The monoamine oxidase-A inhibitor clorgyline promotes a mesenchymal-to-epithelial transition in the MDA-MB-231 breast cancer cell line

doi:10.1016/j.cellsig.2014.08.005

Cellular Signalling

Volume 26, Issue 12, December 2014, Pages 2621-2632

https://doi.org/10.1016/j.cellsig.2014.08.005 Get rights and content

Highlights

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Depression increases the risk of cancers, but the mechanism involved is unknown.
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The contribution of monoamine oxidase-A (MAO-A) to cancers remains ambiguous.
•
Inhibiting MAO-A in post-EMT breast cancer cells promotes metastatic behaviours.
•
MAO-A inhibitors should be avoided in cancer patients with co-morbid depression.

Abstract

Monoamine oxidase-A (MAO-A) dysfunction has been historically associated with depression. Recently, depression as well as altered MAO-A expression have both been associated with a poor prognosis in cancers, although the mechanism involved remains ambiguous. For example, MAO-A mRNA is repressed across cancers, yet MAO-A protein and levels of serotonin, a substrate of MAO-A implicated in depression, are paradoxically increased in malignancies, including breast cancer.

The effect of clorgyline (CLG), a selective inhibitor of MAO-A, on malignant behaviour, expression of transitional markers, and biochemical correlates was examined in two human breast carcinoma cell lines, i.e. the epithelial, oestrogen receptor (ER)-positive MCF-7 cell line and the post-EMT (mesenchymal), ER-negative MDA-MB-231 cell line.

CLG exerted little effect on malignant behaviour in MCF-7 cells, but inhibited proliferation and anchorage-independent growth, and increased invasiveness and active migration of MDA-MB-231 cells. CLG induced the expression of the mesenchymal marker vimentin in MCF-7 cells, but not in MDA-MB-231 cells. In contrast, CLG induced the epithelial protein marker E-cadherin in both cell lines, with a more robust effect in MDA-MB-231 cells (where a nuclear E-cadherin signal was also detected). This effect appears to be independent of any canonical Snai1-mediated regulation of E-cadherin mRNA expression. CLG interfered with the β-catenin/[phospho]GSK-3β complex as well as the E-cadherin/β-catenin complex in both cell lines cells, but, again, the effect was more robust in MDA-MB-231 cells. Parallel studies revealed a general lack of effect of CLG on the ER-negative, epithelial Au565 breast cancer cell line. Thus, any effect of CLG on metastatic behaviours appears to rely on the cell's EMT status rather than on the cell's ER status.

These data suggest that inactivation of MAO-A triggers a mesenchymal-to-epithelial transition in MDA-MB-231 cells via a non-canonical mechanism. This potentially implicates an MAO-A-sensitive step in advanced breast cancer and should be borne in mind when considering pharmacological treatment options for co-morbid depression in breast cancer patients.

Introduction

Serotonin, a neurotransmitter whose levels are often reduced during the course of clinical depression, has also been implicated in the regulation of breast epithelial physiology [1]. Epidemiological studies support an association between the risk of cancer development and the use of serotoninergic antidepressants, with the patient's hormone [oestrogen/progesterone] receptor status potentially contributing a modest influence [2], [3].

Depression has been associated historically with monoamine oxidase-A (MAO-A), the enzyme that degrades serotonin, and inhibitors of MAO-A were the first antidepressant drugs to be used clinically. Although inhibitors of MAO-A can induce severe side effects, which limit the use of these drugs in the clinic, they continue to be used when all other antidepressants fail to provide therapeutic effect. Alterations in MAO-A expression might represent a possible biomarker in cancers [4], yet the exact mechanism involved is not clear. Indeed, there is indication that the inhibition of MAO-A would be beneficial. For example, MAO-A activity is increased in experimental breast cancer [5] and MAO-A protein expression is increased in advanced stages of prostate cancer [6], while inhibition of MAO-A with clorgyline exerts anticancer properties towards cultured prostate cancer cells [7] and resveratrol, a potent inhibitor of MAO-A isolated from red grapes [8], induces MCF-7 breast cancer cell death [9]. Yet in apparent contradiction, MAO-A mRNA is down-regulated in 94% of cancers screened, including breast and prostate cancers [4], and epidemiological studies associate usage of antidepressants, including inhibitors of MAO-A, with an increased risk for cancer [10], [11]. Interestingly, we have demonstrated that overexpression of a catalytically dead MAO-A variant increases proliferation and de novo DNA synthesis in neuronal cells [12], which introduces the potential for a role for the MAO-A protein beyond simple substrate catabolism. This also introduces the possibility that active and inactive pools of MAO-A might exist, and that at any given time a cell's phenotype would be influenced by the predominant pool. This simple notion could certainly be contributing to the ambiguity in the cancer literature regarding risk associated with MAO-A protein expression and paradoxical substrate accumulation [13].

Epithelial-to-mesenchymal transition (EMT) during tumour progression involves loss of epithelial cell adhesion following a reduction in E-cadherin expression [14] that reflects a GSK-3-dependent, Snai1-mediated transcriptional regulation [15]. The oestrogen receptor (ER)-negative, mesenchymal-like MDA-MB-231 cells are a model of a post-EMT cells that are characterized as ‘E-cadherin-null’ as well as by reduced adhesion and increased cell mobility. In contrast, the ER-positive, epithelial-like MCF-7 cells are far less aggressive, express E-cadherin and are often contrasted experimentally with the MDA-MB-231 cell line [16].

We have examined the influence of clorgyline (CLG) — a selective inhibitor of MAO-A — on malignant/metastatic behaviour, on the expression of transitional markers, and on biochemical correlates in MDA-MB-231 and MCF-7 breast cancer cells. Our study strongly supports a role for MAO-A inhibition in triggering a mesenchymal-to-epithelial transition in the MDA-MB-231 breast cancer cell line. The absence of a similar pattern of effects in the ER-negative, epithelial Au565 breast cancer cell line suggests that the effect of MAO-A inhibition in this context is not reliant as much on the cell's ER status as it is on the cell's EMT status.

Section snippets

Reagents and antibodies

Serotonin, 5-bromo-2′-deoxyuridine (BrdU), and the BrdU and β-actin antibodies were purchased from Sigma-Aldrich Co. [¹⁴C]-Serotonin (NEC-225) was purchased from PerkinElmer Life Sciences. The MAO-A (H-70) antibody as well as the GSK-3β (phospho-Ser9 and total) and vimentin antibodies were purchased from Santa Cruz Biotechnology. The E-cadherin and β-catenin antibodies were purchased from Cell Signalling Technology. The AlexaFluor-594 and AlexaFluor-488 labelled donkey anti-mouse/anti-rabbit

MCF-7 and MDA-MB-231 cell lines have different innate MAO-A activities

MCF-7 cells have a significantly lower level of MAO-A catalytic activity than MDA-MB-231 cells (Fig. 1A). The dose-time course of CLG treatment (1 μM, 24 h) effectively inhibited MAO-A activity in MCF-7 cells [P < 0.001] and in MDA-MB-231 cells [P < 0.001] (Fig. 1B).

Inhibition of MAO-A regulates breast cancer cell proliferation

Treatment with CLG reduced MTT conversion in MCF-7 cells [P = 0.003] as well as in MDA-MB-231 cells [P = 0.029] (Fig. 1C). MTT conversion was increased by the overexpression of MAO-A in MCF-7 cells [P < 0.001], but not in MDA-MB-231 cultures [P =

Discussion

The high incidence of depression in patients with breast cancer — with rates of co-morbidity reaching almost 40% [27], [28], [29] — certainly implicates a role for monoaminergic dysfunction and for MAO-A given the historical association of both factors with experimental and clinical depressive phenotypes [13]. This is supported by the increase in MAO-A activity linked to malignancy in experimental breast cancer [5] and by the increase in the risk of breast cancer associated with antidepressants

Conclusions

While it is unclear whether manipulation of the MAO-A system (depression-related or otherwise) could be predisposing to breast cancer metastasis in the clinic, our observations certainly do support this troubling possibility. Comparing the broad range of effects of CLG on MDA-MB-231 (ER-negative, mesenchymal) cells to the limited effects of CLG on MCF-7 (ER-positive, epithelial) and Au-565 (ER-negative, epithelial) cells leads us to propose that inhibition of MAO-A on breast cancer progression

List of abbreviations

MAO-A

monoamine oxidase-A

CLG

clorgyline

EMT

epithelial-to-mesenchymal transition

ME(r)T

mesenchymal-to-epithelial (reverting) transition

GSK-3β

glycogen synthase kinase-3β

PBS

phosphate-buffered saline

MTT

3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide

BrdU

5-bromo-2′-deoxyuridine

SDS-PAGE

sodium dodecyl sulphate-polyacrylamide gel electrophoresis

Authors' contributions

Conceived and designed the experiments: DDM TSM KK JNKN AF. Performed the experiments: TSM KK JNKN KF PRP LT. Analyzed the data: DDM TSM. Contributed reagents/materials/analysis tools: DDM EL AF DHA. Wrote the paper: DDM TSM. Revised the manuscript for critical content: DDM TSM JNKN PRP AF EL DHA. All authors approved the final manuscript.

Competing interests

There are no competing interests.

Acknowledgements

Part of this work was presented at the 2010 Amine Oxidase Workshop (Edmonton, Alberta, Canada). This work was funded by grants from the Canadian Breast Cancer Foundation–Prairies/NWT Region and the Canada-Saskatchewan Western Economic Partnership Agreement (project# 000011361) (both to DDM). We also gratefully acknowledge funding in the form of a University of Saskatchewan (UofS) College of Graduate Studies and Research M.Sc. Scholarship (TSM), and by a UofS Graduate Teaching Fellowship and a

References (46)

J.M. Lizcano et al.
Biochem. Pharmacol.
(1991)
D.M. Peehl et al.
J. Urol.
(2008)
H. Zhao et al.
BMC Med. Genomics
(2009)
M. Yanez et al.
Biochem. Biophys. Res. Commun.
(2006)
J. Luo
Cancer Lett.
(2009)
J.R. Fann et al.
Gen. Hosp. Psychiatry
(2008)
S. Kishida et al.
J. Biol. Chem.
(1998)
M.V. Cespedes et al.
Am. J. Pathol.
(2010)
M. Salmi et al.
FEBS Lett.
(2011)
H.J. Moon et al.
J. Biol. Chem.
(2013)

V.P. Pai et al.

Breast Cancer Res.

(2009)

S. Bahl et al.

Psychother. Psychosom.

(2003)

M. Cotterchio et al.

Cancer Epidemiol. Biomarkers Prev.

(2003)

L.A. Rybaczyk et al.

BMC Genomics

(2008)

G. Filomeni et al.

Genes Nutr.

(2007)

R.B. Wallace et al.

Oncology

(1982)

M. Cotterchio et al.

Am. J. Epidemiol.

(2000)

Z. Wei et al.

J. Neural Transm.

(2012)

D.D. Mousseau et al.

Curr. Top. Med. Chem.

(2012)

G. Berx et al.

Hum. Mutat.

(1998)

R.E. Bachelder et al.

J. Cell Biol.

(2005)

M.J. Hendrix et al.

Breast Cancer Res.

(2000)

X. Cao et al.

J. Neurochem.

(2009)

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The monoamine oxidase-A inhibitor clorgyline promotes a mesenchymal-to-epithelial transition in the MDA-MB-231 breast cancer cell line

Highlights

Abstract

Introduction

Section snippets

Reagents and antibodies

MCF-7 and MDA-MB-231 cell lines have different innate MAO-A activities

Inhibition of MAO-A regulates breast cancer cell proliferation

Discussion

Conclusions

List of abbreviations

Authors' contributions

Competing interests

Acknowledgements

Biochem. Pharmacol.

J. Urol.

BMC Med. Genomics

Biochem. Biophys. Res. Commun.

Cancer Lett.

Gen. Hosp. Psychiatry

J. Biol. Chem.

Am. J. Pathol.

FEBS Lett.

J. Biol. Chem.

Breast Cancer Res.

Psychother. Psychosom.

Cancer Epidemiol. Biomarkers Prev.

BMC Genomics

Genes Nutr.

Oncology

Am. J. Epidemiol.

J. Neural Transm.

Curr. Top. Med. Chem.

Hum. Mutat.

J. Cell Biol.

Breast Cancer Res.

J. Neurochem.