LTBP-2 confers pleiotropic suppression and promotes dormancy in a growth factor permissive microenvironment in nasopharyngeal carcinoma

doi:10.1016/j.canlet.2012.06.005

Cancer Letters

Volume 325, Issue 1, 1 December 2012, Pages 89-98

https://doi.org/10.1016/j.canlet.2012.06.005 Get rights and content

Abstract

This study identified LTBP-2 as a pleiotropic tumor suppressor in nasopharyngeal carcinoma, which safeguards against critical malignant behaviors of tumor cells. LTBP-2 expression was significantly decreased or lost in up to 100% of NPC cell lines (7/7) and 80% of biopsies (24/30). Promoter hypermethylation was found to be involved in LTBP-2 silencing. Using a tetracycline-regulated inducible expression system, we unveiled functional roles of LTBP-2 in suppressing colony formation, anchorage-independent growth, cell migration, angiogenesis, VEGF secretion, and tumorigenicity. Three-dimensional culture studies suggested the involvement of LTBP-2 in maintenance of tumor cell dormancy in a growth factor favorable microenvironment.

Highlights

► LTBP-2 was significantly down-regulated in NPC cell lines and biopsies. ► LTBP-2 silencing is associated with promoter hypermethylation. ► LTBP-2 suppresses malignancy by impairing in vitro & in vivo cell growth properties. ► LTBP-2 pleiotropically inhibits angiogenesis, VEGF secretion and migration. ► LTBP-2 confers tumor cell dormancy in microenvironment favorable for metastasis.

Introduction

Latent TGF-β binding protein 2 (LTBP-2) belongs to the LTBP-fibrillin gene family that encodes LTBPs-1, -2, -3, and -4 and fibrillins-1, -2, and -3. These proteins are extracellular matrix (ECM) glycoproteins sharing a similar overall domain structure for protein–protein interactions. LTBPs mainly consist of cysteine-rich EGF-like and 8-cysteine (8-Cys) repeats. The 8-Cys repeats have so far been found only in fibrillins and LTBPs, which bind to heterologous proteins via disulfide bridges. These repeats mediate the covalent protein complexes formed between LTBPs and TGF-β for all LTBPs except LTBP-2 [1], hence, promoting further investigation of the unique role of LTBP-2.

The expression and distribution of LTBP proteins are closely related to the regulation of latent complexes of TGF-β, raising considerable interest in understanding the involvement of LTBPs in cancers. Down-regulation of LTBP-1 was found in prostate, pancreatic, and ovarian tumors, as well as neuroendocrine tumors of the digestive system [2]. LTBP-4 was also down-regulated in human mammary adenocarcinomas [3]. Similarly, our previous study detected reduced LTBP-2 expression in esophageal squamous cell carcinoma (ESCC) [4], suggesting the importance of LTBP-2 in suppressing tumor development.

The functional impact of LTBP-2 is related to specific cell types, tissue origins, and stromal environments. With regards to cell migration and adhesion, LTBP-2 inhibits cancer cell migration and invasion in ESCC [4] and decreases fibroblast adhesion to fibronectin, revealing an important role of LTBP-2 as an anti-adhesion matrix component [5]. In contrast, LTBP-2 is able to augment melanoma cell adhesion and migration in an integrin-dependent manner [6]. These conflicting data suggest varying functions of LTBP-2 in different cancers. Further studies of LTBP-2 in other cancers will aid our understanding of this important ECM protein.

The Latent TGF-β binding protein 2 (LTBP-2) gene maps to chromosome 14q24, one of the critical tumor suppressive regions identified in nasopharyngeal carcinoma (NPC) [7]. LTBP-2 expression was found to be more crucially involved in NPC development than in ESCC, as evidenced by a dramatic loss of LTBP-2 in clinical samples (80% down-regulation of LTBP-2 in NPC versus 30% in ESCC), suggesting the importance of LTBP-2 in this specific type of cancer. Our previous study in ESCC did not address the potential involvement of LTBP-2 in tumor cell dormancy within the tumor microenvironment. In this study, we employed a more rigorous approach to study LTBP-2. Using a tetracycline-regulated expression system approach, we evaluated the functional roles of LTBP-2 in several common hallmarks of malignancies including cancer cell proliferation and migration, tumorigenicity, and angiogenesis. In addition, we provide insight into the regulation of tumor cell quiescence by LTBP-2 in the tumor microenvironment.

Section snippets

Cell lines and culture conditions

Seven NPC cell lines (HONE1, HNE1 [8], CNE1, CNE2 [9], SUNE1, HK1 [10], and C666-1 [11]), a tetracycline transactivator tTA1-producing cell line (HONE1–2) [12], two immortalized nasopharyngeal epithelial cell lines (NP69 [13] and NP460 [14]), and stable pETE-Bsd-LTBP-2 and pETE-Bsd vector-alone transfectants were maintained as described previously [15].

NPC tissue specimens

Matched normal nasopharyngeal and NPC biopsies from 30 NPC patients were collected at Queen Mary Hospital in Hong Kong from 2006 to 2008, as

Down-regulated LTBP-2 expression in NPC cell lines and tumors

To examine the endogenous expression levels of LTBP-2, quantitative real-time PCR was first performed to determine the transcriptional levels of LTBP-2 in seven NPC cell lines (HK1, HNE1, CNE1, CNE2, SUNE1, HONE1, and C666) and one immortalized nasopharyngeal epithelial cell line (NP460). In all NPC cell lines, the transcriptional level of LTBP-2 was significantly down-regulated to a level of <50%, when compared with the immortalized cell line NP460 (Fig. 1a).

Western blot analysis of the

Discussion

In this study, gene and protein expression analyses have revealed frequent loss of LTBP-2 in NPC cell lines and tumor biopsies, indicating an essential role for LTBP-2 in tumor development. Loss of LTBP-2 expression in NPC biopsies (80% down-regulation) is more profound than previously observed in ESCC (30% down-regulation) [4], strongly suggesting a particularly critical role of LTBP-2 in NPC progression.

Epigenetic inactivation of LTBP-2 was investigated with BGS analysis of a 358 bp region

Acknowledgements

We acknowledge the University Grants Council AoE/M-06/08 for grant support to MLL, the Finnish Cancer Foundation to JK-O, and the Swedish Cancer Society, the Swedish Research Council, the Swedish Institute, and Karolinska Institute to ERZ. We acknowledge the Area of Excellence Hong Kong NPC Research Tissue Bank for NPC specimens.

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LTBP-2 confers pleiotropic suppression and promotes dormancy in a growth factor permissive microenvironment in nasopharyngeal carcinoma

Abstract

Highlights

Introduction

Section snippets

Cell lines and culture conditions

NPC tissue specimens

Down-regulated LTBP-2 expression in NPC cell lines and tumors

Discussion

Acknowledgements

Cytokine & Growth Factor Reviews

Journal of Biological Chemistry

Biochimica et Biophysica Acta

Methods

Biochemical and Biophysical Research Communications

Cell

Differentiation; Research in Biological Diversity

European Journal of Cancer

Matrix Biology: Journal of the International Society for Matrix Biology

Matrix Biology: Journal of the International Society for Matrix Biology

The transforming growth factor b binding protein (LTBP) family

Biochem. J.

Latent transforming growth factor binding protein 4 (LTBP-4) is downregulated in human mammary adenocarcinomas in vitro and in vivo

APMIS

The ECM protein LTBP-2 is a suppressor of esophageal squamous cell carcinoma tumor formation but higher tumor expression associates with poor patient outcome

International Journal of Cancer.

Latent TGF-beta binding protein LTBP-2 decreases fibroblast adhesion to fibronectin

The Journal of Cell Biology

High resolution allelotype of microdissected primary nasopharyngeal carcinoma

Cancer Research

Two epithelial tumor cell lines (HNE-1 and HONE-1) latently infected with epstein-barr virus that were derived from nasopharyngeal carcinomas

Proceedings of the National Academy of Sciences of the United States of America

Detection of epstein-barr virus DNA in well and poorly differentiated nasopharyngeal carcinoma cell lines

Virus Genes

Establishment of a cell line (NPC/HK1) from a differentiated squamous carcinoma of the nasopharynx

International Journal of Cancer.

Nasopharyngeal carcinoma cell line (C666-1) consistently harbouring epstein-barr virus

International Journal of Cancer.