Targeted Differentiation of Primary Head and Neck Squamous Cell Carcinoma Cells

Head and neck squamous cell carcinoma (HNSCC) is a malignant disease; it is the fourth most common cancer in Germany and the sixth most common cancer worldwide, with high fatality rates that have remained essentially unchanged for many years. Especially in the subtype that is negative for human papillomavirus (HPV). Consequently, there is an urgent requirement for new treatment techniques. HNSCC tumors that are negative for HPV have areas of tissue that are terminally differentiated and characterized by a process called cornification. Exploring the intrinsic ability of HNSCC cells to terminally differentiate into non-cancerous cells by losing their self-renewal characteristics has potent tumor-targeting potential. This dissertation created a primary model to reproduce the cornification of HNSCC cells and analyzed the mechanisms underlying differentiation using ATAC and RNA sequencing. Immunofluorescence verified the results using human HNSCC tissue from different anatomical locations. This research discovered that when HNSCC cells are differentiated, they become more adherent, stop proliferating, and show a decreased ability to initiate tumors in immunodeficient mice. Additionally, a signaling program associated with wound healing is activated. Although chromatin overall becomes more compact, an increase in the accessibility of small promoters was observed. During differentiation, KRT17 and the cornification marker SPRR3 are upregulated. KRT17 typically acts as a basal stem-cell marker in normal mucosal tissue. This study confirmed KRT17 as an early marker in HNSCC tissue and dysplastic mucosa. Cornification was frequently observed surrounding necrotic and immune-infiltrated areas in human tumors, indicating an involvement of pro-inflammatory stimuli. Inflammatory mediators were found to activate the HNSCC cell differentiation program. Cell differentiation states create a common tissue architecture in normal mucosa and HNSCCs. Using patient-derived tumor cells, we also created an HNSCC differentiation model of HPV-positive tumor cells from two patients. In differentiating cell culture conditions, a loss of malignant characteristics, including irregularly enlarged cell morphology, cell 4 cycle arrest with downregulation of Ki67, and reduced cell viability, were observed. RNAseq identified myocyte-lineage differentiation with upregulation of markers of myofibril assembly, including TPM1, TAGLN, and ACTA1. Immunofluorescence staining of differentiated and undifferentiated primary HPV+ HNSCC cells confirmed an upregulation of these markers and the formation of parallel actin fibers reminiscent of myoblast-lineage cells. Moreover, immunofluorescence of HPV+ tumor tissue revealed areas of cells co-expressing the identified markers of myofibril assembly, HPV surrogate marker p16, and stress-associated basal keratinocyte marker KRT17, indicating that the observed myocyte-like in vitro differentiation occurs in human tissue. Thus, the same differentiation protocol triggered different phenotypes dependent on HPV status. Moreover, HPV+ HNSCC cells that have undergone induced trans-differentiation exhibit signs of epithelial-to-mesenchymal transition (EMT), which is linked to metastatic spread and represents a hallmark of cancer. In this model, EMT-like transition results in less aggressive behavior of cancer cells, decreasing their proliferation and viability, increasing cell death, and having no effect on cell migration. This work has successfully established primary differentiation models for both subsets of HNSCC and further elucidated their respective mechanistic pathways. However, despite these advancements, we have yet to fully understand the genes and proteins necessary to initiate and maintain terminal differentiation in HNSCC. Our ongoing research aims to address this knowledge gap and provide valuable insights into the mechanisms of terminal HNSCC differentiation. Most importantly, the present work provides a new understanding of the potential and limitations of differentiation therapy in HNSCC.

Level thesis:degree_level

thesis.doctoral

Grantor dc:publisher

Universität Bielefeld

Year

2025

Author dc:creator

• Gendreizig, Sarah

Repository record source_url

https://pub.uni-bielefeld.de/record/3002486

OAI identifier oai:identifier

oai:pub.uni-bielefeld.de:3002486