NEW YORK – A University of North Carolina at Chapel Hill-led team has assembled a collection of single-cell sequence and chromatin accessibility maps for cancers found in the endometrium or ovaries, and started untangling ties between regulatory, transcriptional, and clinical features in gynecological malignancies.
"This dataset reveals the complex cellular heterogeneity of these tumors and enabled us to quantitatively link variation in chromatin accessibility to gene expression," senior author Hector Franco, a cancer, genetics, bioinformatics, and computational biology researcher at the University of North Carolina at Chapel Hill, and his colleagues explained in a study published in Molecular Cell on Thursday.
For their analyses, the researchers did lipid droplet-based single-cell RNA sequencing and single-cell ATAC-seq on 75,523 individual cells and 74,621 single cells, respectively, isolated immediately from 11 surgically resected, previously untreated endometrial or ovarian tumors. One of the cases was marked by endometrial cancer metastasis to the ovary and another involved a metastatic gastrointestinal tumor in the ovary, while the remaining cases involved primary tumors, they noted.
"We show that malignant cells acquire previously unannotated regulatory elements to drive hallmark cancer pathways," the authors reported, noting that "malignant cells from the same patients show substantial variation in chromatin accessibility linked to transcriptional output, highlighting the importance of intra-tumoral heterogeneity."
With cell marker clues and gene expression clustering, the investigators tracked down 10 cell types and three-dozen subtype clusters, along with transcription factor activity that corresponded with the specific tumor, immune, or normal muscle/fibroblast/stromal cell types. Likewise, gene expression and enhancer usage within cells varied by cell type.
Using a computational method known as total functional score of enhancer element, or TFSEE, analyses, the team flagged transcription factors with more associations than usual to new and known distal regulatory elements in malignant cells from the five endometrial and six ovarian tumors considered, while untangling several other transcription factor expression and enhancer relationships.
The cancer-specific distal regulatory elements detected fell in noncoding parts of the genome implicated in the regulation of cancer-related genes such as RHEB and other components of the mTOR signaling pathway, for example, which has been targeted in combination treatment clinical trials in ovarian cancer.
"Our TFSEE analysis is a powerful tool that facilitates integration of scRNA-seq and scATAC-seq datasets to interrogate complex mechanisms of gene regulation," the authors suggested. "This helps prioritize [transcription factors] for follow up investigation and could help inspire novel therapeutic avenues in gynecologic malignancies."
The gynecological cancer types considered for the study are often detected at an advanced stage and tend to be aggressive, the investigators noted. Combined with limited targeted treatment options, these characteristics contribute to the poor prognoses observed in many ovarian and endometrial cancer cases.
"To date, the standard of care for [ovarian cancer] and [endometrial cancer] is a combination of surgery, chemotherapy, and radiation," they wrote. "Despite these aggressive treatments, most women with advanced stage [ovarian cancer] and [endometrial cancer] will succumb to their disease, highlighting the need to develop better targeted therapies."
In a survival analysis that included data for more than 600 ovarian cancer patients, the researchers found that enhanced expression of the mTOR regulatory gene RHEB is associated with worse outcomes, consistent with poor prognoses linked to high expression of the gene in the past. Their results also suggested that chromatin accessibility in the RHEB gene promoter tended to increase in malignant cells.
Among the study's limitations, they cautioned that their findings relied on bulk tumor data for a relatively limited number of gynecological cancer patients. Even so, they argued that "these data and the analyses described herein represent a true baseline for these cancers, serving as a foundation for defining the regulatory logic of malignant cells at singe-cell resolution."