NYMC Student Theses and Dissertations

Date of Award

5-20-2025

Document Type

Doctoral Dissertation - Restricted (NYMC/Touro only) Access

Degree Name

Doctor of Philosophy

Department

Basic Medical Sciences

First Advisor

Raj K. Tiwari

Abstract

Anaplastic thyroid cancer (ATC) is a rare and highly aggressive subset of thyroid cancer, characterized by its refractory nature and poor prognosis. Conventional cancer treatment modalities, including chemotherapy and radiotherapy, have proven ineffective in managing ATC. At the same time, targeted therapies show limited promise due to the absence of identifiable targetable genetic alterations, culminating in a dismal five-year survival rate of approximately 4%.

The low mutational burden observed in ATC prompted exploration of differential gene expression patterns through analysis of publicly available transcriptomic datasets from the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GSE33630, GSE85457). Bioinformatic analysis was conducted using GEO2R software, comparing ATC tissue samples to PTC tissues or normal thyroid tissues. Strikingly, the differentiation-associated gene Homeobox D4 (HOXD4) was significantly upregulated in ATC patient tissue samples compared to papillary thyroid cancer (PTC) and normal tissues. Immunohistochemistry analysis of ATC, PTC, and normal thyroid tissues obtained from the National Institute of Health: Cooperative Human Tissue Network confirmed that HOXD4 expression was significantly elevated in ATC patient tissues. Kaplan-Meier survival analysis indicated that ATC patients with high levels of HOXD4 expression exhibited significantly reduced survival rates. ATC is unique because it lacks differentiation, resulting in the loss of thyroid-like morphology and function. Thus, the marked upregulation of HOXD4, a gene typically associated with differentiation, warranted further in vitro exploration. HOXD4 RNA and protein expression were significantly upregulated in ATC cell line T238 and SW1736 compared to PTC cell line K1 and immortalized “normal” thyroid epithelial cell line Nthy-ori-3-1. Bioinformatic analysis of predicted HOXD4 binding partners revealed six binding partners that were concurrently upregulated in ATC, revealing the ATC-associated

HOXD4 protein interactome. This protein interactome is associated with the main hallmarks of ATC that contribute to its highly aggressive nature: loss of thyroid functional genes, loss of thyroid signaling (including Notch signaling), and loss of adherens junctions, characteristic of epithelial- to-mesenchymal transition.

To assess the functional role of HOXD4 in ATC, a CRISPRi knockdown was employed to transcriptionally repress HOXD4 expression in both T238 and SW1736 ATC cell lines, generating HOXD4 knockdown cell lines (T238-HOX and SW1736-HOX) as well as a control cell line treated with NS oligonucleotides. Functional assays revealed a significant reduction in cell proliferation, increased cellular senescence, and reduced self-renewal capacity in the HOXD4 knockdown lines. HOXD4 was found to mediate differentiation in ATC, as knockdown cells had increased thyroid functional gene expression and increased Notch signaling, both of which contribute to the refractory nature of this malignancy. Repression of HOXD4 resulted in reduced invasion, migration, and epithelial-to-mesenchymal transition in ATC cells in both two- dimensional and three-dimensional contexts. Considering that ATC is a highly metastatic and treatment-refractory disease, repression of HOXD4 presents as a potential therapeutic avenue.

To address the hypothesis of HOXD4 repression as a potential therapeutic target in ATC, an inducer of differentiation, all-trans retinoic acid (ATRA), was investigated to determine its potential to abrogate ATC’s aggressive phenotype. ATRA significantly reduced HOXD4 expression at the RNA and protein levels in ATC cell lines T238 and SW1736 while not affecting NThy-ori-3-1 HOXD4 levels. The modulation of HOXD4 using ATRA treatment was related to the classical retinoic acid pathway, leading to the subsequent induction of differentiation in ATC cells. Thus, the restoration of thyroid functional genes and Notch signaling suggests the potential for successful radioiodine therapy. ATC cells treated with ATRA displayed a significant reduction in proliferation, cellular senescence, and self-propagation capacity. Furthermore, ATRA-treated T238 and SW1736 cells exhibited a significant decrease in invasion and migration capacities in both two-dimensional and three-dimensional contexts as well as a reversal of epithelial-to- mesenchymal signatures. By contrast, NTHY-ori-3-1 cells demonstrated no significant alteration in proliferation, senescence, invasion, or migration following ATRA administration, indicating that ATRA treatment is specific to ATC cells.

These findings highlight the novel role of HOXD4 as a putative oncogenic driver in ATC, underscoring its potential as both a diagnostic and prognostic biomarker for the disease. Additionally, these data suggest that HOXD4 may serve as a viable therapeutic target in future treatments for ATC. Considering the efficacy of the differentiation-inducing agent all-trans retinoic acid in mitigating this deadly malignancy, it may prove useful as a standalone treatment or in combination with other therapeutic strategies to develop more effective clinical applications for ATC.

Keywords

anaplastic thyroid cancer, HOXD4, differentiation, therapeutic target, prognostic marker

Disciplines

Medicine and Health Sciences

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