LRP4 promotes proliferation, migration, and invasion in papillary thyroid cancer

https://doi.org/10.1016/j.bbrc.2018.06.012Get rights and content

Abstract

Dysregulation of cell proliferation and death is considered the foundation of the malignant biological characteristics of cancer. In this study, we conducted a comprehensive analysis of a massively parallel whole transcriptome resequencing of paired papillary thyroid cancer and normal thyroid tissues from 19 patients. In addition, we found that LRP4, a member of the low-density lipoprotein receptor-related protein family, is significantly overexpressed in thyroid carcinoma. We demonstrated through quantitative real-time polymerase chain reaction (qRT-PCR) that LRP4 is upregulated in papillary thyroid cancer (PTC) tissues. This observation was also consistent with data analyzed from The Cancer Genome Atlas (TCGA) cohort. Thus, the biological role of LRP4 in the thyroid cancer in the present study was investigated using the PTC cell lines TPC1, BCPAP and KTC-1. In these cell lines, the mRNA level of LRP4 was higher than normal thyroid cancer cell named HTORI3. In vitro experiments demonstrated that LRP4 downregulation significantly inhibited the colony formation, proliferation, migration, and invasion of the three PTC cell lines. Knockdown of LRP4 by small interfering RNA (siRNA) in those cell lines decreased the protein expression of N-cadherin, Enhancer of zeste homolog 2 (EZH2), and Zinc finger E-box-binding home-box 1 (ZEB1). Furthermore, LRP4 knockdown significantly reduced the levels of phosphorylated PI3K in the PTC cell lines. In conclusion, the present study indicated that LRP4 is a gene associated with PTC and might become a potential therapeutic target.

Introduction

Thyroid cancer is one of the most predominant endocrine malignancies with an increasing prevalence of morbidity. The U. S. National Cancer Institute estimated that the number of new thyroid cancers in 2017 was 56,870, with an estimated 2010 deaths [1]. When referring to the incidence of thyroid cancer in China, the annual estimated number is 90,000, while the number of estimated deaths is 6800 [2]. The majority of the thyroid cancers included papillary (PTC) and follicular thyroid carcinoma (FTC), with PTC comprising 85–95% of all thyroid cancers [3]. The prognosis of PTC is often favorable, and its 5-year survival are as high as 90% [4]. Nevertheless, approximately 20% of PTC patients had lymph node metastasis, and the number of those who underwent total thyroidectomy and experienced regional recurrence is approximately 10% [5]. There are certain clinicopathologic factors, such as age, extrathyroidal spread (ETS), lymph nodes metastasis and distant metastasis, that contribute to poor prognoses [6]. Therefore, early diagnosis and treatment are necessary to improve the patients' quality of life. Currently, fine-needle aspiration cytology with cytological evaluation remains the gold standard, but this procedure fails to discriminate benign and malignant cancer in up to one-third of cases. Additionally, molecular testing of thyroid nodules has evolved rapidly over the past decade and not only helps improve the accuracy of thyroid cytology for indeterminate cases but also potentially guides the extent of surgery as initial therapy for suspected thyroid malignancies [7,8]. Considerable studies have been made in understanding the molecular mechanisms of PTC over the past two decades. Among those studies, B-type Raf kinase (BRAF) V600E mutations could promote PTC tumorigenesis and progression by aberrantly activating the mitogen-activated pathway kinase(MAPK) pathway [9]. In addition, the BRAF V600E mutation in PTC has been widely used as a biological marker to diagnose and predict the prognosis of PTC due to its specificity. Furthermore, TERT, RAS and PI3K gene mutations also play a critical role in thyroid cancer progression [10,11]. Although some studies have made some progress regarding thyroid cancer diagnosis, the mechanism of thyroid cancer progression is complex and much less understood. LRP4, a member of the low-density lipoprotein receptor-related protein family, plays a significant role in the ischemic brain injury response, and its deficiency seems to be a protective factor in response to ischemic brain injury via the release of ATP and adenosine as well as A2AR signaling [12]. Remarkably, in the progression of neuromuscular junctions, LRP4 can be a key factor in connection with neuromuscular disorders such as myasthenia gravis, Lambert-Eaton myasthenic syndrome, Isaacs' syndrome, congenital myasthenic syndromes, Fukuyama-type congenital muscular dystrophy, amyotrophic lateral sclerosis, and sarcopenia [13,14]. Furthermore, LRP4 could also regulate synapse formation during CNS development [15]. Several human cancers are also associated with LRP4 gene, for example, hepatocellular cancer (HCC) [16], neuroendocrine tumors [17]. In HCC, Agrin binds to LRP4 to form a critical oncogenic axis [16]. Several findings in PTC have been revealed through sequencing technology and a common database. Hucz J et al. conducted microarray studies in differentiated papillary carcinoma and normal tissues and found that LRP4 is significantly overexpressed in PTC tissue compared with normal thyroid tissues [18]. The same conclusion was confirmed in Jarzab B and Hucz J's work [18,19].

In our study, 19 paired PTC tissue samples and adjacent noncancerous samples were subjected to whole transcriptome sequencing and bioinformatics analysis to assess the mRNA expression profiles. We showed that LRP4 is one of the most significantly upregulated genes, and 43 pairs of tumor samples with matched adjacent noncancerous tissues were subjected to qRT-PCR to validate the abnormal expression of LRP4. Cell culture experiments and western blot were performed to demonstrate the role of LRP4 in PTC. Our study was designed to explore the role of LRP4 in the proliferation and metastasis of thyroid carcinoma and to analyze the relationship of LRP4 expression with the clinical and molecular features of PTC.

In a conclusion, we demonstrated that LRP4 is a gene associated with PTC and might become a potential therapeutic target.

Section snippets

Patients and samples

In all, 43 tissue samples, including primary PTC samples and paired parathyroid tissues, were obtained from patients who underwent surgical treatment at the First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China. In addition, none of the included patients underwent preoperative treatments, such as chemotherapy or radiotherapy. The samples were snap-frozen in liquid nitrogen immediately after resection and then stored at −80 °C before RNA extraction. The cases were

The LRP4 gene is overexpressed in PTC

To validate the results of whole transcriptome resequencing, we conducted qRT-PCR to detect the mRNA expression of the LRP4 gene in 43 paired PTC and adjacent normal thyroid tissues from patients, and LRP4 gene expression was remarkably elevated in the PTC samples compared with that in the matched normal tissues (Fig. 1A) (T: N = 12.33 ± 14.06: 1.04 ± 1.37, P < 0.001). This conclusion is consistent with the data shown in the TCGA cohort (Fig. 1B). The relationship between LRP4 expression and

Discussion

The number of annual thyroid cancer cases had increased by 4% globally, and, based on its incidence, thyroid cancer would surpass colorectal as the fourth most common cancer diagnosis by 2030 [22]. Although the current work presents data based on genetic assessments, the molecular mechanisms of PTC are still unclear. Previously, 19 pairs of PTC tumors and adjacent normal tissues were subjected to whole transcriptome resequencing, and we found that LRP4 is differentially expressed; this result

Ethical approval and consent to participate

Ethical approval for this study was obtained from the Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University. Written informed consent was obtained from each individual participant.

Consent for publication

None.

Availability of data and materials

The data sets supporting the conclusions of this article are included within this article and its, additional images. Raw data are available on the main electronic data storage system of First Affiliated Hospital of Wenzhou Medical University and access can be provided upon request to the authors.

Funding

This study was funded by Natural Science Foundation of Zhejiang Province(LY18H160053, LY17H160053 and LY18H160053)and the Science And Technology Project of Wenzhou (Y20170030).

Author contributions statement

Adheesh Bhandari wrote the manuscript. Xiaofen Zhou and Erjie Xia did the main experiment. Xiaohua Zhang designed the whole work. Chen Zheng, Jingjing Xiang and Yaoyao Guan helped to revise the article. All authors read and approved the final manuscript.

Competing interests statement

The authors state that they have no competing interests.

Acknowledgments

We would like to thank Prof.Dr. Ouchen Wang for his support. Without his help, this article would not have been possible.

References (33)

  • M.Y. Roth et al.

    Molecular testing for thyroid nodules: review and current state

    Cancer

    (2018)
  • E.S. Cibas et al.

    The bethesda system for reporting thyroid cytopathology

    Thyroid

    (2009)
  • M. Xing

    BRAF mutation in thyroid cancer

    Endocr. Relat. Canc.

    (2005)
  • H.G. Vuong et al.

    Role of molecular markers to predict distant metastasis in papillary thyroid carcinoma: promising value of TERT promoter mutations and insignificant role of BRAF mutations-a meta-analysis

    Tumour Biol

    (2017)
  • M. Decaussin-Petrucci et al.

    Molecular testing of BRAF, RAS and TERT on thyroid FNAs with indeterminate cytology improves diagnostic accuracy

    Cytopathology

    (2017)
  • X.C. Ye et al.

    Astrocytic Lrp4 (Low-Density lipoprotein receptor-related protein 4) contributes to ischemia-induced brain injury by regulating ATP release and adenosine-A2AR (adenosine A2A receptor) signaling

    Stroke

    (2018)
  • Cited by (21)

    View all citing articles on Scopus
    1

    Xiaofen Zhou and Erjie Xia contributed equally to this work.

    View full text