ATF2 protein may play a crucial role in cancer's ability to resist chemotherapy and radiation treatment

Researchers at Purdue University have discovered a molecular mechanism that may play a crucial role in cancer's ability to resist chemotherapy and radiation treatment and that also may be involved in Alzheimer's and heart disease.

The scientists, using an innovative imaging technique invented at Purdue, have learned that a protein previously believed to be confined to the nucleus of healthy cells actually shuttles between the nucleus and cytoplasm, the region of the cell surrounding the nucleus. Moreover, the protein's shuttling is controlled by the presence of another protein in the nucleus and its attachment to that second protein.

"Our findings may provide a new avenue for the development of innovative treatments for certain cancers and other conditions," said Chang-Deng Hu, an assistant professor in Purdue's Department of Medicinal Chemistry and Molecular Pharmacology and an investigator at the Walther Cancer Institute in Indianapolis.

The experiments were done using a line of "teratocarcinoma" malignant tumor cells from mice called F9, which, when subjected to the right biochemical signals, have the ability to alter their properties and are considered to be "cancer stem cells." The hypothetical cancer-resistance role of cancer stem cells could explain why tumors return after treatment. If stem cells prove to be critical to cancer's resistance to treatment, new medications might be developed to target cancer stem cells while chemotherapy or radiation is administered, Hu said.

Research findings are detailed in a paper appearing this month in the EMBO Journal, published by the European Molecular Biology Organization. The paper was written by postdoctoral research associate Han Liu, laboratory technician Xuehong Deng and graduate student Y. John Shyu, all in the Department of Medicinal Chemistry and Molecular Pharmacology; Jian Jian Li, an associate professor in the Department of Health Sciences; Elizabeth J. Taparowsky, a professor in the Department of Biological Sciences; and Hu.

The research focuses on two proteins called c-Jun and ATF2, which are key components of a protein complex called activating protein-1, or AP-1. AP-1 is a major "transcription factor" that binds to DNA, activating the "expression" of genes required to produce proteins needed for vital cellular processes. The proteins that make up AP-1, including ATF2 and c-Jun, often join together in the nucleus, forming either "homodimers," when two of the same proteins join, or "heterodimers," when two different proteins come together.

"Current thinking is that all of these AP-1 proteins in healthy cells are localized, or confined, to the nucleus," Hu said. "But in this work we found for the first time that ATF2 constantly shuttles between the cytoplasm and the nucleus."

The researchers found that the ATF2 protein possesses "nuclear export" and "nuclear localization" signals, which enable it to travel out of and back into the nucleus, respectively. The researchers also discovered that if ATF2 attaches to c-Jun in the nucleus, forming a heterodimer, the nuclear export signal is blocked, preventing ATF2 from traveling from the nucleus to the cytoplasm.

Researchers had already known that chemotherapy and radiation cause cancer cells to increase production of ATF2. The Purdue researchers found that "overexpressed," or overproduced, ATF2 is predominantly located in the cytoplasm because of an inadequate amount of c-Jun in the nucleus, suggesting it is likely that overexpressed ATF2 also may be localized in the cytoplasm in cancer cells, Hu said.

The Purdue researchers not only discovered that ATF2 is localized in the cytoplasm of the mouse cancer stem cells, but also that exposing the cells to ultraviolet light induced more production of c-Jun protein in the nucleus, causing the ATF2 to bind with c-Jun, stopping the shuttling process and causing cell death. The c-Jun-ATF2 heterodimers cause more c-Jun protein to be produced, attracting more ATF2 and reinforcing the localization of ATF2 in the nucleus.

Because it has been reported that ATF2 overexpression causes the resistance of cancer cells to chemotherapy and radiation, the ATF2 shuttling might play a key role in the ability of cancer cells to resist cancer treatments, and preventing the ATF2 from moving into the cytoplasm might improve the effectiveness of anticancer treatments.

"Ultimately, we are trying to figure out how to make the cancer cells more sensitive to chemotherapy and radiation treatment by keeping the ATF2 in the nucleus," Hu said.

The Purdue researchers tracked ATF2 and other proteins using a fluorescent imaging technique developed by Hu called bimolecular fluorescence complementation. The procedure involves breaking a fluorescent protein into two fragments and then fusing each fragment to different AP-1 proteins, including c-Jun and ATF2. When the proteins later bind to form a heterodimer, the fluorescent-protein fragments are reunited, causing them to glow green when illuminated with a light source. The fluorescence enabled the researchers to pinpoint the location of c-Jun-ATF2 heterodimers and discover the shuttling movements of ATF2. The ATF2 protein also has been found in the cytoplasm of diseased brain cells in Alzheimer's disease and muscle cells in the heart, suggesting the same shuttling mechanism might be involved in those conditions.

"These findings suggest a new avenue to study how ATF2 is implicated in the pathogenesis of these diseases," Hu said.