Metastasis suppressor 1 (MTSS1) demonstrates prognostic value and anti-metastatic properties in breast cancer

doi:10.1016/j.ejca.2009.02.019

European Journal of Cancer

Volume 45, Issue 9, June 2009, Pages 1673-1683

https://doi.org/10.1016/j.ejca.2009.02.019 Get rights and content

Abstract

Metastasis suppressor 1 (MTSS1) may play an important role in cancer metastasis. Firstly, this study assessed MTSS1 expression levels within breast cancer patients to reveal any clinical relevance. Secondly, we aimed to clarify the cellular function of MTSS1 in breast cancer cells. MTSS1 expression levels were assessed in a cohort of breast cancer specimens (normal n = 33; cancer n = 127), through quantitative PCR analysis and immuno-histochemical techniques. The influence of MTSS1 was further examined via biological overexpression and knockdown within breast cancer cell lines. We report that patients with tumours expressing reduced levels of MTSS1 had a poorer prognosis (p = 0.042). High levels of MTSS1 correlated with an increased patient overall survival (p = 0.0108) and disease-free survival (p = 0.012). Furthermore, overexpression of MTSS1 significantly suppressed (p < 0.01) the invasive, migratory, growth and adherence properties of a human breast cancer cell line. In contrast, knockdown of MTSS1 dramatically enhanced these properties. We conclude that MTSS1 is a prognostic indicator of disease-free survival in breast cancer patients and demonstrates the ability to play a role in governing the metastatic nature of breast cancer cells.

Introduction

The most deadly aspect of cancer is its ability to metastasise from the primary tumour to secondary sites. The metastatic cascade consists of a series of sequential, interrelated steps that are not as yet completely understood. However, it is known that these metastatic events are modulated by many factors, including metastasis activators and suppressors. Metastasis suppressors, by definition, inhibit metastasis at any step of the metastatic cascade without blocking tumourigenicity.¹

Metastasis suppressor 1 (MTSS1), also known as MIM or MIM-B (missing-in-metastasis) was originally identified as a potential metastasis suppressor gene that was present in non-metastatic bladder cancer cell lines, but was not expressed in a metastatic bladder cancer cell line.² This first study suggested that MIM could be a potential metastasis suppressor. It was also demonstrated that MIM-B induced actin-rich protrusions resembling microspikes and lamellipodia at the plasma membrane and promoted disassembly of actin stress fibres.³ Actin filament assembly is associated with cytoskeletal structure organisation and many forms of cell motility.⁴ This alteration in actin dynamics will have serious consequences on the metastatic ability of cancer cells.5, 6 This may help explain the involvement of MTSS1 in the metastasis of cancer cells. However, further evidence is required to substantiate this preliminary claim and to elucidate any potential role of MTSS1 in the regulation of tumour cell behaviour.

To date, there are contrasting views to whether or not MTSS1 is in fact a metastasis suppressor. MTSS1 has been reported to play a role as a metastasis suppressor in both prostate cancer7, 8 and bladder cancer2, 8, 9; however, some reports suggest that MTSS1 is unlikely to be metastasis suppressor and instead acts as a scaffold protein that interacts with actin-associated proteins to modulate lamellipodia formation.¹⁰ Recently, evidence suggests that MTSS1 is a regulator of carcinogenesis in hepatocellular carcinoma,¹¹ and is a member of the sonic hedgehog (SHH) signalling pathway that modulates Gli responses during growth and carcinogenesis.12, 13

Therefore, a role for MTSS1 in tumourigenesis and metastasis is yet to be established. All these controversial data are due in part to the fact that the study of MTSS1 has been restricted to a limited of cancer types, with little support from the clinical aspect. Studies suggest that further analysis of MTSS1 expression or inactivation in tissue samples and its association with different human malignancies will define a novel candidate to be used as a marker of primary tumours or metastasis.⁸

In an attempt to clarify the situation, we sought to determine the relevance of MTSS1 in breast cancer. Here, we provide new insights into the biological functions of MTSS1 and its role in breast cancer. In the present study, we examined MTSS1 expression a cohort of human breast cancer patient specimens and demonstrated an inverse correlation between MTSS1 and patient prognosis and survival. Importantly, we further analysed MTSS1 through a series of expression and inactivation studies to clarify MTSS1 function in breast cancer cells.

Section snippets

Cell lines and culture

All cell lines used in this study were obtained from the European Collection for Animal Cell Culture (ECACC, Porton Down, Salisbury, UK), unless otherwise stated. Cells were routinely cultured with Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% foetal calf serum, penicillin and streptomycin (Gibco BRC, Paisley, Scotland).

This study used human breast cancer cells (MDA-MB-157, MDA-MB-231, MDA-MB-435s, MDA-MB-436, MDA-MB-453, MCF-7, BT474, BT549 and ZR-751), human colorectal cancer

MTSS1 mRNA expression in human cell lines

A variety of 21 human normal and cancer cell lines were examined for the presence of MTSS1 through RT-PCR (Fig. 1A) and Quantitative-PCR (Fig. 1B). Of the nine human breast cancer cells examined, MTSS1 was most strongly expressed in the BT474 and MCF-7 cell lines, which are known for having low-invasive potential.²⁰ The MDA-MB-157, MDA-MB-231, MDA-MB-435s, MDA-MB-436 and ZR-751 breast cancer cell lines were negative for MTSS1 transcript. The HRT-18 colon cancer cell line and the PLC/PRF/5 liver

Discussion

Although metastasis suppressor 1 (MTSS1) is suggested to be an exciting new metastasis suppressor; there is little information available on this factor. The knowledge on MTSS1 is particularly weak from a clinical aspect. Supported by a series of cellular function tests, the present study indicates for the first time that MTSS1 acts as a powerful inhibitor to the aggressiveness of breast cancer cells and has strong prognostic and survival relevance in patients with breast cancer.

Our initial

Conflict of interest statement

None declared.

Acknowledgements

We wish to thank Cancer Research Wales and the Albert Hung Foundation for funding this research, Dr. Anthony Douglas-Jones for his expert evaluation of histology slides, and Mr. Gareth Watkins for his technical assistance.

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A lack of MTSS1 expression showed tumour aggressiveness, but surprisingly, mainly MTSS1 intensity was correlated with a worse patient outcome. There was a significant correlation between higher MTSS1 intensity and an increased risk for lymph node metastasis (P = .027) and a significant increased risk for extracapsular growth (P = .016). Furthermore, disease-specific survival was worse in cases with higher MTSS1 intensity (P = .001).
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Alternagin-C, an alpha2beta1 integrin ligand, attenuates collagen-based adhesion, stimulating the metastasis suppressor 1 expression in triple-negative breast tumor cells
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Triple-negative breast cancer has an aggressive clinical course and its treatment has been challenging due to high metastatic risk. Molecular targets have been sought to provide better strategies for this type of cancer. Integrins are cell adhesion receptors involved in tumor progression and α₂β₁ integrin, a collagen receptor, has a key role in breast metastasis. Disintegrins, a family of proteins from snake venoms, selectively block the function of integrin receptors. Alternagin-C (ALT-C), a disintegrin-like protein purified from Bothrops alternatus venom, binds to α₂β₁ integrin, attenuating inflammation and angiogenesis, and decreasing metalloprotease levels in the tumor microenvironment, which suggests anti-metastatic effects. However, its mechanisms of action in metastatic tumor cells have not been fully explored. Here, we investigated ALT-C effects in a triple-negative breast cancer cell line (MDA-MB-231) to elucidate how α₂β₁ integrin affects cellular adhesion, migration and gene expression related to metastasis. We observed that ALT-C attenuated cell adhesion of MDA-MB-231 cells to collagen I. α₂ integrin subunit silencing in MDA-MB-231 cells did not inhibit cell adhesion and migration to collagen I, indicating that other integrins play a crucial role in cell motility for this cell line. ALT-C also stimulated the metastasis suppressor 1 (MTSS1) expression and decreased metalloproteases MMP9 and MMP2. Therefore, we suggest that ALT-C contributes to impair metastasis, preventing extracellular matrix degradation and tumor attachment to collagen I, increasing MTSS1. This study is the first to elucidate the anti-metastatic mechanism involving a disintegrin-like protein from snake venom targeting α₂β₁ integrin and stimulating a metastasis suppressor.
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^☆: Grant Support: Cancer Research Wales.

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Metastasis suppressor 1 (MTSS1) demonstrates prognostic value and anti-metastatic properties in breast cancer☆

Abstract

Introduction

Section snippets

Cell lines and culture

MTSS1 mRNA expression in human cell lines

Discussion

Conflict of interest statement

Acknowledgements

Metastasis suppressors genes in cancer

Int J Biochem Cell Biol

MIM, a potential metastasis suppressor gene in bladder cancer

Neoplasia

MIM-B, a putative metastasis suppressor protein, binds to actin and to protein tyrosine phosphatase delta

Biochem J

Cellular motility driven by assembly and disassembly of actin filaments

Cell

Rho-stimulated contractility contributes to the fibroblastic phenotype of Ras-transformed epithelial cells

Mol Biol Cell

An essential role for Rac in Ras transformation

Nature

Differential expression analysis of MIM (MTSS1) splice variants and a functional role of MIM in prostate cancer cell biology

Int J Oncol

The expression of metastasis suppressor MIM/MTSS1 is regulated by DNA methylation

Int J Cancer

Expression and regulation of MIM (missing in metastasis), a novel putative metastasis suppressor gene, and MIM-B, in bladder cancer cell lines

Cancer Lett

Involvement of Rac in actin cytoskeleton rearrangements induced by MIM-B