High Protein Levels Bypass Functional BRCA Defenses to Generate Toxic Double-Strand Genomic Breaks

When it comes to cancer, tumor suppressor genes are usually thought of as the "good guys." These genes make proteins that protect and repair DNA in cells. If they stop functioning or there's not enough, cancer risk goes up. But there can be too much of a good thing: When cells overexpress the gene EXO1 - meaning that they make more of the protein than they should - it can degrade the DNA it's supposed to repair. This causes damage that can disrupt the genome, which is a hallmark of cancer, according to a team of researchers from Penn State College of Medicine.

In a study published in Nature Communications, the researchers found that the EXO1 gene is overexpressed in 20% to 30% of breast and ovarian cancers as well as in melanoma, testicular, cervical and hepatobiliary cancers, which develop in the liver, gall bladder and bile duct. Tumor cells with high levels of EXO1 protein exhibit characteristics similar to cells with a BRCA mutation, a genetic code change known for its link to hereditary breast and ovarian cancers. That means that these tumor cells behave like BRCA-mutant cells - including their response to the chemotherapies and other drugs - even when there is no BRCA mutation present, a finding the researchers said hasn't previously been established.

"EXO1 doesn't predict cancer risk, but it could potentially serve as a biomarker to help predict which patients are more likely to respond to certain chemotherapy treatments, leading to more personalized therapies," said George-Lucian Moldovan, professor of molecular and precision medicine and senior author on the study.

The same drugs that are reserved for treating BRCA-mutant tumors and that have fewer side effects could potentially be used to treat EXO1 overexpressing tumors, which don't have BRCA mutations. It would expand the applicability of those drugs."

George-Lucian Moldovan, The Pennsylvania State University

For this study, the research team analyzed The Cancer Genome Atlas, a cancer genomic program of the National Cancer Institute, for EXO1 alterations in tumor samples. They found that EXO1 was overproduced in several tumors, including those in the breast, skin, liver and cervix, which aligns with previous studies in the field. Specifically, high levels of EXO1 were linked to basal-like breast cancers, an aggressive subtype of breast cancer.

The research team conducted laboratory studies with commercially available human cancer cells. The researchers overexpressed the EXO1 gene in the cells to see how an overabundance affected the cell's DNA. They also overexpressed a biochemically disabled version of the EXO1 gene, meaning the proteins it produced were present but not interacting with other cells, to confirm that any DNA damage observed was specifically caused by protein activity and not just the presence of the protein.

In normal cells, the EXO1 protein acts like a pair of molecular scissors, trimming and repairing damaged DNA. When there's too much EXO1 in cells, the researchers found that it started cutting things that it shouldn't. They observed that EXO1 destabilizes newly synthesized DNA in two primary ways - by expanding single-stranded DNA gaps and degrading reversed replication forks. Both processes chew up DNA and leads to localized loss of DNA sequences, Moldovan explained.

"Regardless of which pathway, EXO1 overexpression leads to the generation and accumulation of toxic lesions in DNA, such as double strand breaks, which we ultimately think is what makes the tumor more sensitive to chemotherapy and increases cell death," said Alexandra Nusawardhana, the lead author of the study and who earned her doctorate in biomedical sciences this year from Penn State College of Medicine.

Normally, BRCA genes produce proteins that protect these genomic structures; when there's a mutation in the BRCA genes, cells can't protect its DNA during replication, which can eventually lead to cancer development. However, in this study, high levels of EXO1 protein bypassed BRCA's defenses, even in cells where BRCA is fully functional and there is no mutation. The researchers found that EXO1 worked in concert with another protein called MRE11 to expand gaps in DNA and create dangerous breaks.

"Mechanistically, this overexpression does exactly what the loss of the BRCA pathway does in BRCA-mutant tumor cells," Moldovan said. But unlike BRCA mutations, EXO1 overexpression isn't inherited and it's not known if it causes cancer, he explained.

Because of the similar behavior, the researchers wanted to know if tumor cells that overexpressed EXO1 would also respond to cancer treatments the same way that BRCA-mutant tumors, too. They tested how EXO1 overexpressing tumors responded to olaparib, an existing medication used to treat BRCA-mutant cancers by targeting the cellular repair system. They found that the tumors were hypersensitive to the drug, responding like the BRCA-mutant cancers. The finding suggests that tumors that overexpress EXO1, but don't have a BRCA mutation, might also benefit from the same kind of cellular repair-targeted therapy. The researchers also found that EXO1-overexpressing tumors responded to cisplatin, a widely used chemotherapy drug, suggesting that lower doses of cisplatin could potentially achieve the same tumor shrinkage with less side effects.

Since EXO1 overexpression is more common across tumors than the BRCA mutation, its presence in cancer cells could potentially be a useful biomarker to identify more personalized treatment options, according to Moldovan.

"We shouldn't treat cancers based on what tissue they come from but based on the landscape of the genetic mutations present in the tumors," Moldovan said. "That would result in high efficiency treatment. That's the future of cancer treatment."

The research team plans to continue this line of research with the goal of eventually conducting clinical trials with cancer patients with tumors that exhibit EXO1 overexpression.

Claudia Nicolae, assistant professor of molecular and precision medicine at Penn State College of Medicine, also contributed to the paper.

Funding from the National Institutes of Health and Four Diamonds supported this work.