A signature of circadian rhythm genes in driving anaplastic thyroid carcinoma malignant progression

Highlights

• Circadian rhythm genes drives anaplastic thyroid carcinoma malignant progression.

• A dynamic 9-DE-CRGs signature in ATC is identified in the malignant progression of ATC.

• NPAS2 regulates cell cycle and focal adhesion signaling to promote ATC progression.

Abstract

Background

Anaplastic thyroid carcinoma (ATC) was a rare and extremely malignant endocrine cancer. Recently, dysregulation of circadian rhythm genes was demonstrated to play an essential role in tumor progression, while its exact role and mechanism in ATC remained poorly clear.

Methods

4 ATC-related datasets were integrated to screen for differentially expressed circadian rhythm genes (DE-CRGs). Thereafter, Multiscale Embedded Gene Co-expression Network Analysis (MEGENA) and network enrichment analysis were conducted to investigate the dynamic characteristics of circadian rhythm genes. Next, Lasso-logistic model and immunohistochemistry were applied for determining the candidates. Finally, cell biological experiments and gene set enrichment analysis (GSEA) were used to confirm the roles of NPAS2 in ATC.

Results

25 DE-CRGs were firstly identified in ATC. These DE-CRGs mainly regulated mitotic nuclear division, cytokinesis and DNA replication signals. Notably, NPAS2, CSNK1E, NAMPT, TYMS, SERPINE1, TOP2A, JUN, EGR3 and HEBP1 were identified as the dynamic signature in the malignant progression of ATC, which were confirmed by prognostic analysis. Furthermore, NPAS2 was found to be significantly up-regulated in ATC through clinical samples and cell experiments. Silencing NPAS2 effectively inhibited the proliferation, migration and invasion of ATC cells. GSEA showed that high expression of NPAS2 was mainly associated with cell cycle and focal adhesion, and silencing of NPAS2 suppressed these signals in our experiments.

Conclusions

In summary, we found a dynamic 9-DE-CRGs signature in ATC. And the aberrant expression of NPAS2 drove the malignant phenotypes of ATC, which facilitated to deepen our understanding of the roles of circadian rhythm genes in ATC.

Introduction

Anaplastic thyroid carcinoma (ATC) accounted for 2% to 3% of all thyroid cancers and was a rare and extremely malignant cancer of the endocrine system [1,2]. ATC progressed rapidly, with a median survival period of less than 6 months, occuring with distant metastases usually and showed poor clinical treatment results, approaching half of the deaths from thyroid cancers [[3], [4], [5]]. The clinical treatment drugs of ATC mainly included doxorubicin, docetaxel, paclitaxel and platinum drugs, as well as inhibitors targeting polytyrosine kinases [6,7]. Currently, there was no targeted therapy drug that could improve the regrettable survival rate of ATC. Therefore, it was urgent to explore the key molecules that affect the malignant progression of ATC.

The circadian rhythm was a complex transcription-translation auto-regulatory network produced by the oscillation of clock gene expression [8,9]. As we all know, there was an inseparable close connection between circadian rhythm and thyroid functions. The hypothalamic–pituitary– thyroid axis was controlled by the circadian rhythm so that the disorder of the circadian rhythm could cause abnormal secretion of TSH, so the TSH level of people who had sleepless-night was significantly higher than that of people who sleep normally [10,11]. The study by Kim et al. showed that the serum TSH level of night shift workers were higher than that of daytime workers, and they had a greater risk of subclinical hypothyroidism [12]. In turn, surgical removal of the thyroid gland shortened the circadian rhythm period in rats and affects the expression of the circadian rhythm gene PER2 [13,14]. And researchers had observed drastic changes of circadian rhythm genes in primary cultured thyroid cells established from thyroid tissue and benign nodules [15]. It was worth noting that the “circadian rhythm disorder” had been listed as a possible human carcinogen by the International Agency for Research on Cancer [16]. Recent studies had revealed the disorder of circadian gene expression in thyroid cancer. Compared with normal thyroid tissue, BMAL1 and CRY2 in papillary and follicular thyroid cancer were significantly up-regulated and down-regulated [15,[17], [18], [19]]. Up to now, the role of circadian rhythm genes in ATC had not been explained and clarified yet. Therefore, characterizing the profiles of circadian rhythm genes in ATC was beneficial to explore the relationship between the circadian rhythm and the carcinogenic transformation of thyroid cells, and further developed new anti-tumor strategies.

The rarity of ATC and extremely difficult collection of clinical patient samples had resulted in few and limited datasets currently available. Therefore, we have integrated the datasets from multiple laboratories or array-platforms for large-scale cohort gene expression studies, thereby greatly reducing the system bias between different datasets and overcoming shortages. In this study, we integrated and analyzed 4 ATC-related GEO datasets to identify the unique and specifically altered circadian rhythm genes in ATC. This was currently the largest cohort in the world, including 52 ATC, 69 PTC, 17 PDTC and 78 normal thyroid tissue samples. We showed significant circadian rhythm genes in ATC, and constructed the gene co-expression network. Further, we identified a signature of 9 circadian rhythm genes to reveal pivotal signals that affect the malignant progression of ATC. We found that upregulation of NPAS2 significantly promoted the cell cycle and focal adhesion signals, driving ATC progression. To the best of our knowledge, this was the first study to investigate the functions of circadian rhythm genes in ATC. These results indicated that the disorder of circadian rhythm genes is the more aggressive driving factor of ATC, and the intervention of NPAS2 may be beneficial to the clinical treatment of ATC.