Transcription Factor Prospero Homeobox 1 (PROX1) as a Potential Angiogenic Regulator of Follicular Thyroid Cancer Dissemination.

Magdalena Rudzińska,Ewa Gajda,Barbara Czarnocka,Katarzyna D Arczewska,Tomasz Stępień,Jerzy Ostrowski,Stanisława Sabalińska,Michał Mikula

doi:10.3390/ijms20225619

Abstract

It is well known that Prospero homeobox 1 (PROX1) is a crucial regulator of lymphangiogenesis, that reprograms blood endothelial cells to lymphatic phenotype. However, the role of PROX1 in tumor progression, especially in angiogenesis remains controversial. Herein, we studied the role of PROX1 in angiogenesis in cell lines derived from follicular thyroid cancer (FTC: FTC-133) and squamous cell carcinoma of the thyroid gland (SCT: CGTH-W-1) upon PROX1 knockdown. The genes involved in angiogenesis were selected by RNA-seq, and the impact of PROX1 on vascularization potential was investigated using human umbilical vein endothelial cells (HUVECs) cultured in conditioned medium collected from FTC- or SCT-derived cancer cell lines after PROX1 silencing. The angiogenic phenotype was examined in connection with the analysis of focal adhesion and correlated with fibroblast growth factor 2 (FGF2) levels. Additionally, the expression of selected genes involved in angiogenesis was detected in human FTC tissues. As a result, we demonstrated that PROX1 knockdown resulted in upregulation of factors associated with vascularization, such as metalloproteinases (MMP1 and 3), FGF2, vascular endothelial growth factors C (VEGFC), BAI1 associated protein 2 (BAIAP2), nudix hydrolase 6 (NUDT6), angiopoietin 1 (ANGPT1), and vascular endothelial growth factor receptor 2 (KDR). The observed molecular changes resulted in the enhanced formation of capillary-like structures by HUVECs and upregulated focal adhesion in FTC-133 and CGTH-W-1 cells. The signature of selected angiogenic genes’ expression in a series of FTC specimens varied depending on the case. Interestingly, PROX1 and FGF2 showed opposing expression levels in FTC tissues and seven thyroid tumor-derived cell lines. In summary, our data revealed that PROX1 is involved in the spreading of thyroid cancer cells by regulation of angiogenesis.

Highlights

Thyroid cancer malignancies are divided into three major types: (1) differentiated thyroid carcinoma (DTC) arising from follicular cells of the thyroid, (2) anaplastic thyroid carcinoma, and (3) medullary thyroid carcinoma arising from parafollicular C cells
These observations are in agreement with our previously published data [24,26], and here we again confirm the effect of silencing of Prospero homeobox 1 (PROX1) protein using Western blotting for siRNAs targeting PROX1 (siPROX1)
Representative images show the results of Western blotting analysis using the anti-vascular endothelial growth factors C (VEGFC) and anti-fibroblast growth factor 2 (FGF2) primary antibodies; β-actin served as an internal loading control

Summary

Introduction

Thyroid cancer malignancies are divided into three major types: (1) differentiated thyroid carcinoma (DTC) arising from follicular cells of the thyroid, (2) anaplastic thyroid carcinoma, and (3) medullary thyroid carcinoma arising from parafollicular C cells. DTC is the most common thyroid malignancy and accounts for 90%–95% of all thyroid cancer cases [1]. Squamous cell carcinoma of the thyroid (SCT), constituting less than 1% of all thyroid malignancies, is an aggressive neoplasm thought to arise as a primary tumor or as a component of an anaplastic or undifferentiated carcinoma and gives the distant metastasis [3]. DTCs spread through different pathways and PTCs have a propensity to disseminate via lymphatic vessels to the neck regional lymph nodes, whereas FTCs tend to metastasize to remote organs by the hematogenous route [4]. Metastases from primary SCT are common and mostly its vascular invasion occurs in the lungs, bones, liver, kidney, and heart [3]

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