Cancer incidence rates in 2024 are estimated to increase, according to the latest statistics from the American Cancer Society, with cancer-related death rates declining overall due to improved intervention and treatment strategies. However, these strategies remain only partially effective for some cancers due to their sub-clonal evolution and associated molecular heterogeneity with added complexities related to socioeconomic status. One such type comprises head and neck squamous cell carcinomas (HNSCCs) that arise from the mucosal epithelium in the upper aerodigestive tract with the oral cavity representing a major subsite that presents with oral squamous cell carcinoma (OSCC) pathology. The five-year survival rate for OSCC is ~66% for localized stages and reduces to ~35% upon locoregional spread and metastasis. OSCC etiology is associated with tobacco usage, excessive alcohol consumption, and the usage of carcinogens-containing substances. Other HNSCC subsites, such as oropharynx and nasopharynx are associated with human papillomavirus (HPV) and Epstein-Barr viruses infections, respectively. The HPV-negative OSCC subtype is highly heterogeneous in its molecular and cellular composition and remains understudied compared to other solid tumor types; hence, there is a need for early detection strategies in addition to effective targeted therapeutics for improving treatment outcomes. With the advances in computational methodologies to study human diseases, multi-omic data modalities are becoming a crucial tool to improve patient care and have already led to remarkable discoveries. In my thesis, bioinformatics, statistical, and machine learning methodologies were leveraged to study the transcriptome-wide changes across different modalities (bulk and single-cell) of pre-cancer oral conditions in humans and of treatment effects on OSCC tumors in murine models.
In my first aim, the transcriptional changes of early oral lesions and their progression to OSCC were studied. OSCC arises from oral epithelial dysplasia through a series of clinical and histopathological changes. A subset of OSCC develops from oral leukoplakia, classified as “oral potentially malignant disorders” (OPMD), clinically defined as either localized homogenous leukoplakia (LL), erythroplakia, or proliferative verrucous leukoplakia (PVL), with a malignant transformation ranging between 15% to 90%. The association of leukoplakia with dysplasia is a strong predictor of its capacity to progress to OSCC. In addition, leukoplakia with hyperkeratosis/hyperplasia or hyperkeratosis non-reactive (HkNR) can develop into OSCC. While these lesions have been characterized clinically, a rigorous molecular characterization of these lesions and of the concomitant microbiota are lacking. In this project, I leveraged bulk RNA sequencing to study the molecular profiles of oral lesions at different stages of transformation, and to characterize their defining pathways and host/microbiome interactions. To this end, I used data collected from tissue biopsies isolated from a cohort of 66 human patients harboring distinct histopathology groups consisting of healthy oral mucosa, PMLs comprising HkNR and dysplasia, and OSCC. These samples were profiled using total RNA-sequencing technology, allowing us to study the global transcriptome, including host and microbiome of the oral mucosa from these groups. Our data revealed that PMLs were enriched in gene signatures associated with cellular plasticity, such as partial EMT (p-EMT) phenotypes, and with immune response. Integrated analyses of the host transcriptome and microbiome further highlighted a significant association between differential microbial abundance and PML pathway activity, suggesting a contribution of the oral microbiome to the evolution of PML to OSCC. Collectively, this study revealed molecular processes associated with PML progression that may help early diagnosis and disease interception at an early stage.
My second aim was focused on elucidating the mechanisms of action of candidate therapeutics. As the efficacy rate of FDA-approved drugs for HNSCC tumors remains relatively low due to a combination of factors, including tumor heterogeneity, late diagnosis, and drug resistance, the need to develop targeted therapies focusing on selected cancer pathways represents a promising avenue. In previous studies, our laboratories have shown that pharmacological blockade of Wnt/β-catenin/CBP activity with small molecule inhibitors effectively abolished oncogenic cell phenotypes in OSCC. To determine the identity of additional cell subtypes contributing to OSCC progression to advanced disease, we induced oral tumors in immunocompetent mice using a tobacco-associated carcinogen, 4-Nitroquinoline 1-oxide (4-NQO), that causes DNA damage and recapitulates the genomic and histopathological changes observed with the progressive development of OSCC in human oral epithelia. Following 4-NQO treatment, mice were treated with E7386, an orally active small molecule modulator of β-catenin/CBP activity, at two concentrations — 25 mg/kg and 50 mg/kg. We generated a single-cell RNA sequencing dataset with ~50K cells to explore the E7386 treatment-associated effects on OSCC development and to better understand the underlying molecular and cellular changes that might provide novel insights into its mechanisms of action. Our analyses revealed shifts in cellular diversity between treatment groups, with the proportion of epithelial cells decreasing upon treatment — consistent with greatly diminished tumor volumes – while endothelial and fibroblast populations increased compared to the 4NQO control group, providing further evidence for the antitumor activity of E7386. In the immune compartment, we found enrichment of effector T-cell (CD8+ and CD4+) activity and a decrease in disease-associated neutrophil activity in the inhibitor-treated profiles. Additionally, epithelial sub-typing using curated markers of cell identities revealed a decrease in basal cancer stem-like cells (Krt5+, Krt14+) concomitant with an increase in cycling cells (Top2a+, Cdc20+). Finally, we identified a decrease in a stress cell phenotype associated with the AP-1 complex (Jun+, Fos+), and decreased cell plasticity in response to E7386, confirmed by functional validations through immunofluorescence staining experiments. Overall, this study provides cellular characterizations of murine oral tumors and presents further evidence of Wnt/β-catenin/CBP inhibition as a promising therapeutic strategy in OSCC.
In summary, my studies have contributed to advancing early detection and treatment strategies and to unveil molecular mechanisms of treatment response in OSCC. / 2025-05-17T00:00:00Z
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/48784 |
| Date | 17 May 2024 |
| Creators | Khan, Mohammed Muzamil |
| Contributors | Monti, Stefano |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
| Rights | Attribution 4.0 International, http://creativecommons.org/licenses/by/4.0/ |
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