Source: vishnukuma/Fotolia
Source: vishnukuma/Fotolia

Scientists in the U.S. have uncovered a previously unknown molecular pathway in ovarian cancer that could lead to new therapeutic strategies that target a cancer-promoting mutant form of the tumor suppressor protein p53, known as p53-R175H.  The team at Baylor College of Medicine and the University of Texas MD Anderson Cancer Center has shown that inhibiting a deubiquitinase (DUB) known as USP15 allows the clump-forming mutant p53 to be tagged for degradation, which leads to increased cancer cell death.

“Our findings offer a new opportunity for regulating mutant p53-R175H by developing drugs that inhibit USP15,” says lead researcher Joanne Richards, Ph.D., professor of molecular and cellular biology and member of the Dan L Duncan Comprehensive Cancer Center at Baylor College of Medicine. “A possible future scenario in the clinic could be to use DNA analysis to determine whether this p53 mutation is present in a patient's tumor. If so, then we may use our approach to treat it or combine it with other anticancer drugs and take advantage of the fact that treatment with inhibitors of USP15 results in the cancer cells becoming more susceptible to chemotherapy.”

The researchers report on their discoveries in Nature Communications, in a paper entitled “USP15-Dependent Lysosomal Pathway Controls p53-R175H Turnover in Ovarian Cancer Cells.”

Mutations in the tumor suppressor protein p53 are found in more than 50% of human cancers, and genome sequencing has identified p53 gene mutations in more than 96% of high-grade serous ovarian cancers, the most malignant and common ovarian cancer subtype, the researchers explain. Some p53 mutations cause loss of the normal protein’s tumor suppressive activity, which allows cancer to develop, but other mutations known as gain-of-function (GOF) mutations, actively promote cancer formation. Normal, or wild-type, p53 is broken down quickly inside most healthy cells, but p53 mutants, including p53-R175H, persist as clusters. “These gain-of-function (GOF) oncogenic p53 mutant proteins (mutp53) accumulate to high levels in cells, form stable protein aggregates, activate alternative gene expression programs, and contribute to carcinogenesis as well as drug resistance,” the team writes.

Scientists have already shown that reducing the amount of GOF mutant p53 in cancer cells can induce cell death, “underscoring the merit of developing strategies tht selectively target mutp53 in cancer cells,” the team writes. “Researchers have discovered that if we remove the mutant p53 forms from cancer cells, the cells will enter a path toward cell death and become more sensitive to chemotherapy,” comments Achuth Padmanabhan, Ph.D., instructor of molecular and cell biology at Baylor College of Medicine. “This is very valuable from the clinical point of view.”

The team had previously identified a small-molecule compound called MCB-613, which on delivery to p53-R175H-carrying ovarian cancer cell lines led to significant and sustainable reductions in levels of the usually stable GOF mutant, including aggregates. Interestingly, tests in different cell lines demonstrated that MCB-613 administration led to slightly increased levels of normal p53 but had little effect on other GOF p53 mutants. Further analyses indicated that treating the ovarian cancer cells with MCB-613 resulted in rapid export of the p53-R175H mutant from the nucleus to the cell’s cytoplasm, and its subsequent degradation through the lysosome system. This contrasts with removal of normal p53, which is broken down by the cell’s proteasome system.

Further studies showed that rather than cause destruction of the mutant p53-R175H protein directly, MCB-613 acted to reduce levels of a deubiquitinase molecule that removes a specific tag from the p53 mutant that targets the protein for destruction. “…we hypothesized that MCB-613 might be acting through pathways that either augment the activity of specific ubiquitin ligases, or inhibit the activity of DUBs [deubiquitinases],” the authors explain. They found that treating cells with deubiquitinase inhibitors mimicked the effect of MCB-613 on p53-R175H, which led to the identification of USP15 as a deubiquitinse upstream regulator of p53-R175H in the ovarian cancer cells. Knocking down USP15 in p53-R175H-carrying ovarian cancer cells similarly led to decreased cell viability. “Having fewer molecules of USP15 results in more mutant p53-R175H protein tagged for degradation in the cell,” Padmanabhan says. “As a result, the balance tips.”

“Using state-of-the-art genetic and chemical approaches, we identified the deubiquitinase USP15 as the mediator of MCB-613’s effect on p53-R175H, and established USP15 as a selective upstream regulator of p53-R175H in ovarian cancer cells,” the authors conclude. “…this study implicates USP15 as a previously unknown clinically important regulator of p53-R175H mutant protein in ovarian cancer cells….These results confirm that distinct pathways regulate the turnover of p53-WT and the different p53 mutants and open new opportunities to selectively target them.”

Public databases show that USP15 expression is upregulated in a range of cancers, including ovarian serous cystadenoacrcinoma, lobular breast carcinomas, prostate cancer, cervical squamous cell carcinomas, and glioblastomas. Studies have separately shown that USP15 deficiency also promotes T-cell activation and boosts immune responses to tumors in specific mouse models, while USP15 gene knockout mice have higher levels of effector T cells that promote resistance to transplanted tumor growth. “Thus targeting USP15 in tumors containing p53-R175H mutant appears to provide the dual advantage of targeting oncogenic mutp53 and enhancing cancer immunotherapy,” the team suggests.

The researchers have also found that mutant p53-R175H is more sensitive to ovarian steroids, so they are looking to see whether drug combinations could represent a new approach to ovarian cancer therapy. “We are combining ways of regulating steroid receptor and coactivator action, as well as p53,” notes co-author Bert O'Malley, Ph.D., chair and professor of molecular and cellular biology, Thomas C. Thompson Chair in Cell Biology and associate director of basic research in the Dan L Duncan Comprehensive Cancer Center. “Maybe by manipulating both of those pathways we may have a better chance of regulating cancer growth.”


 








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