Activating PTEN Tumor Suppressor Expression with the CRISPR/dCas9 System

Colette Moses, Fiona Nugent, Charlene Babra Waryah, Benjamin Garcia-Bloj, Alan R. Harvey, Pilar Blancafort

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

PTEN expression is lost in many cancers, and even small changes in PTEN activity affect susceptibility and prognosis in a range of highly aggressive malignancies, such as melanoma and triple-negative breast cancer (TNBC). Loss of PTEN expression occurs via multiple mechanisms, including mutation, transcriptional repression and epigenetic silencing. Transcriptional repression of PTEN contributes to resistance to inhibitors used in the clinic, such as B-Raf inhibitors in BRAF mutant melanoma. We aimed to activate PTEN expression using the CRISPR system, specifically dead (d) Cas9 fused to the transactivator VP64-p65-Rta (VPR). dCas9-VPR was directed to the PTEN proximal promoter by single-guide RNAs (sgRNAs), in cancer cells that exhibited low levels of PTEN expression. The dCas9-VPR system increased PTEN expression in melanoma and TNBC cell lines, without transcriptional regulation at predicted off-target sgRNA binding sites. PTEN activation significantly repressed downstream oncogenic pathways, including AKT, mTOR, and MAPK signaling. BRAF V600E mutant melanoma cells transduced with dCas9-VPR displayed reduced migration, as well as diminished colony formation in the presence of B-Raf inhibitors, PI3K/mTOR inhibitors, and with combined PI3K/mTOR and B-Raf inhibition. CRISPR-mediated targeted activation of PTEN may provide an alternative therapeutic approach for highly aggressive cancers that are refractory to current treatments.

Original languageEnglish
Pages (from-to)287-300
Number of pages14
JournalMolecular Therapy - Nucleic Acids
Volume14
DOIs
Publication statusPublished - 1 Mar 2019

Fingerprint

Clustered Regularly Interspaced Short Palindromic Repeats
Melanoma
Triple Negative Breast Neoplasms
Phosphatidylinositol 3-Kinases
Epigenetic Repression
Neoplasms
Guide RNA
Trans-Activators
Binding Sites
Cell Line
Mutation

Cite this

@article{b4b069e082364388a9bd292f20d021e7,
title = "Activating PTEN Tumor Suppressor Expression with the CRISPR/dCas9 System",
abstract = "PTEN expression is lost in many cancers, and even small changes in PTEN activity affect susceptibility and prognosis in a range of highly aggressive malignancies, such as melanoma and triple-negative breast cancer (TNBC). Loss of PTEN expression occurs via multiple mechanisms, including mutation, transcriptional repression and epigenetic silencing. Transcriptional repression of PTEN contributes to resistance to inhibitors used in the clinic, such as B-Raf inhibitors in BRAF mutant melanoma. We aimed to activate PTEN expression using the CRISPR system, specifically dead (d) Cas9 fused to the transactivator VP64-p65-Rta (VPR). dCas9-VPR was directed to the PTEN proximal promoter by single-guide RNAs (sgRNAs), in cancer cells that exhibited low levels of PTEN expression. The dCas9-VPR system increased PTEN expression in melanoma and TNBC cell lines, without transcriptional regulation at predicted off-target sgRNA binding sites. PTEN activation significantly repressed downstream oncogenic pathways, including AKT, mTOR, and MAPK signaling. BRAF V600E mutant melanoma cells transduced with dCas9-VPR displayed reduced migration, as well as diminished colony formation in the presence of B-Raf inhibitors, PI3K/mTOR inhibitors, and with combined PI3K/mTOR and B-Raf inhibition. CRISPR-mediated targeted activation of PTEN may provide an alternative therapeutic approach for highly aggressive cancers that are refractory to current treatments.",
keywords = "breast neoplasms, CRISPR/Cas systems, human PTEN protein, melanoma, tumor suppressor genes",
author = "Colette Moses and Fiona Nugent and Waryah, {Charlene Babra} and Benjamin Garcia-Bloj and Harvey, {Alan R.} and Pilar Blancafort",
year = "2019",
month = "3",
day = "1",
doi = "10.1016/j.omtn.2018.12.003",
language = "English",
volume = "14",
pages = "287--300",
journal = "Molecular Therapy-Nucleic Acids",
issn = "2162-2531",
publisher = "Nature Publishing Group",

}

Activating PTEN Tumor Suppressor Expression with the CRISPR/dCas9 System. / Moses, Colette; Nugent, Fiona; Waryah, Charlene Babra; Garcia-Bloj, Benjamin; Harvey, Alan R.; Blancafort, Pilar.

In: Molecular Therapy - Nucleic Acids, Vol. 14, 01.03.2019, p. 287-300.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Activating PTEN Tumor Suppressor Expression with the CRISPR/dCas9 System

AU - Moses, Colette

AU - Nugent, Fiona

AU - Waryah, Charlene Babra

AU - Garcia-Bloj, Benjamin

AU - Harvey, Alan R.

AU - Blancafort, Pilar

PY - 2019/3/1

Y1 - 2019/3/1

N2 - PTEN expression is lost in many cancers, and even small changes in PTEN activity affect susceptibility and prognosis in a range of highly aggressive malignancies, such as melanoma and triple-negative breast cancer (TNBC). Loss of PTEN expression occurs via multiple mechanisms, including mutation, transcriptional repression and epigenetic silencing. Transcriptional repression of PTEN contributes to resistance to inhibitors used in the clinic, such as B-Raf inhibitors in BRAF mutant melanoma. We aimed to activate PTEN expression using the CRISPR system, specifically dead (d) Cas9 fused to the transactivator VP64-p65-Rta (VPR). dCas9-VPR was directed to the PTEN proximal promoter by single-guide RNAs (sgRNAs), in cancer cells that exhibited low levels of PTEN expression. The dCas9-VPR system increased PTEN expression in melanoma and TNBC cell lines, without transcriptional regulation at predicted off-target sgRNA binding sites. PTEN activation significantly repressed downstream oncogenic pathways, including AKT, mTOR, and MAPK signaling. BRAF V600E mutant melanoma cells transduced with dCas9-VPR displayed reduced migration, as well as diminished colony formation in the presence of B-Raf inhibitors, PI3K/mTOR inhibitors, and with combined PI3K/mTOR and B-Raf inhibition. CRISPR-mediated targeted activation of PTEN may provide an alternative therapeutic approach for highly aggressive cancers that are refractory to current treatments.

AB - PTEN expression is lost in many cancers, and even small changes in PTEN activity affect susceptibility and prognosis in a range of highly aggressive malignancies, such as melanoma and triple-negative breast cancer (TNBC). Loss of PTEN expression occurs via multiple mechanisms, including mutation, transcriptional repression and epigenetic silencing. Transcriptional repression of PTEN contributes to resistance to inhibitors used in the clinic, such as B-Raf inhibitors in BRAF mutant melanoma. We aimed to activate PTEN expression using the CRISPR system, specifically dead (d) Cas9 fused to the transactivator VP64-p65-Rta (VPR). dCas9-VPR was directed to the PTEN proximal promoter by single-guide RNAs (sgRNAs), in cancer cells that exhibited low levels of PTEN expression. The dCas9-VPR system increased PTEN expression in melanoma and TNBC cell lines, without transcriptional regulation at predicted off-target sgRNA binding sites. PTEN activation significantly repressed downstream oncogenic pathways, including AKT, mTOR, and MAPK signaling. BRAF V600E mutant melanoma cells transduced with dCas9-VPR displayed reduced migration, as well as diminished colony formation in the presence of B-Raf inhibitors, PI3K/mTOR inhibitors, and with combined PI3K/mTOR and B-Raf inhibition. CRISPR-mediated targeted activation of PTEN may provide an alternative therapeutic approach for highly aggressive cancers that are refractory to current treatments.

KW - breast neoplasms

KW - CRISPR/Cas systems

KW - human PTEN protein

KW - melanoma

KW - tumor suppressor genes

UR - http://www.scopus.com/inward/record.url?scp=85059850212&partnerID=8YFLogxK

U2 - 10.1016/j.omtn.2018.12.003

DO - 10.1016/j.omtn.2018.12.003

M3 - Article

VL - 14

SP - 287

EP - 300

JO - Molecular Therapy-Nucleic Acids

JF - Molecular Therapy-Nucleic Acids

SN - 2162-2531

ER -