Identifying therapies to combat epithelial mesenchymal plasticity-associated chemoresistance to conventional breast cancer therapies using an shrna library screen

Sugandha Bhatia; Tony Blick; Cletus Pinto; Mark Waltham; James Monkman; Ekaterina Ivanova; Pamela M. Pollock; Shivashankar H. Nagaraj; Adrian P. Wiegmans; Izhak Haviv; Kaylene J. Simpson; Erik W. Thompson

doi:10.3390/cancers12051123

Identifying therapies to combat epithelial mesenchymal plasticity-associated chemoresistance to conventional breast cancer therapies using an shrna library screen

Sugandha Bhatia
, Tony Blick
, Cletus Pinto
, Mark Waltham
, James Monkman
, Ekaterina Ivanova
, Pamela M. Pollock
, Shivashankar H. Nagaraj
, Adrian P. Wiegmans
, Izhak Haviv
, Kaylene J. Simpson
, Erik W. Thompson

Research output: Contribution to journal › Article › peer-review

7 Scopus citations

Abstract

Background: Breast cancer (BC) is a heterogeneous disease for which the commonly used chemotherapeutic agents primarily include the anthracyclines (doxorubicin, epirubicin), microtubule inhibitors (paclitaxel, docetaxel, eribulin), and alkylating agents (cyclophosphamide). While these drugs can be highly effective, metastatic tumours are frequently refractory to treatment or become resistant upon tumour relapse. Methods: We undertook a cell polarity/epithelial mesenchymal plasticity (EMP)-enriched short hairpin RNA (shRNA) screen in MDA-MB-468 breast cancer cells to identify factors underpinning heterogeneous responses to three chemotherapeutic agents used clinically in breast cancer: Doxorubicin, docetaxel, and eribulin. shRNA-transduced cells were treated for 6 weeks with the EC₁₀ of each drug, and shRNA representation assessed by deep sequencing. We first identified candidate genes with depleted shRNA, implying that their silencing could promote a response. Using the Broad Institute’s Connectivity Map (CMap), we identified partner inhibitors targeting the identified gene families that may induce cell death in combination with doxorubicin, and tested them with all three drug treatments. Results: In total, 259 shRNAs were depleted with doxorubicin treatment (at p < 0.01), 66 with docetaxel, and 25 with eribulin. Twenty-four depleted hairpins overlapped between doxorubicin and docetaxel, and shRNAs for TGFB2, RUNX1, CCDC80, and HYOU1 were depleted across all the three drug treatments. Inhibitors of MDM/TP53, TGFBR, and FGFR were identified by CMap as the top pharmaceutical perturbagens and we validated the combinatorial benefits of the TGFBR inhibitor (SB525334) and MDM inhibitor (RITA) with doxorubicin treatment, and also observed synergy between the inhibitor SB525334 and eribulin in MDA-MB-468 cells. Conclusions: Taken together, a cell polarity/EMP-enriched shRNA library screen identified relevant gene products that could be targeted alongside current chemotherapeutic agents for the treatment of invasive BC.

Original language	English
Article number	1123
Journal	Cancers
Volume	12
Issue number	5
DOIs	https://doi.org/10.3390/cancers12051123
State	Published - 30 Apr 2020
Externally published	Yes

Bibliographical note

Publisher Copyright:
© 2020 by the authors. Licensee MDPI, Basel, Switzerland.

Funding

Acknowledgments: This work E.W.T. and I.H. were supported in part by US-DOD IDEA grant BC 084667 and a National Breast Cancer Foundation National Collaborative Research Program grant (CG-10-04) to E.W.T. The Translational Research Institute (TRI) receives support from the Australian Government. We would like to thank the core facility teams based at TRI for their technical assistance and Aleksandra Rajapakse for the validation of an experimental assay. The Victorian Centre for Functional Genomics (K.J.S.) is funded by the Australian Cancer Research Foundation (ACRF), the Australian Phenomics Network (APN) through funding from the Australian Government’s National Collaborative Research Infrastructure Strategy (NCRIS) program, the Peter MacCallum Cancer Centre Foundation and the University of Melbourne Research Collaborative Infrastructure Program. During the course of the study, S.B. was supported by a QUTPRA scholarship. This work E.W.T. and I.H. were supported in part by US-DOD IDEA grant BC 084667 and a National Breast Cancer Foundation National Collaborative Research Program grant (CG-10-04) to E.W.T. The Translational Research Institute (TRI) receives support from the Australian Government. We would like to thank the core facility teams based at TRI for their technical assistance and Aleksandra Rajapakse for the validation of an experimental assay. The Victorian Centre for Functional Genomics (K.J.S.) is funded by the Australian Cancer Research Foundation (ACRF), the Australian Phenomics Network (APN) through funding from the Australian Government?s National Collaborative Research Infrastructure Strategy (NCRIS) program, the Peter MacCallum Cancer Centre Foundation and the University of Melbourne Research Collaborative Infrastructure Program. During the course of the study, S.B. was supported by a QUTPRA scholarship.

Funders	Funder number
Australian Government
Australian Phenomics Network
Peter MacCallum Cancer Centre Foundation
US-DOD IDEA	BC 084667
Australian Government
Australian Cancer Research Foundation
National Breast Cancer Foundation	CG-10-04
University of Melbourne
Peter Maccallum Cancer Centre

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

SDG 3 Good Health and Well-being

Keywords

Chemotherapy resistance
Combination chemotherapy
Docetaxel
Doxorubicin
Eribulin
FGFR
MDM
ShRNA library screening
TGFBR
TP53

Access to Document

10.3390/cancers12051123

Cite this

Bhatia, S., Blick, T., Pinto, C., Waltham, M., Monkman, J., Ivanova, E., Pollock, P. M., Nagaraj, S. H., Wiegmans, A. P., Haviv, I., Simpson, K. J., & Thompson, E. W. (2020). Identifying therapies to combat epithelial mesenchymal plasticity-associated chemoresistance to conventional breast cancer therapies using an shrna library screen. Cancers, 12(5), Article 1123. https://doi.org/10.3390/cancers12051123

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abstract = "Background: Breast cancer (BC) is a heterogeneous disease for which the commonly used chemotherapeutic agents primarily include the anthracyclines (doxorubicin, epirubicin), microtubule inhibitors (paclitaxel, docetaxel, eribulin), and alkylating agents (cyclophosphamide). While these drugs can be highly effective, metastatic tumours are frequently refractory to treatment or become resistant upon tumour relapse. Methods: We undertook a cell polarity/epithelial mesenchymal plasticity (EMP)-enriched short hairpin RNA (shRNA) screen in MDA-MB-468 breast cancer cells to identify factors underpinning heterogeneous responses to three chemotherapeutic agents used clinically in breast cancer: Doxorubicin, docetaxel, and eribulin. shRNA-transduced cells were treated for 6 weeks with the EC10 of each drug, and shRNA representation assessed by deep sequencing. We first identified candidate genes with depleted shRNA, implying that their silencing could promote a response. Using the Broad Institute{\textquoteright}s Connectivity Map (CMap), we identified partner inhibitors targeting the identified gene families that may induce cell death in combination with doxorubicin, and tested them with all three drug treatments. Results: In total, 259 shRNAs were depleted with doxorubicin treatment (at p < 0.01), 66 with docetaxel, and 25 with eribulin. Twenty-four depleted hairpins overlapped between doxorubicin and docetaxel, and shRNAs for TGFB2, RUNX1, CCDC80, and HYOU1 were depleted across all the three drug treatments. Inhibitors of MDM/TP53, TGFBR, and FGFR were identified by CMap as the top pharmaceutical perturbagens and we validated the combinatorial benefits of the TGFBR inhibitor (SB525334) and MDM inhibitor (RITA) with doxorubicin treatment, and also observed synergy between the inhibitor SB525334 and eribulin in MDA-MB-468 cells. Conclusions: Taken together, a cell polarity/EMP-enriched shRNA library screen identified relevant gene products that could be targeted alongside current chemotherapeutic agents for the treatment of invasive BC.",

keywords = "Chemotherapy resistance, Combination chemotherapy, Docetaxel, Doxorubicin, Eribulin, FGFR, MDM, ShRNA library screening, TGFBR, TP53",

author = "Sugandha Bhatia and Tony Blick and Cletus Pinto and Mark Waltham and James Monkman and Ekaterina Ivanova and Pollock, \{Pamela M.\} and Nagaraj, \{Shivashankar H.\} and Wiegmans, \{Adrian P.\} and Izhak Haviv and Simpson, \{Kaylene J.\} and Thompson, \{Erik W.\}",

note = "Publisher Copyright: {\textcopyright} 2020 by the authors. Licensee MDPI, Basel, Switzerland.",

year = "2020",

month = apr,

day = "30",

doi = "10.3390/cancers12051123",

language = "אנגלית",

volume = "12",

journal = "Cancers",

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Bhatia, S, Blick, T, Pinto, C, Waltham, M, Monkman, J, Ivanova, E, Pollock, PM, Nagaraj, SH, Wiegmans, AP, Haviv, I, Simpson, KJ & Thompson, EW 2020, 'Identifying therapies to combat epithelial mesenchymal plasticity-associated chemoresistance to conventional breast cancer therapies using an shrna library screen', Cancers, vol. 12, no. 5, 1123. https://doi.org/10.3390/cancers12051123

TY - JOUR

T1 - Identifying therapies to combat epithelial mesenchymal plasticity-associated chemoresistance to conventional breast cancer therapies using an shrna library screen

AU - Bhatia, Sugandha

AU - Blick, Tony

AU - Pinto, Cletus

AU - Waltham, Mark

AU - Monkman, James

AU - Ivanova, Ekaterina

AU - Pollock, Pamela M.

AU - Nagaraj, Shivashankar H.

AU - Wiegmans, Adrian P.

AU - Haviv, Izhak

AU - Simpson, Kaylene J.

AU - Thompson, Erik W.

PY - 2020/4/30

Y1 - 2020/4/30

N2 - Background: Breast cancer (BC) is a heterogeneous disease for which the commonly used chemotherapeutic agents primarily include the anthracyclines (doxorubicin, epirubicin), microtubule inhibitors (paclitaxel, docetaxel, eribulin), and alkylating agents (cyclophosphamide). While these drugs can be highly effective, metastatic tumours are frequently refractory to treatment or become resistant upon tumour relapse. Methods: We undertook a cell polarity/epithelial mesenchymal plasticity (EMP)-enriched short hairpin RNA (shRNA) screen in MDA-MB-468 breast cancer cells to identify factors underpinning heterogeneous responses to three chemotherapeutic agents used clinically in breast cancer: Doxorubicin, docetaxel, and eribulin. shRNA-transduced cells were treated for 6 weeks with the EC10 of each drug, and shRNA representation assessed by deep sequencing. We first identified candidate genes with depleted shRNA, implying that their silencing could promote a response. Using the Broad Institute’s Connectivity Map (CMap), we identified partner inhibitors targeting the identified gene families that may induce cell death in combination with doxorubicin, and tested them with all three drug treatments. Results: In total, 259 shRNAs were depleted with doxorubicin treatment (at p < 0.01), 66 with docetaxel, and 25 with eribulin. Twenty-four depleted hairpins overlapped between doxorubicin and docetaxel, and shRNAs for TGFB2, RUNX1, CCDC80, and HYOU1 were depleted across all the three drug treatments. Inhibitors of MDM/TP53, TGFBR, and FGFR were identified by CMap as the top pharmaceutical perturbagens and we validated the combinatorial benefits of the TGFBR inhibitor (SB525334) and MDM inhibitor (RITA) with doxorubicin treatment, and also observed synergy between the inhibitor SB525334 and eribulin in MDA-MB-468 cells. Conclusions: Taken together, a cell polarity/EMP-enriched shRNA library screen identified relevant gene products that could be targeted alongside current chemotherapeutic agents for the treatment of invasive BC.

AB - Background: Breast cancer (BC) is a heterogeneous disease for which the commonly used chemotherapeutic agents primarily include the anthracyclines (doxorubicin, epirubicin), microtubule inhibitors (paclitaxel, docetaxel, eribulin), and alkylating agents (cyclophosphamide). While these drugs can be highly effective, metastatic tumours are frequently refractory to treatment or become resistant upon tumour relapse. Methods: We undertook a cell polarity/epithelial mesenchymal plasticity (EMP)-enriched short hairpin RNA (shRNA) screen in MDA-MB-468 breast cancer cells to identify factors underpinning heterogeneous responses to three chemotherapeutic agents used clinically in breast cancer: Doxorubicin, docetaxel, and eribulin. shRNA-transduced cells were treated for 6 weeks with the EC10 of each drug, and shRNA representation assessed by deep sequencing. We first identified candidate genes with depleted shRNA, implying that their silencing could promote a response. Using the Broad Institute’s Connectivity Map (CMap), we identified partner inhibitors targeting the identified gene families that may induce cell death in combination with doxorubicin, and tested them with all three drug treatments. Results: In total, 259 shRNAs were depleted with doxorubicin treatment (at p < 0.01), 66 with docetaxel, and 25 with eribulin. Twenty-four depleted hairpins overlapped between doxorubicin and docetaxel, and shRNAs for TGFB2, RUNX1, CCDC80, and HYOU1 were depleted across all the three drug treatments. Inhibitors of MDM/TP53, TGFBR, and FGFR were identified by CMap as the top pharmaceutical perturbagens and we validated the combinatorial benefits of the TGFBR inhibitor (SB525334) and MDM inhibitor (RITA) with doxorubicin treatment, and also observed synergy between the inhibitor SB525334 and eribulin in MDA-MB-468 cells. Conclusions: Taken together, a cell polarity/EMP-enriched shRNA library screen identified relevant gene products that could be targeted alongside current chemotherapeutic agents for the treatment of invasive BC.

KW - Chemotherapy resistance

KW - Combination chemotherapy

KW - Docetaxel

KW - Doxorubicin

KW - Eribulin

KW - FGFR

KW - MDM

KW - ShRNA library screening

KW - TGFBR

KW - TP53

UR - https://www.scopus.com/pages/publications/85084974784

U2 - 10.3390/cancers12051123

DO - 10.3390/cancers12051123

M3 - ???researchoutput.researchoutputtypes.contributiontojournal.article???

C2 - 32365878

AN - SCOPUS:85084974784

SN - 2072-6694

VL - 12

JO - Cancers

JF - Cancers

IS - 5

M1 - 1123

ER -

Identifying therapies to combat epithelial mesenchymal plasticity-associated chemoresistance to conventional breast cancer therapies using an shrna library screen

Abstract

Bibliographical note

Funding

UN SDGs

Keywords

Access to Document

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