Epigenetic Mechanisms of Gene Expression in Thoracic Malignancies

Division Of Basic Sciences - Nci

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Abstract

Novel in-vitro models and correlative experiments with primary tumor/normal tissue specimens have been utilized to identify epigenomic alterations which contribute to initiation and progression of lung and esophageal cancers and malignant pleural mesotheliomas. A steady stream of manuscripts have been published by our group describing the epigenomic effects of cigarette smoke and other environmental carcinogens in normal aerodigestive tract epithelial cells and thoracic cancer cells in-vitro and in-vivo. Our most recent efforts have included experiments demonstrating that cigarette smoke dysregulates a novel feed-forward regulatory loop involving LncRNA ZFAS1 and a poorly characterized tumor suppressor gene, ZNFX1 during pulmonary carcinogenesis (published), as well as delineation of a unified inflammatory signaling cascade by which diverse, environmental carcinogens induce clinically relevant epigenetic perturbations during mesothelioma development (in preparation). Recently a series of experiments have been performed to examine epigenetic mechanisms governing dissemination of lung cancers. Briefly, 24 stem-like clones were isolated from the murine Lewis Lung Cancer (LLC) line under non-adherent culture conditions, of which 12 were randomly selected for further analysis. qRT-PCR, flow cytometry, cell count, RNA-Seq, and DNA methylation array techniques were used to compare stem cell and cancer-germline gene expression, drug resistance, and genome-wide transcriptome/DNA methylome signatures of selected clones. Metastatic potentials of various clones were evaluated by bioluminescence and necropsy following tail vein or subcutaneous inoculation of luciferase-tagged cells into syngeneic, immunocompetent C57BL/6 mice. LLC clones exhibited variable stemness properties including sphere formation, up-regulation of stem cell and cancer-germline genes, drug resistance, enhanced tumorigenicities, and diverse, highly reproducible organ-specific metastases (ie, lung only; liver/lung only; pleura only; lung/endocrine organs only) which persisted despite serial isolation of the metastases and propagation as 2-D cultures. Predilections for organ-specific metastases of the various clones coincided with distinct transcriptome and DNA methylome, and ATAC-seq signatures which were enriched in oncogene signaling, extracellular matrix interactions, cell cycle regulation, angiogenesis, and chromatin remodeling; a novel gene signature correlating with high metastatic potential of murine lung cancer stem cells was highly associated with PFS and OS of patients with lung adenocarcinomas- particularly those with early-stage disease. To further examine mechanisms mediating metastatic potential of lung cancer cells, we initiated a collaboration with Dr. Jennifer Jones from the Radiation Oncology Branch whose translational research is focused on extracellular vesicles (ECVs). Our preliminary experiments indicate that relative to the lung cancer stem cell clone that is organotrophic only to the lungs, widely metastatic lung cancer stem cell clones release ECVs which contain high levels of DNA including portions of c-myc and other oncogenes; these ECVs appear to reprogram normal cells within the tumor microenvironment to create a pro-metastatic niche. Confirmatory, mechanistic studies are in progress and a full manuscript pertaining to our novel findings is being prepared for peer review. During the past year, we have expanded our translational research efforts related to subjects with germline BAP1 Cancer Syndrome (BCS). Whereas mesotheliomas are the most common malignancies identified in BCS, the prevalence and natural history of these neoplasms have not been elucidated. In 2022, I initiated protocol NCT04431024 to prospectively examine if novel high resolution computed tomographic (CT) imaging and minimally invasive surgical evaluation could facilitate early detection and surveillance of mesotheliomas in subjects with germline BAP1 mutations. Exploratory objectives included examination of the epigenomic effects of germline BAP1 mutations in normal and tumor cells, and identification of potential biomarkers of malignancy in subjects with BCS. To date, more than 80 subjects have enrolled on this protocol, which represents the only such efforts in the world at this time. Results of the first 60 consecutive subjects of whom 55 were evaluable have recently been submitted for peer review. 55 subjects (26 males, 29 females; median age: 50.6 years (33.8-73.3) with 35 different germline BAP1 mutations underwent CT imaging followed by surgical evaluation between March 2021 and March 2025. Median follow-up was 18.9 months (0.99 - 48.2). 15 patients had a prior history of mesothelioma involving 16 compartments treated with surgery, HIPEC, and/or systemic chemotherapy; known sites of disease were excluded from analysis. Surgical evaluation identified a spectrum of histologically verified, clinically occult, diffuse mesotheliomas in 48 of 55 (87%) subjects, including 116 of 142 (82%) compartments. CT scans proved unreliable for either detecting or ruling out disease (sensitivity: 33%; specificity: 62%, positive predictive value: 77%; negative predictive value: 19%). Germline BAP1 mutant mesotheliomas had uniform histologic features that were distinctly different than sporadic (asbestos-induced) mesotheliomas and exhibited very slow evolution without standard-of-care (SOC) therapy. Common as well as mutation-specific, cancer-associated alterations in DNA methylomes were identified in dermal fibroblasts and PBMC from all subjects. A 10 gene DNA methylation signature in normal fibroblasts and matched PBMC was highly associated with tumor predilection in subjects with BCS. Collectively, our efforts are the first demonstration that adult subjects with BCS exhibit a high prevalence of subclinical, multicompartment mesotheliomas for which surveillance alone may be a safe alternative to reflexive SOC interventions. Furthermore our data provide first proof of concept that epigenetic burdens in histologically normal, non-target tissues reflect malignancy in subjects with BCS. These findings establish a paradigm for longitudinal assessment of subjects with germline BAP1 mutations and support further efforts to validate epigenetic biomarkers of cancer risk and identify novel targets for the treatment and possible prevention of mesotheliomas in these individuals. A manuscript pertaining to our findings has been tentatively accepted to the Journal of Thoracic Oncology, and we have been invited to resent our findings in plenary talks at the International Mesothelioma Interest Group Meeting and the AATS Thoracic Surgical Oncology Summit, as well as the Weinman Cancer Symposium, which will convene during fall of 2025 and January 2026, respectively. Our current imaging/surveillance protocol provides unparalleled opportunities to study fundamental epigenetic mechanisms of malignancy and stemness of mesotheliomas (and other malignancies) associated with germline BAP1 mutations. We have established an active collaboration with Drs. Michele Carbone and Haining Yang at the University of Hawaii Cancer Center (UHCC). Clinical histories, family pedigrees, tumor biopsies, DNA, RNA, longitudinal serum/blood samples, primary and hTERT immortalized fibroblast cultures established from skin biopsies from patients at UHCC and NCI collectively constitute the world's largest repository of clinical records and biospecimens from BAP1+/- carriers. Elucidation of the mechanisms by which germline BAP1 mutations predispose to mesotheliomas yet appear to mitigate lethality of these neoplasms may provide novel insights into how to treat more common, sporadic thoracic cancers irrespective of tumor histology.