the factors outside the cancer cell that drive it to invasion and metastasis or keep it contained could provide an entirely new avenue of therapy. She argued in a 2003 International Journal of Cancer review that the power of tissue context to overcome the malignant potential of cells could be harnessed to treat or even prevent cancers. But to do that, it will be crucial to develop more sophisticated cell culture models, she says.
"I argue that we should be doing drug testing in three dimensions," said Bissell. "I think we need to make more and more ambitious culture models. The context is going to change what you get. People say working in three-dimensional models is difficult, but I think working in two dimensions is difficult because cells growing in plastic can do anything, and you don't know if it has any relevance in vivo."
Bissell is taking the lead in developing ever more complex culture systems, and others are starting to climb on the bandwagon.
"What you see happening is a handful of leaders in the field moving in the direction of studying tissue microenvironment because they know this is where the action is," said Suresh Mohla, Ph.D., chief of tumor biology and metastasis at the National Cancer Institute. "It is almost as if the community as a whole is now agreed that this is the way to go."
In a Dec. 27, 2002, commentary in Cell, Tyler Jacks, Ph.D., and Robert A. Weinberg, Ph.D., a pioneer in the study of the molecular basis of cancer, both from the Whitehead Institute at the Massachusetts Institute of Technology, Cambridge, argue that three-dimensional culture systems have the potential to bridge the knowledge gap between detailed in vitro molecular studies and whole-animal studies using transgenic mice.
"Suddenly," they write, "the study of cancer cells in two dimensions seems quaint, if not archaic."
Mark Ewen, Ph.D, an oncologist at Dana-Farber Cancer Institute, Boston, agrees. His research on the role of the oncogene cyclin D1 in the development of cancer has led him to the hypothesis that cyclin D1 exerts its oncogenic effects by altering the differentiation programming of the epithelial cell. But to test his ideas, Ewen has now turned to Bissell's 3-D culture system.
"We are trying to dissect the function of naturally occurring tumors," Ewen said. "We felt the need to study cyclin D1 in its natural setting. The stroma is supplying factors, and to see the communication between the stroma and epithelial cells, you have to work in 3-D."
Brian Druker, M.D., the developer of Gleevec, says that if you talked to people 10 years ago they would have said anyone pursuing a kinase inhibitor is nuts and they would have given you three or four reasons why it would not have worked.
"Once you start to move people in one direction, you can't ignore the possibility of exploring other areas that might be complementary or that might be even better," he said.
"I don't think targeted therapy is going to be the be-all and end-all," he continued. "There's the seed and the soil. Going after the seed is the molecularly targeted approach, but there's a supporting, enriching soil that is permissive for the development of cancer, so maybe when we start to think about new strategies we really need to start thinking about the soil."
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