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Differential expression and prognostic relevance of autophagy-related markers ATG4B, GABARAP, and LC3B in breast cancer

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Abstract

Purpose

Previous studies indicate that breast cancer molecular subtypes differ with respect to their dependency on autophagy, but our knowledge of the differential expression and prognostic significance of autophagy-related biomarkers in breast cancer is limited.

Methods

Immunohistochemistry (IHC) was performed on tissue microarrays from a large population of 3992 breast cancer patients divided into training and validation cohorts. Consensus staining scores were used to evaluate the expression levels of autophagy proteins LC3B, ATG4B, and GABARAP and determine the associations with clinicopathological variables and molecular biomarkers. Survival analyses were performed using the Kaplan–Meier function and Cox proportional hazards regression models.

Results

We found subtype-specific expression differences for ATG4B, with its expression lowest in basal-like breast cancer and highest in Luminal A, but there were no significant associations with patient prognosis. LC3B and GABARAP levels were highest in basal-like breast cancers, and high levels were associated with worse outcomes across all subtypes (DSS; GABARAP: HR 1.43, LC3B puncta: HR 1.43). High ATG4B levels were associated with ER, PR, and BCL2 positivity, while high LC3B and GABARAP levels were associated with ER, PR, and BCL2 negativity, as well as EGFR, HER2, HER3, CA-IX, PD-L1 positivity, and high Ki67 index (p < 0.05 for all associations). Exploratory multi-marker analysis indicated that the combination of ATG4B and GABARAP with LC3B could be useful for further stratifying patient outcomes.

Conclusions

ATG4B levels varied across breast cancer subtypes but did not show prognostic significance. High LC3B expression and high GABARAP expression were both associated with poor prognosis and with clinicopathological characteristics of aggressive disease phenotypes in all breast cancer subtypes.

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Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Abbreviations

AIC:

Akaike information criterion

ATG:

Autophagy-related

ATG4B:

Autophagy-related 4B

BCL2:

B-cell lymphoma 2

CA-IX:

Carbonic anhydrase 9

DSS:

Disease-specific survival

EGFR:

Epidermal growth factor receptor

ER:

Estrogen receptor

FDA:

Food and Drug Administration

GABARAP:

GABA type A receptor-associated protein

GATE-16:

Golgi-associated ATPase enhancer of 16 kDa

GABARAPL2:

GABA type A receptor-associated protein like 2

HER2:

Human epidermal growth factor receptor 2

HER3:

Human epidermal growth factor receptor 3

HR:

Hazard ratio

IHC:

Immunohistochemistry

LC3B:

Microtubule associated protein 1 light chain 3 beta

OS:

Overall survival

PD-L1:

Programmed death-ligand 1

PE:

Phosphatidylethanolamine

PR:

Progesterone receptor

RFS:

Relapse-free survival

TMA:

Tissue microarray

TNBC:

Triple-negative breast cancer

TNP:

Triple-negative phenotype

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Acknowledgements

We thank Christine Chow, Angela Cheng, and Rebecca Wu for technical support and members of the Gorski laboratory for helpful comments and discussions.

Funding

This study was funded by CIHR in partnership with Avon Foundation for Women-Canada grant OBC127216 and CIHR PJT-159536 Grant to SMG. SBo was supported by a CIHR Frederick Banting and Charles Best Canada Graduate Scholarship Doctoral Award.

Author information

Authors and Affiliations

  1. Canada’s Michael Smith Genome Sciences Centre, BC Cancer, Vancouver, BC, Canada

    Svetlana Bortnik, Jing Xu & Sharon M. Gorski

  2. Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada

    Basile Tessier-Cloutier, Samuel Leung, Karama Asleh, Samantha Burugu, Jamie Magrill, Kendall Greening, Fatemeh Derakhshan, Stephen Yip, Tony Ng & Torsten O. Nielsen

  3. Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, Canada

    Jing Xu & Sharon M. Gorski

  4. Medical Oncology, BC Cancer, Vancouver, BC, Canada

    Karen A. Gelmon

  5. Department of Medicine, UBC, Vancouver, BC, Canada

    Karen A. Gelmon

Authors
  1. Svetlana Bortnik
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  2. Basile Tessier-Cloutier
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  3. Samuel Leung
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  4. Jing Xu
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  6. Samantha Burugu
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  11. Tony Ng
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  14. Sharon M. Gorski
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Contributions

SBo, KAG, and SMG conceived the study; SBo, BTC, and FD scored slides with assistance of JM and KG; SL, KA, and SBu performed statistical analyses; JX created KO lines and performed GABARAP antibody validation; SY and TN supervised scoring; KAG, TON, and SMG supervised and directed the study; SBo, BTC, and SL wrote the manuscript; all authors read, edited, and approved the final manuscript.

Corresponding author

Correspondence to Sharon M. Gorski.

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Conflict of interest

TON reports a licensing agreement with Veracyte, Inc. regarding the PAM50 intrinsic subtype classifier, not used in this study. The other authors declare that they have no conflict of interest.

Ethical approval

This study and our access to the de-identified human data were approved by the Research Ethics Board of the University of British Columbia, British Columbia Cancer Agency and Simon Fraser University. This article does not contain any studies with animals performed by any of the authors.

Informed consent

No informed consent was needed as anonymized archival specimens were used in this study, according to the Canadian Tri-Council Policy Statement for ethical research involving human subjects.

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Bortnik, S., Tessier-Cloutier, B., Leung, S. et al. Differential expression and prognostic relevance of autophagy-related markers ATG4B, GABARAP, and LC3B in breast cancer. Breast Cancer Res Treat 183, 525–547 (2020). https://doi.org/10.1007/s10549-020-05795-z

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