NYMC Student Theses and Dissertations

Date of Award

5-23-2024

Document Type

Doctoral Dissertation - Restricted (NYMC/Touro only) Access

Degree Name

Doctor of Philosophy

Department

Basic Medical Sciences

First Advisor

Jan Geliebter, PhD

Abstract

Papillary thyroid cancer (PTC) is the most common cancer in young women and has been increasing in incidence over the last several decades. While PTC is often indolent and treatable, recurrence years to decades later occurs in ~30% of patients (now only in their 40s/50s), decreasing survival ~60%. Therefore, the identification of prognostic biomarkers and actionable therapeutic targets for this high-risk PTC is a critically important, unmet need. RNA-Sequencing identified collagen 26A1 (COL26A1) as significantly upregulated in patient samples with extrathyroidal extension (ETE), lymph node metastasis (LNM), and multifocal tumors, indicating high-risk for recurrence. The Cancer Genome Atlas (TCGA) data demonstrated increased COL26A1 expression decreases survival probability by 25% and correlated with MACIS score, thyroid differentiation score (TDS), ERK score, tumor stage, and ETE, further denoting high-risk PTC. Additionally, COL26A1 correlated with known metastatic signature genes for PTC, genes involved in stiff tumor texture, and with that, the strong positive correlation with cancer-associated fibroblast infiltration, all of which suggest a poor prognosis.

To establish a manipulable in vitro cell model, PTC cell lines were investigated for COL26A1 expression. COL26A1 expression was significantly higher in PTC cell lines K1 and TPC1 compared to normal thyroid epithelial cells, NThy-ori-3-1. CRISPR inhibition using two independent short guides successfully repressed COL26A1 expression at both the RNA and protein level in both K1 and TPC1. RNA-Sequencing of knock-down compared to control cells demonstrated significantly differentially expressed biological processes including apoptosis, cell adhesion, cell migration, and cell proliferation, specifically with modulations to MAPK, Wnt, and PI3K signaling. Exploration of the effects of COL26A1 knock-down demonstrated that metastatic phenotypes were significantly decreased, including invasion, migration, motility, gelatin degradation, cell-cell and -matrix adhesion, anchorage-dependent and -independent clonogenicity, and proliferation compared to controls. This coincided with the effect of collagens on epithelial-mesenchymal transition (EMT) including reduced anoikis resistance, altered matrix metalloprotease secretion, and decreased mesenchymal markers with increases in epithelial markers and restoration of TDS. The partial induction of EMT is a characteristic of an intermediate population of cells that are more effective at undergoing metastasis through survival signals with collective migration. Notably, E-cadherin expression, while an epithelial marker, was reduced in the knock-down cells. Functionally, the ability of cells to maintain their cell-cell contact and promote their metastatic phenotype was assessed through hanging-droplet 3D spheroid formation. 3D spheroids were smaller in size and number, which further demonstrated a reduction in their Matrigel growth and 3D collagen dissemination. This confirmed COL26A1 promotes the invasive and metastatic abilities of PTC in vitro. COL26A1 secretion in conditioned media from K1 and TPC1 was seen at higher levels compared to NThy. COL26A1 repression decreased levels of secretion in the knock-down cells to that of NThy. Treatment of NThy and knock-down cells with control cell conditioned media was performed to assess the effect of COL26A1 expression and the secreteome changes on metastatic phenotypes. Treatment of knock-down cells with control media restored the migration ability of the cells and treatment of NThy increased both migration and anoikis resistance. This in vitro validation of secreted COL26A1 in conditioned media prompted the identification of protease cleavage sites that could uncover pathologically relevant fragments serving as non-invasive biomarkers.

Qiagen Ingenuity Pathway Analysis (IPA) elucidated a link with androgen receptor (AR), coinciding with the known effects of sex hormones on collagen expression and organization, and the sex disparity in PTC. In TCGA-THCA, COL26A1 expression significantly negatively correlated with AR expression and furthermore, COL26A1 expression strongly positively correlated with genes repressed by AR activation. Physiological levels of androgen decrease COL26A1 RNA and protein expression in K1 cells expressing functional AR. This coincided with the demonstrated protective effects of AR on PTC through induction of senescence. Bioinformatic analysis also identified oncogenic lncRNA NEAT1 as a potential positive regulator of COL26A1 expression through complementary binding to miRNA targets. Thus, COL26A1 may serve as a novel therapeutic target and prognostic biomarker, identifying patients with a Stage I or II tumor at diagnosis whose tumor has a propensity to become Stage III or IV, and hence requiring more aggressive therapeutic approaches and follow-up.

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