Newly Identified Oncogene Can Substitute for EGFR Activation
By Anette Breindl
By using a forward genetics approach, two separate teams of researchers have identified related genes as potential oncogenes and mediators of receptor tyrosine kinase inhibitors. Their findings were published back-to-back in the Aug. 13, 2012, online issue of the Journal of Clinical Investigation.
Forward genetics, Mark Jackson told BioWorld Today, is the opposite of a candidate gene approach to identify genes that are related to a trait of interest. The method "encompasses all unbiased screening strategies" such as shRNA and cDNA libraries. Genes are disrupted one by one to see which cells (or animals) show the behavior of interest, and those cells are then dissected in greater detail.
In their paper, Jackson, who is at Case Western Reserve University, and his team used a modified form of insertional mutagenesis to identify genes whose expression caused cells to become more tumor-like in their behavior. That method – which allows researchers to increase the expression of individual genes – has long been used in animals, but Jackson and his colleagues optimized it for use in cell culture.
The immortalized cell lines that Jackson's team used in their experiments are already on their way to becoming tumor cells. "They have at least three of the four hits we need to get the phenotypes of interest," namely, rapid growth and so-called anchorage-independent growth, which means the cells no longer need to be in contact with the culture dish to keep dividing.
In tumors, one change that is capable of providing such a fourth hit is an activating EGFR mutation. Many breast cancer patients have mutations in EGFR, and those who do have a poor prognosis. But EGFR inhibitors such as Tarceva (erlotinib) often do not work well in such patients – often, Jackson said, breast cancer cells seem to be "inherently resistant" to such treatment. In their work, Jackson said, his team was looking for signals that could substitute for EGFR signaling to drive tumor growth.
They identified the FAM83B gene as one gene whose activation could provide the crucial fourth hit that allowed cells to start growing uncontrollably. Depleting cells of FAM83B protein, on the other hand, stopped such growth and made the cells become less tumorigenic.
The team also looked at patient samples and found that a subset of such tissues, too, had high levels of FAM83B expression, and that such high expression boded poorly for the patients in question.
In the second paper, Mina Bissell and her team used a related forward genetics method, to look for genes that might mediate resistance to endothelial growth factor receptor, and receptor tyrosine kinase inhibitors more generally, in breast tumors. In their experiments, Bissell and her team tested whether resistance was due to the fact that signaling pathways downstream of EGFR were already activated in such patients. By overexpressing proteins in cell lines through the use of cDNAs, they came across a cousin of the protein that Jackson's group had identified: FAM83A.
They found that FAM83A appeared to function both as an oncogene and as a resistance gene to receptor tyrosine kinase inhibitor. FAM83A overexpression, too, led to anchorage-independent growth of cells, and to tumor formation in animals, and its depletion could prevent those effects. Depleting the protein also made cells more sensitive to the effects of receptor tyrosine kinase inhibitors.
Both FAM83A and FAM83B belong to an eight-member family of proteins. Some of those proteins had been found to be overexpressed in certain tumor types in previous work, but what if anything such overexpression meant in functional terms had not been clear. The two new studies suggested that the proteins "are turning on signaling downstream of the EGF receptor," Jackson summarized, and ultimately activating the MAP kinase pathway.
Jackson said that his team are preparing to do small-molecule screens to identify inhibitors of FAM83A and B, though he was circumspect about the ultimate prospects of the work in terms of practical applications. Drug development, he noted, "is a very long and difficult process even if it were to work."
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