Cell & Bioscience

Liu et al. Cell Biosci (2016) 6:24 DOI 10.1186/s13578-016-0091-9

Open Access

RESEARCH

SOSTDC1 is down‑regulated in non‑small cell lung cancer and contributes to cancer cell proliferation Lei Liu1,2†, Shanshan Wu1,2†, Yi Yang1,3, Junchao Cai1,2, Xun Zhu1,2, Jueheng Wu1,2, Mengfeng Li1,2 and Hongyu Guan4*

Abstract  Background:  Non-small cell lung cancer (NSCLC) is the most commonly diagnosed and fatal cancer worldwide. Sclerostin domain containing protein 1 (SOSTDC1) has been found to be tumor-suppressive in several types of cancers. However, the expression level and biological functions of SOSTDC1 in NSCLC remain unknown. Our current study aimed to identify the biological significance of SOSTDC1 in NSCLC. Results:  We found that SOSTDC1 was significantly down-regulated in NSCLC. Moreover, patients with higher expression of SOSTDC1 had a significant better prognosis than those with lower SOSTDC1 expression. Ectopic expression of SOSTDC1 in NSCLC cell lines A549 and NCI-H520 could inhibit proliferation as shown by MTT, colony formation, soft agar and EdU incorporation assays in vitro. Furthermore, A549 cells stably expressing ectopic SOSTDC1 grew more slowly and formed smaller tumors than vector-control cells in vivo. Mechanistic studies demonstrated that SOSTDC1 over-expression led to increased p21Cip and p27Kip levels, thereby decreasing Rb phosphorylation status and E2F transcription activity. Conclusions:  SOSTDC1 is down-regulated in NSCLC, and its expression level is indicative of clinical outcome of patients with the disease. SOSTDC1 might represent a tumor suppressor through inhibiting the proliferation of NSCLC cells by regulating p21Cip and p27Kip, which in turn affects Rb-E2F signaling. Keywords:  SOSTDC1, Non-small cell lung cancer, Proliferation, p21Cip, p27Kip Background Lung cancer is one of the most commonly diagnosed cancer types worldwide and the leading cause of cancerrelated death [1, 2]. Non-small cell lung cancer (NSCLC), which includes squamous cell carcinoma (SCC), adenocarcinoma (AD), large cell carcinoma (LCC), and other less frequently diagnosed histological types, accounts for about 80  % of lung cancer cases [3]. While various therapeutic approaches, including surgical resection, chemo- and radio-therapies, have been applied in the *Correspondence: [email protected] † Lei Liu and Shanshan Wu contributed equally to this study 4 Department of Endocrinology and Diabetes Center, The First Affiliated Hospital of Sun Yat-sen University, 58 Zhongshan Road II, Guangzhou 510080, Guangdong, China Full list of author information is available at the end of the article

management of NSCLC, the overall 5-year survival rate of NSCLC patients still remains at only 15  % [4]. Better understanding  of the genetic events and key molecules involved in NSCLC development and progression is needed for developing effective therapeutic strategies against the disease. Sclerostin domain-containing protein 1 (SOSTDC1), an important regulator of cell signaling, has been found to contribute to several physiological and pathological processes [5, 6]. Accumulating evidence has revealed that SOSTDC1 might act as a tumor suppressor in many cancers. In wilms tumor, SOSTDC1 is lost as a result of a 7p21 homozygous deletion, which led to accelerate angiogenesis and activation of Wnt signaling [7]. The expression of SOSTDC1 is down-regulated in gastric cancer, and ectopic over-expression of SOSTDC1 in gastric

© 2016 Liu et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/ zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Liu et al. Cell Biosci (2016) 6:24

cancer cells suppressed cell proliferation, cell cycle progression and anchorage-independent growth [8]. Clausen et al. found that the expression of SOSTDC1 reduced in breast cancer and such a reduction correlated   to poor prognosis of patients with the disease [9]. They also demonstrated that SOSTDC1 interfered with the signaling mediated by the Wnt3a, BMP-2, and BMP-7 pathways in breast cancer cells [10]. Moreover, down-regulation of SOSTDC1 was observed in renal cancer, and SOSTDC1 could suppress the proliferation of renal cancer cells via regulating BMP and Wnt3a signaling [11]. Nevertheless, the expression pattern and biological significance of SOSTDC1 in NSCLC remains largely unknown. Deregulation of cell cycle progression represents a key characteristic in many types of cancer [12]. Under physiologically normal conditions, the cell cycle progression is elegantly regulated by cyclins and cyclin-dependent kinases (CDK) [13]. Of note, the activity of these molecules is restricted by the Cip/Kip family members [14–16], among which p21Cip and p27Kip are key functional cell cycle inhibitors containing an N-terminal CDK inhibitory domain and prevent CDKs from phosphorylating Rb, thus restricting the E2F transcription activity [14–17]. Given that E2F is an essential transcription factor for the production of cell cycle regulatory genes, p21Cip and p27Kip induces G1 arrest. Moreover, p21Cip could directly interfere with DNA replication by inhibiting PCNA [18]. Therefore, p21Cip and p27Kip are widely recognized as tumor suppressors, and their inactivation is frequently observed in various types of cancer, including NSCLC [19, 20]. While functional inactivation of p21Cip and p27Kip occur frequently in cancer cells, the mechanisms involved in the reduction or loss of p21Cip and p27Kip functions remain to be further understood. In the context of understanding the possible role of SOSTDC1 in NSCLC development and progression, our current study led to findings that the protein is down-regulated in NSCLC and might play a tumor-suppressive role.

Results The expression of SOSTDC1 is down‑regulated in NSCLC

To determine whether the expression of SOSTDC1 in NSCLC is altered, we first analyzed 99 pairs of primary tumor (T) versus normal tissues (N) using RNASeqV2 data sets for NSCLC deposited on the TCGA website (https://tcga-data.nci.nih.gov/tcga) [21] for SOSTDC1 expression. Our results showed that SOSTDC1 was down-regulated in 90 tumor tissues as compared to their adjacent non-cancerous lung tissues (Fig.  1a). To further assess the expression level of SOSTDC1 in NSCLC, we examined the level of SOSTDC1 in 18 pairs of tumors and their adjacent non-cancerous lung tissues. In agreement with the TCGA data, decrease in SOSTDC1 mRNA levels

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was observed in most tumor tissues as compared with that in their adjacent non-tumorous lung tissues (Fig. 1b). Next, we investigated the protein levels of SOSTDC1 in the 128 clinical specimens by performing immunohistochemistry (IHC) (Fig.  1c; Table  1). While no significant correlation between SOSTDC1 protein level and patient age (p  =  0.732), gender (p  =  0.150), pathologic type (p = 0.765), or N classification (p = 0.204) was found, the expression of SOSTDC1 significantly correlated to clinical staging (p  =  0.010), and T classification (p  56

44

18

0.732

Gender  Male

66

24

 Female

23

15

0.150

Pathologic type  Squamous cell carcinoma

32

12

 Adenocarcinoma

51

25

6

2

 Adenosquamous carcinoma

0.765

Clinical staging  I

30

25

 II

23

8

 III

30

5

 IV

6

1

0.010

T classification  T1

10

17

 T2

46

18

 T3

29

4

 T4

4

0

SOSTDC1 is down-regulated in non-small cell lung cancer and contributes to cancer cell proliferation.

Non-small cell lung cancer (NSCLC) is the most commonly diagnosed and fatal cancer worldwide. Sclerostin domain containing protein 1 (SOSTDC1) has bee...
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