SPOCK1 is a novel transforming growth factor-β target gene that regulates lung cancer cell epithelial-mesenchymal transition

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Highlights

  • SPOCK1 is upregulation in lung cancer and associated with metastasis and survival.

  • Silencing of SPOCK1 inhibits lung cancer cell growth and colony formation.

  • Silencing of SPOCK1 inhibits lung cancer cell invasion.

  • SPOCK1 induces EMT and is a novel target of TGF-β in lung cancer.

Abstract

Lung cancer is the leading cause of cancer related death worldwide and the prognosis is still poor with 5-year survival of approximately 15%. Metastasis is the leading cause of death by cancer. Recent researches have demonstrated that epithelial-to-mesenchymal transition (EMT) plays a key role in the early process of metastasis of cancer cells. Here, we identified that SPARC/osteonectin, cwcv and kazal-like domains proteoglycan 1 (SPOCK1) is a novel transforming growth factor-β1 (TGF-β) target gene that regulates lung cancer cell EMT. TGF-β has been reported as a major inductor of EMT. We observed that the expression of SPOCK1 in lung cancer tumor tissues is significantly higher than matched normal lung tissues. Moreover, the expression of SPOCK1 was also significantly higher in metastasis tumor tissues than non-metastasis tumor tissues. Levels of SPOCK1 mRNA were increased among patients with shorter disease-free survival times, indicating the potential role of SPOCK1 in lung cancer progression and metastasis. Silencing SPOCK1 expression with endoribonuclease-prepared small interfering RNA (esiRNA) in lung cells inhibits lung cancer cell growth, colony formation and invasion in vitro. Interestingly, ectopic expression of SPOCK1 in epithelial lung cancer cells induced EMT with increased expression of the mesenchymal marker Vimentin and decreased expression of epithelial marker E-cadherin. We also found that the expression of SPOCK1 was increased under treatment of TGF-β, indicating that SPOCK1 is a novel downstream target of TGF-β. Taken together, our study showed that SPOCK1 is a novel metastasis related biomarker in lung cancer and may be new diagnostic and therapeutic target for lung cancer.

Introduction

Lung cancer is the leading cause of cancer-related death, both in the United States and worldwide; however, the prognosis is still poor with a 5-year survival rate of approximately 15% [1]. Despite improvements in early diagnostic modalities, around 50% of lung cancer patients present with local or systemic advanced disease [2]. Moreover, the majority of cancer patients die of metastasis rather than the primary disease.

Recent studies have found that epithelial-to-mesenchymal transitions (EMT) play a key role in the early process of tumor metastasis. EMTs are trans-differentiation programs that are required for tissue morphogenesis during embryonic development [3], [4]. The EMT process can be regulated by a number of cytokines and growth factors, such as transforming growth factor (TGF)-β, whose expressions are dysregulated during malignant tumor progression [5].

At the transcriptional level, a diverse list of embryonic transcription factors, such as E-cadherin repressing Zinc finger proteins Snail (SNAI1), and Slug (SNAI2), ZEB1 (δEF1) and ZEB2 (SIP1), the basic helix-loop-helix proteins TWIST1 and TWIST2, and the winged-forkhead transcription factor FOXC2, can be potently activated by TGF-β, resulting in epithelial-to-mesenchymal transitions. However, other EMT core signature makers, such as SPOCK1, have never been reported in lung cancer cells. Even less is known about the function and mechanism by which SPOCK1 contributes to the growth and progression of lung cancer cells.

SPOCK1 encodes a matricellular glycoprotein family member. Other members of this family include SPARC, TESTICAN-2 and TESTICAN-3. Among these, SPARC has been well documented; it has been demonstrated to regulate proliferation, apoptosis, adhesion and cell–matrix interaction in various cancers [6]. In view of the similarity between SPOCK1 and SPARC in structure, it is of great interest to investigate the role of SPOCK1 in the development and progression of cancer. More interestingly, a number of studies have demonstrated that SPOCK1 plays a critical role in prostate cancer recurrence, glioblastoma invasion and HCC progression [7], [8], [9], indicating that SPOCK1 may also be involved in the invasion of lung cancer. Here, we reported that the silencing of SPOCK1 can inhibit the lung cancer cell growth rate and colony formation in soft agar. Furthermore, the silencing of this protein decreases the invasion ability of lung cancer cells. We also found that the inductor of EMT–TGF-β treatment can induce the expression of SPOCK1, which indicates that SPOCK1 may play a role in epithelial-to-mesenchymal transition in lung cancer.

Section snippets

Patients, specimens and cell lines

NSCLC samples (40 samples) and their adjacent non-tumor lung tissues were collected from patients who underwent lobectomy at Drum Tower Hospital Affiliated to Nanjing University (Nanjing, China). None of these patients received preoperative chemotherapy or radiotherapy. The samples used in this study were approved by the Committees for Ethical Review of Research Involving Human Subjects at the Drum Tower Hospital Affiliated to Nanjing University. The mean age of the 40 NSCLC patients included

SPOCK1 is significantly upregulation in lung cancer and associated with metastasis and survival

To investigate the clinical significance of SPOCK1 in NSCLC, expression of SPOCK1 mRNA in 40 examples of NSCLCs (tumor and corresponding non-tumor tissues) was compared by qRT-PCR. The relative expression level of SPOCK1 was significantly higher in tumor tissues compared with their non-tumor counterparts (P < 0.001, paired Student’s t-test; Fig. 1A). In another study, SPOCK1 was found to be significantly associated with advanced clinical stage and metastasis [8]. Thus, we detected the expression

Discussion

SPOCK1 is a proteoglycan protein that was first isolated in human testes and initially called “TESTICAN”. It is dysregluated in many organs and tissues, including the brain cartilage, vascular endothelium, lymphocytes, and neuromuscular junctions [10], [11], [12]. Recently, SPOCK1 was also identified to be over-expressed in HCCs [8]. In our findings, SPOCK1 was also highly expressed in tumor tissues compared with normal lung tissues, indicating that it may play a role in lung cancer

Acknowledgment

This work was supported by Grants from the National Natural Science Foundation of China; Grant No.: 81301882.

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