Antitumoral actions of the anti-obesity drug orlistat (Xenical™) in breast cancer cells: Blockade of cell cycle progression, promotion of apoptotic cell death and PEA3-mediated transcriptional repression of Her2/neu (erbB-2) oncogene

J. A. Menendez, L. Vellon, Ruth Lupu

Research output: Contribution to journalArticle

110 Citations (Scopus)

Abstract

Background: Orlistat (Xenical™), a US Food and Drug Administration (FDA)-approved drug for bodyweight loss, has recently been demonstrated to exhibit antitumor properties towards prostate cancer cells by virtue of its ability to block the lipogenic activity of fatty acid synthase (FAS). FAS (oncogenic antigen-519) is up-regulated in about 50% of breast cancers, is an indicator of poor prognosis, and has recently been functionally associated with the Her2/neu (erbB-2) oncogene. Materials and methods: We assessed the antitumoral effects of orlistat against the human breast cancer cell line SK-Br3, an in vitro paradigm of FAS and Her2/neu overexpression in breast cancer. Results: Cell cycle analyses revealed that micromolar concentrations of orlistat induced, in a time- and dose-dependent manner, significant changes in the distribution of cell populations including a complete loss of G2-M phase, S-phase accumulation and a concomitant increase in the emerging sub-G1 (apoptotic) cells. Poly (ADP-ribose) polymerase (PARP) cleavage, an early event required for cells committed to apoptosis, was more predominant in orlistat-treated G1 phase cells. When we characterized signaling molecules participating in the cellular events following orlistat-induced inhibition of FAS activity and preceded inhibition of breast cancer cell proliferation, a dramatic down-regulation of Her2/neu-coded p185Her2/neu oncoprotein was found in orlistat-treated SK-Br3 cells (>90% reduction). Interestingly, a significant accumulation of the DNA-binding protein PEA3, a member of the Ets transcription factor family that specifically targets a PEA3-binding motif present on the Her2/neu gene promoter and down-regulates its activity, was observed in orlistat-treated SK-Br3 cells. When a Luciferase reporter gene driven by the Her2/neu promoter was transiently transfected in SK-Br3 cells, orlistat exposure was found to dramatically repress the promoter activity of Her2/neu gene, whereas a Her2/neu promoter bearing a mutated binding DNA sequence was not subject to negative regulation by orlistat, thus demonstrating that the intact PEA3 binding site on the Her2/neu promoter is required for the orlistat-induced transcriptional repression of Her2/neu overexpression. RNA interference (RNAi)-mediated silencing of FAS gene expression similarly repressed Her2/neu gene expression in a PEA3-dependent manner, thus ruling out a role for non-FAS orlistat-mediated effects. When the combination of orlistat and the anti-Her2/neu antibody trastuzumab (Herceptin™) in either concurrent (orlistat + trastuzumab) or sequential (orlistat → trastuzumab; trastuzumab → orlistat) schedules was tested for synergism, addition or antagonism using the combination index (CI) method of Chou-Talalay, co-exposure of orlistat and trastuzumab demonstrated strong synergistic effects (CI10-90 =0.110-0.847), whereas sequential exposure to orlistat followed by trastuzumab (CI10-90=0.380-1.210) and trastuzumab followed by orlistat (CI10-90=0.605-1.278) mainly showed additive or antagonistic interactions. Indeed, orlistat-induced FAS inhibition synergistically promoted apoptotic cell death when concurrently combined with trastuzumab as determined by an ELISA for histone-associated DNA fragments. Importantly, the degree of FAS expression in a panel of human breast cancer cell lines was predictive of sensitivity to orlistat-induced anti-proliferative effects as determined by a MTT-based characterization of metabolically viable breast cancer cells. Moreover, hypersensitivity to orlistat-induced cytotoxicity was observed in MCF-7 breast cancer cells engineered to overexpress Her2/neu (MCF-7/Her2-18 cells), which exhibit a significant up-regulation of FAS expression and activity. Conclusions: These findings reveal that the development of more potent and/or bioavailable orlistat's variants targeting the lipogenic activity of FAS may open a novel therapeutic avenue for treating Her2/ neu-overexpressing breast carcinomas.

Original languageEnglish (US)
Pages (from-to)1253-1267
Number of pages15
JournalAnnals of Oncology
Volume16
Issue number8
DOIs
StatePublished - Aug 2005
Externally publishedYes

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Anti-Obesity Agents
Oncogenes
Cell Cycle
Cell Death
Breast Neoplasms
Fatty Acid Synthases
erbB-2 Genes
orlistat

Keywords

  • Breast cancer
  • Fatty acid synthase
  • Her2/neu
  • Orlistat
  • PEA3
  • Trastuzumab

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

@article{3bbec87c35254fd9b65cbfdad6b37dcf,
title = "Antitumoral actions of the anti-obesity drug orlistat (Xenical™) in breast cancer cells: Blockade of cell cycle progression, promotion of apoptotic cell death and PEA3-mediated transcriptional repression of Her2/neu (erbB-2) oncogene",
abstract = "Background: Orlistat (Xenical™), a US Food and Drug Administration (FDA)-approved drug for bodyweight loss, has recently been demonstrated to exhibit antitumor properties towards prostate cancer cells by virtue of its ability to block the lipogenic activity of fatty acid synthase (FAS). FAS (oncogenic antigen-519) is up-regulated in about 50{\%} of breast cancers, is an indicator of poor prognosis, and has recently been functionally associated with the Her2/neu (erbB-2) oncogene. Materials and methods: We assessed the antitumoral effects of orlistat against the human breast cancer cell line SK-Br3, an in vitro paradigm of FAS and Her2/neu overexpression in breast cancer. Results: Cell cycle analyses revealed that micromolar concentrations of orlistat induced, in a time- and dose-dependent manner, significant changes in the distribution of cell populations including a complete loss of G2-M phase, S-phase accumulation and a concomitant increase in the emerging sub-G1 (apoptotic) cells. Poly (ADP-ribose) polymerase (PARP) cleavage, an early event required for cells committed to apoptosis, was more predominant in orlistat-treated G1 phase cells. When we characterized signaling molecules participating in the cellular events following orlistat-induced inhibition of FAS activity and preceded inhibition of breast cancer cell proliferation, a dramatic down-regulation of Her2/neu-coded p185Her2/neu oncoprotein was found in orlistat-treated SK-Br3 cells (>90{\%} reduction). Interestingly, a significant accumulation of the DNA-binding protein PEA3, a member of the Ets transcription factor family that specifically targets a PEA3-binding motif present on the Her2/neu gene promoter and down-regulates its activity, was observed in orlistat-treated SK-Br3 cells. When a Luciferase reporter gene driven by the Her2/neu promoter was transiently transfected in SK-Br3 cells, orlistat exposure was found to dramatically repress the promoter activity of Her2/neu gene, whereas a Her2/neu promoter bearing a mutated binding DNA sequence was not subject to negative regulation by orlistat, thus demonstrating that the intact PEA3 binding site on the Her2/neu promoter is required for the orlistat-induced transcriptional repression of Her2/neu overexpression. RNA interference (RNAi)-mediated silencing of FAS gene expression similarly repressed Her2/neu gene expression in a PEA3-dependent manner, thus ruling out a role for non-FAS orlistat-mediated effects. When the combination of orlistat and the anti-Her2/neu antibody trastuzumab (Herceptin™) in either concurrent (orlistat + trastuzumab) or sequential (orlistat → trastuzumab; trastuzumab → orlistat) schedules was tested for synergism, addition or antagonism using the combination index (CI) method of Chou-Talalay, co-exposure of orlistat and trastuzumab demonstrated strong synergistic effects (CI10-90 =0.110-0.847), whereas sequential exposure to orlistat followed by trastuzumab (CI10-90=0.380-1.210) and trastuzumab followed by orlistat (CI10-90=0.605-1.278) mainly showed additive or antagonistic interactions. Indeed, orlistat-induced FAS inhibition synergistically promoted apoptotic cell death when concurrently combined with trastuzumab as determined by an ELISA for histone-associated DNA fragments. Importantly, the degree of FAS expression in a panel of human breast cancer cell lines was predictive of sensitivity to orlistat-induced anti-proliferative effects as determined by a MTT-based characterization of metabolically viable breast cancer cells. Moreover, hypersensitivity to orlistat-induced cytotoxicity was observed in MCF-7 breast cancer cells engineered to overexpress Her2/neu (MCF-7/Her2-18 cells), which exhibit a significant up-regulation of FAS expression and activity. Conclusions: These findings reveal that the development of more potent and/or bioavailable orlistat's variants targeting the lipogenic activity of FAS may open a novel therapeutic avenue for treating Her2/ neu-overexpressing breast carcinomas.",
keywords = "Breast cancer, Fatty acid synthase, Her2/neu, Orlistat, PEA3, Trastuzumab",
author = "Menendez, {J. A.} and L. Vellon and Ruth Lupu",
year = "2005",
month = "8",
doi = "10.1093/annonc/mdi239",
language = "English (US)",
volume = "16",
pages = "1253--1267",
journal = "Annals of Oncology",
issn = "0923-7534",
publisher = "Oxford University Press",
number = "8",

}

TY - JOUR

T1 - Antitumoral actions of the anti-obesity drug orlistat (Xenical™) in breast cancer cells

T2 - Blockade of cell cycle progression, promotion of apoptotic cell death and PEA3-mediated transcriptional repression of Her2/neu (erbB-2) oncogene

AU - Menendez, J. A.

AU - Vellon, L.

AU - Lupu, Ruth

PY - 2005/8

Y1 - 2005/8

N2 - Background: Orlistat (Xenical™), a US Food and Drug Administration (FDA)-approved drug for bodyweight loss, has recently been demonstrated to exhibit antitumor properties towards prostate cancer cells by virtue of its ability to block the lipogenic activity of fatty acid synthase (FAS). FAS (oncogenic antigen-519) is up-regulated in about 50% of breast cancers, is an indicator of poor prognosis, and has recently been functionally associated with the Her2/neu (erbB-2) oncogene. Materials and methods: We assessed the antitumoral effects of orlistat against the human breast cancer cell line SK-Br3, an in vitro paradigm of FAS and Her2/neu overexpression in breast cancer. Results: Cell cycle analyses revealed that micromolar concentrations of orlistat induced, in a time- and dose-dependent manner, significant changes in the distribution of cell populations including a complete loss of G2-M phase, S-phase accumulation and a concomitant increase in the emerging sub-G1 (apoptotic) cells. Poly (ADP-ribose) polymerase (PARP) cleavage, an early event required for cells committed to apoptosis, was more predominant in orlistat-treated G1 phase cells. When we characterized signaling molecules participating in the cellular events following orlistat-induced inhibition of FAS activity and preceded inhibition of breast cancer cell proliferation, a dramatic down-regulation of Her2/neu-coded p185Her2/neu oncoprotein was found in orlistat-treated SK-Br3 cells (>90% reduction). Interestingly, a significant accumulation of the DNA-binding protein PEA3, a member of the Ets transcription factor family that specifically targets a PEA3-binding motif present on the Her2/neu gene promoter and down-regulates its activity, was observed in orlistat-treated SK-Br3 cells. When a Luciferase reporter gene driven by the Her2/neu promoter was transiently transfected in SK-Br3 cells, orlistat exposure was found to dramatically repress the promoter activity of Her2/neu gene, whereas a Her2/neu promoter bearing a mutated binding DNA sequence was not subject to negative regulation by orlistat, thus demonstrating that the intact PEA3 binding site on the Her2/neu promoter is required for the orlistat-induced transcriptional repression of Her2/neu overexpression. RNA interference (RNAi)-mediated silencing of FAS gene expression similarly repressed Her2/neu gene expression in a PEA3-dependent manner, thus ruling out a role for non-FAS orlistat-mediated effects. When the combination of orlistat and the anti-Her2/neu antibody trastuzumab (Herceptin™) in either concurrent (orlistat + trastuzumab) or sequential (orlistat → trastuzumab; trastuzumab → orlistat) schedules was tested for synergism, addition or antagonism using the combination index (CI) method of Chou-Talalay, co-exposure of orlistat and trastuzumab demonstrated strong synergistic effects (CI10-90 =0.110-0.847), whereas sequential exposure to orlistat followed by trastuzumab (CI10-90=0.380-1.210) and trastuzumab followed by orlistat (CI10-90=0.605-1.278) mainly showed additive or antagonistic interactions. Indeed, orlistat-induced FAS inhibition synergistically promoted apoptotic cell death when concurrently combined with trastuzumab as determined by an ELISA for histone-associated DNA fragments. Importantly, the degree of FAS expression in a panel of human breast cancer cell lines was predictive of sensitivity to orlistat-induced anti-proliferative effects as determined by a MTT-based characterization of metabolically viable breast cancer cells. Moreover, hypersensitivity to orlistat-induced cytotoxicity was observed in MCF-7 breast cancer cells engineered to overexpress Her2/neu (MCF-7/Her2-18 cells), which exhibit a significant up-regulation of FAS expression and activity. Conclusions: These findings reveal that the development of more potent and/or bioavailable orlistat's variants targeting the lipogenic activity of FAS may open a novel therapeutic avenue for treating Her2/ neu-overexpressing breast carcinomas.

AB - Background: Orlistat (Xenical™), a US Food and Drug Administration (FDA)-approved drug for bodyweight loss, has recently been demonstrated to exhibit antitumor properties towards prostate cancer cells by virtue of its ability to block the lipogenic activity of fatty acid synthase (FAS). FAS (oncogenic antigen-519) is up-regulated in about 50% of breast cancers, is an indicator of poor prognosis, and has recently been functionally associated with the Her2/neu (erbB-2) oncogene. Materials and methods: We assessed the antitumoral effects of orlistat against the human breast cancer cell line SK-Br3, an in vitro paradigm of FAS and Her2/neu overexpression in breast cancer. Results: Cell cycle analyses revealed that micromolar concentrations of orlistat induced, in a time- and dose-dependent manner, significant changes in the distribution of cell populations including a complete loss of G2-M phase, S-phase accumulation and a concomitant increase in the emerging sub-G1 (apoptotic) cells. Poly (ADP-ribose) polymerase (PARP) cleavage, an early event required for cells committed to apoptosis, was more predominant in orlistat-treated G1 phase cells. When we characterized signaling molecules participating in the cellular events following orlistat-induced inhibition of FAS activity and preceded inhibition of breast cancer cell proliferation, a dramatic down-regulation of Her2/neu-coded p185Her2/neu oncoprotein was found in orlistat-treated SK-Br3 cells (>90% reduction). Interestingly, a significant accumulation of the DNA-binding protein PEA3, a member of the Ets transcription factor family that specifically targets a PEA3-binding motif present on the Her2/neu gene promoter and down-regulates its activity, was observed in orlistat-treated SK-Br3 cells. When a Luciferase reporter gene driven by the Her2/neu promoter was transiently transfected in SK-Br3 cells, orlistat exposure was found to dramatically repress the promoter activity of Her2/neu gene, whereas a Her2/neu promoter bearing a mutated binding DNA sequence was not subject to negative regulation by orlistat, thus demonstrating that the intact PEA3 binding site on the Her2/neu promoter is required for the orlistat-induced transcriptional repression of Her2/neu overexpression. RNA interference (RNAi)-mediated silencing of FAS gene expression similarly repressed Her2/neu gene expression in a PEA3-dependent manner, thus ruling out a role for non-FAS orlistat-mediated effects. When the combination of orlistat and the anti-Her2/neu antibody trastuzumab (Herceptin™) in either concurrent (orlistat + trastuzumab) or sequential (orlistat → trastuzumab; trastuzumab → orlistat) schedules was tested for synergism, addition or antagonism using the combination index (CI) method of Chou-Talalay, co-exposure of orlistat and trastuzumab demonstrated strong synergistic effects (CI10-90 =0.110-0.847), whereas sequential exposure to orlistat followed by trastuzumab (CI10-90=0.380-1.210) and trastuzumab followed by orlistat (CI10-90=0.605-1.278) mainly showed additive or antagonistic interactions. Indeed, orlistat-induced FAS inhibition synergistically promoted apoptotic cell death when concurrently combined with trastuzumab as determined by an ELISA for histone-associated DNA fragments. Importantly, the degree of FAS expression in a panel of human breast cancer cell lines was predictive of sensitivity to orlistat-induced anti-proliferative effects as determined by a MTT-based characterization of metabolically viable breast cancer cells. Moreover, hypersensitivity to orlistat-induced cytotoxicity was observed in MCF-7 breast cancer cells engineered to overexpress Her2/neu (MCF-7/Her2-18 cells), which exhibit a significant up-regulation of FAS expression and activity. Conclusions: These findings reveal that the development of more potent and/or bioavailable orlistat's variants targeting the lipogenic activity of FAS may open a novel therapeutic avenue for treating Her2/ neu-overexpressing breast carcinomas.

KW - Breast cancer

KW - Fatty acid synthase

KW - Her2/neu

KW - Orlistat

KW - PEA3

KW - Trastuzumab

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U2 - 10.1093/annonc/mdi239

DO - 10.1093/annonc/mdi239

M3 - Article

C2 - 15870086

AN - SCOPUS:23044480300

VL - 16

SP - 1253

EP - 1267

JO - Annals of Oncology

JF - Annals of Oncology

SN - 0923-7534

IS - 8

ER -