Research ArticleImmunologyOncology Open Access | 10.1172/JCI180983

Michela Perego,1 Minjeong Yeon,1 Ekta Agarwal,1 Andrew T. Milcarek,1 Irene Bertolini,1 Chiara Camisaschi,2 Jagadish C. Ghosh,1 Hsin-Yao Tang,3,4 Nathalie Grandvaux,5,6 Marcus Ruscetti,7 Andrew V. Kossenkov,3,8 Sarah Preston-Alp,9 Italo Tempera,9 Noam Auslander,3,10 and Dario C. Altieri1

1Immunology, Microenvironment and Metastasis Program, The Wistar Institute, Philadelphia, Pennsylvania, USA.

2Flow Cytometry Core, IRCCS Humanitas Research Hospital, Rozzano, Milan, Italy.

3Center for Systems and Computational Biology and

4Proteomics and Metabolomics Shared Resource, The Wistar Institute, Philadelphia, Pennsylvania, USA.

5CRCHUM — Centre de Recherche du Centre Hospitalier de l’Université de Montréal, Montréal, Québec, Canada.

6Department of Biochemistry and Molecular Medicine, Faculty of Medicine, Université de Montréal, Montréal, Québec, Canada.

7Department of Molecular, Cell and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, Massachusetts, USA.

8Bioinformatics Shared Resource,

9Gene Expression and Regulation Program,

10Molecular and Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, Pennsylvania, USA.

Address correspondence to: Dario C. Altieri or Michela Perego, The Wistar Institute, 3601 Spruce Street, Philadelphia, Pennsylvania 19104, USA. Phone: 215.495.6970; Email: daltieri@wistar.org (DCA); Phone: 215.495.6974; Email: mperego@wistar.org (MP).

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1Immunology, Microenvironment and Metastasis Program, The Wistar Institute, Philadelphia, Pennsylvania, USA.

2Flow Cytometry Core, IRCCS Humanitas Research Hospital, Rozzano, Milan, Italy.

3Center for Systems and Computational Biology and

4Proteomics and Metabolomics Shared Resource, The Wistar Institute, Philadelphia, Pennsylvania, USA.

5CRCHUM — Centre de Recherche du Centre Hospitalier de l’Université de Montréal, Montréal, Québec, Canada.

6Department of Biochemistry and Molecular Medicine, Faculty of Medicine, Université de Montréal, Montréal, Québec, Canada.

7Department of Molecular, Cell and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, Massachusetts, USA.

8Bioinformatics Shared Resource,

9Gene Expression and Regulation Program,

10Molecular and Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, Pennsylvania, USA.

Address correspondence to: Dario C. Altieri or Michela Perego, The Wistar Institute, 3601 Spruce Street, Philadelphia, Pennsylvania 19104, USA. Phone: 215.495.6970; Email: daltieri@wistar.org (DCA); Phone: 215.495.6974; Email: mperego@wistar.org (MP).

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1Immunology, Microenvironment and Metastasis Program, The Wistar Institute, Philadelphia, Pennsylvania, USA.

2Flow Cytometry Core, IRCCS Humanitas Research Hospital, Rozzano, Milan, Italy.

3Center for Systems and Computational Biology and

4Proteomics and Metabolomics Shared Resource, The Wistar Institute, Philadelphia, Pennsylvania, USA.

5CRCHUM — Centre de Recherche du Centre Hospitalier de l’Université de Montréal, Montréal, Québec, Canada.

6Department of Biochemistry and Molecular Medicine, Faculty of Medicine, Université de Montréal, Montréal, Québec, Canada.

7Department of Molecular, Cell and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, Massachusetts, USA.

8Bioinformatics Shared Resource,

9Gene Expression and Regulation Program,

10Molecular and Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, Pennsylvania, USA.

Address correspondence to: Dario C. Altieri or Michela Perego, The Wistar Institute, 3601 Spruce Street, Philadelphia, Pennsylvania 19104, USA. Phone: 215.495.6970; Email: daltieri@wistar.org (DCA); Phone: 215.495.6974; Email: mperego@wistar.org (MP).

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1Immunology, Microenvironment and Metastasis Program, The Wistar Institute, Philadelphia, Pennsylvania, USA.

2Flow Cytometry Core, IRCCS Humanitas Research Hospital, Rozzano, Milan, Italy.

3Center for Systems and Computational Biology and

4Proteomics and Metabolomics Shared Resource, The Wistar Institute, Philadelphia, Pennsylvania, USA.

5CRCHUM — Centre de Recherche du Centre Hospitalier de l’Université de Montréal, Montréal, Québec, Canada.

6Department of Biochemistry and Molecular Medicine, Faculty of Medicine, Université de Montréal, Montréal, Québec, Canada.

7Department of Molecular, Cell and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, Massachusetts, USA.

8Bioinformatics Shared Resource,

9Gene Expression and Regulation Program,

10Molecular and Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, Pennsylvania, USA.

Address correspondence to: Dario C. Altieri or Michela Perego, The Wistar Institute, 3601 Spruce Street, Philadelphia, Pennsylvania 19104, USA. Phone: 215.495.6970; Email: daltieri@wistar.org (DCA); Phone: 215.495.6974; Email: mperego@wistar.org (MP).

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1Immunology, Microenvironment and Metastasis Program, The Wistar Institute, Philadelphia, Pennsylvania, USA.

2Flow Cytometry Core, IRCCS Humanitas Research Hospital, Rozzano, Milan, Italy.

3Center for Systems and Computational Biology and

4Proteomics and Metabolomics Shared Resource, The Wistar Institute, Philadelphia, Pennsylvania, USA.

5CRCHUM — Centre de Recherche du Centre Hospitalier de l’Université de Montréal, Montréal, Québec, Canada.

6Department of Biochemistry and Molecular Medicine, Faculty of Medicine, Université de Montréal, Montréal, Québec, Canada.

7Department of Molecular, Cell and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, Massachusetts, USA.

8Bioinformatics Shared Resource,

9Gene Expression and Regulation Program,

10Molecular and Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, Pennsylvania, USA.

Address correspondence to: Dario C. Altieri or Michela Perego, The Wistar Institute, 3601 Spruce Street, Philadelphia, Pennsylvania 19104, USA. Phone: 215.495.6970; Email: daltieri@wistar.org (DCA); Phone: 215.495.6974; Email: mperego@wistar.org (MP).

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1Immunology, Microenvironment and Metastasis Program, The Wistar Institute, Philadelphia, Pennsylvania, USA.

2Flow Cytometry Core, IRCCS Humanitas Research Hospital, Rozzano, Milan, Italy.

3Center for Systems and Computational Biology and

4Proteomics and Metabolomics Shared Resource, The Wistar Institute, Philadelphia, Pennsylvania, USA.

5CRCHUM — Centre de Recherche du Centre Hospitalier de l’Université de Montréal, Montréal, Québec, Canada.

6Department of Biochemistry and Molecular Medicine, Faculty of Medicine, Université de Montréal, Montréal, Québec, Canada.

7Department of Molecular, Cell and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, Massachusetts, USA.

8Bioinformatics Shared Resource,

9Gene Expression and Regulation Program,

10Molecular and Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, Pennsylvania, USA.

Address correspondence to: Dario C. Altieri or Michela Perego, The Wistar Institute, 3601 Spruce Street, Philadelphia, Pennsylvania 19104, USA. Phone: 215.495.6970; Email: daltieri@wistar.org (DCA); Phone: 215.495.6974; Email: mperego@wistar.org (MP).

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1Immunology, Microenvironment and Metastasis Program, The Wistar Institute, Philadelphia, Pennsylvania, USA.

2Flow Cytometry Core, IRCCS Humanitas Research Hospital, Rozzano, Milan, Italy.

3Center for Systems and Computational Biology and

4Proteomics and Metabolomics Shared Resource, The Wistar Institute, Philadelphia, Pennsylvania, USA.

5CRCHUM — Centre de Recherche du Centre Hospitalier de l’Université de Montréal, Montréal, Québec, Canada.

6Department of Biochemistry and Molecular Medicine, Faculty of Medicine, Université de Montréal, Montréal, Québec, Canada.

7Department of Molecular, Cell and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, Massachusetts, USA.

8Bioinformatics Shared Resource,

9Gene Expression and Regulation Program,

10Molecular and Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, Pennsylvania, USA.

Address correspondence to: Dario C. Altieri or Michela Perego, The Wistar Institute, 3601 Spruce Street, Philadelphia, Pennsylvania 19104, USA. Phone: 215.495.6970; Email: daltieri@wistar.org (DCA); Phone: 215.495.6974; Email: mperego@wistar.org (MP).

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1Immunology, Microenvironment and Metastasis Program, The Wistar Institute, Philadelphia, Pennsylvania, USA.

2Flow Cytometry Core, IRCCS Humanitas Research Hospital, Rozzano, Milan, Italy.

3Center for Systems and Computational Biology and

4Proteomics and Metabolomics Shared Resource, The Wistar Institute, Philadelphia, Pennsylvania, USA.

5CRCHUM — Centre de Recherche du Centre Hospitalier de l’Université de Montréal, Montréal, Québec, Canada.

6Department of Biochemistry and Molecular Medicine, Faculty of Medicine, Université de Montréal, Montréal, Québec, Canada.

7Department of Molecular, Cell and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, Massachusetts, USA.

8Bioinformatics Shared Resource,

9Gene Expression and Regulation Program,

10Molecular and Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, Pennsylvania, USA.

Address correspondence to: Dario C. Altieri or Michela Perego, The Wistar Institute, 3601 Spruce Street, Philadelphia, Pennsylvania 19104, USA. Phone: 215.495.6970; Email: daltieri@wistar.org (DCA); Phone: 215.495.6974; Email: mperego@wistar.org (MP).

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1Immunology, Microenvironment and Metastasis Program, The Wistar Institute, Philadelphia, Pennsylvania, USA.

2Flow Cytometry Core, IRCCS Humanitas Research Hospital, Rozzano, Milan, Italy.

3Center for Systems and Computational Biology and

4Proteomics and Metabolomics Shared Resource, The Wistar Institute, Philadelphia, Pennsylvania, USA.

5CRCHUM — Centre de Recherche du Centre Hospitalier de l’Université de Montréal, Montréal, Québec, Canada.

6Department of Biochemistry and Molecular Medicine, Faculty of Medicine, Université de Montréal, Montréal, Québec, Canada.

7Department of Molecular, Cell and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, Massachusetts, USA.

8Bioinformatics Shared Resource,

9Gene Expression and Regulation Program,

10Molecular and Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, Pennsylvania, USA.

Address correspondence to: Dario C. Altieri or Michela Perego, The Wistar Institute, 3601 Spruce Street, Philadelphia, Pennsylvania 19104, USA. Phone: 215.495.6970; Email: daltieri@wistar.org (DCA); Phone: 215.495.6974; Email: mperego@wistar.org (MP).

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1Immunology, Microenvironment and Metastasis Program, The Wistar Institute, Philadelphia, Pennsylvania, USA.

2Flow Cytometry Core, IRCCS Humanitas Research Hospital, Rozzano, Milan, Italy.

3Center for Systems and Computational Biology and

4Proteomics and Metabolomics Shared Resource, The Wistar Institute, Philadelphia, Pennsylvania, USA.

5CRCHUM — Centre de Recherche du Centre Hospitalier de l’Université de Montréal, Montréal, Québec, Canada.

6Department of Biochemistry and Molecular Medicine, Faculty of Medicine, Université de Montréal, Montréal, Québec, Canada.

7Department of Molecular, Cell and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, Massachusetts, USA.

8Bioinformatics Shared Resource,

9Gene Expression and Regulation Program,

10Molecular and Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, Pennsylvania, USA.

Address correspondence to: Dario C. Altieri or Michela Perego, The Wistar Institute, 3601 Spruce Street, Philadelphia, Pennsylvania 19104, USA. Phone: 215.495.6970; Email: daltieri@wistar.org (DCA); Phone: 215.495.6974; Email: mperego@wistar.org (MP).

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1Immunology, Microenvironment and Metastasis Program, The Wistar Institute, Philadelphia, Pennsylvania, USA.

2Flow Cytometry Core, IRCCS Humanitas Research Hospital, Rozzano, Milan, Italy.

3Center for Systems and Computational Biology and

4Proteomics and Metabolomics Shared Resource, The Wistar Institute, Philadelphia, Pennsylvania, USA.

5CRCHUM — Centre de Recherche du Centre Hospitalier de l’Université de Montréal, Montréal, Québec, Canada.

6Department of Biochemistry and Molecular Medicine, Faculty of Medicine, Université de Montréal, Montréal, Québec, Canada.

7Department of Molecular, Cell and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, Massachusetts, USA.

8Bioinformatics Shared Resource,

9Gene Expression and Regulation Program,

10Molecular and Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, Pennsylvania, USA.

Address correspondence to: Dario C. Altieri or Michela Perego, The Wistar Institute, 3601 Spruce Street, Philadelphia, Pennsylvania 19104, USA. Phone: 215.495.6970; Email: daltieri@wistar.org (DCA); Phone: 215.495.6974; Email: mperego@wistar.org (MP).

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1Immunology, Microenvironment and Metastasis Program, The Wistar Institute, Philadelphia, Pennsylvania, USA.

2Flow Cytometry Core, IRCCS Humanitas Research Hospital, Rozzano, Milan, Italy.

3Center for Systems and Computational Biology and

4Proteomics and Metabolomics Shared Resource, The Wistar Institute, Philadelphia, Pennsylvania, USA.

5CRCHUM — Centre de Recherche du Centre Hospitalier de l’Université de Montréal, Montréal, Québec, Canada.

6Department of Biochemistry and Molecular Medicine, Faculty of Medicine, Université de Montréal, Montréal, Québec, Canada.

7Department of Molecular, Cell and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, Massachusetts, USA.

8Bioinformatics Shared Resource,

9Gene Expression and Regulation Program,

10Molecular and Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, Pennsylvania, USA.

Address correspondence to: Dario C. Altieri or Michela Perego, The Wistar Institute, 3601 Spruce Street, Philadelphia, Pennsylvania 19104, USA. Phone: 215.495.6970; Email: daltieri@wistar.org (DCA); Phone: 215.495.6974; Email: mperego@wistar.org (MP).

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1Immunology, Microenvironment and Metastasis Program, The Wistar Institute, Philadelphia, Pennsylvania, USA.

2Flow Cytometry Core, IRCCS Humanitas Research Hospital, Rozzano, Milan, Italy.

3Center for Systems and Computational Biology and

4Proteomics and Metabolomics Shared Resource, The Wistar Institute, Philadelphia, Pennsylvania, USA.

5CRCHUM — Centre de Recherche du Centre Hospitalier de l’Université de Montréal, Montréal, Québec, Canada.

6Department of Biochemistry and Molecular Medicine, Faculty of Medicine, Université de Montréal, Montréal, Québec, Canada.

7Department of Molecular, Cell and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, Massachusetts, USA.

8Bioinformatics Shared Resource,

9Gene Expression and Regulation Program,

10Molecular and Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, Pennsylvania, USA.

Address correspondence to: Dario C. Altieri or Michela Perego, The Wistar Institute, 3601 Spruce Street, Philadelphia, Pennsylvania 19104, USA. Phone: 215.495.6970; Email: daltieri@wistar.org (DCA); Phone: 215.495.6974; Email: mperego@wistar.org (MP).

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1Immunology, Microenvironment and Metastasis Program, The Wistar Institute, Philadelphia, Pennsylvania, USA.

2Flow Cytometry Core, IRCCS Humanitas Research Hospital, Rozzano, Milan, Italy.

3Center for Systems and Computational Biology and

4Proteomics and Metabolomics Shared Resource, The Wistar Institute, Philadelphia, Pennsylvania, USA.

5CRCHUM — Centre de Recherche du Centre Hospitalier de l’Université de Montréal, Montréal, Québec, Canada.

6Department of Biochemistry and Molecular Medicine, Faculty of Medicine, Université de Montréal, Montréal, Québec, Canada.

7Department of Molecular, Cell and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, Massachusetts, USA.

8Bioinformatics Shared Resource,

9Gene Expression and Regulation Program,

10Molecular and Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, Pennsylvania, USA.

Address correspondence to: Dario C. Altieri or Michela Perego, The Wistar Institute, 3601 Spruce Street, Philadelphia, Pennsylvania 19104, USA. Phone: 215.495.6970; Email: daltieri@wistar.org (DCA); Phone: 215.495.6974; Email: mperego@wistar.org (MP).

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1Immunology, Microenvironment and Metastasis Program, The Wistar Institute, Philadelphia, Pennsylvania, USA.

2Flow Cytometry Core, IRCCS Humanitas Research Hospital, Rozzano, Milan, Italy.

3Center for Systems and Computational Biology and

4Proteomics and Metabolomics Shared Resource, The Wistar Institute, Philadelphia, Pennsylvania, USA.

5CRCHUM — Centre de Recherche du Centre Hospitalier de l’Université de Montréal, Montréal, Québec, Canada.

6Department of Biochemistry and Molecular Medicine, Faculty of Medicine, Université de Montréal, Montréal, Québec, Canada.

7Department of Molecular, Cell and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, Massachusetts, USA.

8Bioinformatics Shared Resource,

9Gene Expression and Regulation Program,

10Molecular and Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, Pennsylvania, USA.

Address correspondence to: Dario C. Altieri or Michela Perego, The Wistar Institute, 3601 Spruce Street, Philadelphia, Pennsylvania 19104, USA. Phone: 215.495.6970; Email: daltieri@wistar.org (DCA); Phone: 215.495.6974; Email: mperego@wistar.org (MP).

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Published August 30, 2024 - More info

Published in Volume 134, Issue 22 on November 15, 2024
J Clin Invest. 2024;134(22):e180983. https://doi.org/10.1172/JCI180983.
© 2024 Perego et al. This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Published August 30, 2024 - Version history
Received: March 8, 2024; Accepted: August 20, 2024 View PDF Related article:

Abstract

Parkin, a ring-between-ring-type E3 ubiquitin ligase, first shown to play a critical role in autosomal recessive juvenile Parkinsonism, has recently emerged as a key player in cancer biology. Parkin is now known to serve as a tumor suppressor, and its deregulation frequently promotes tumorigenesis. In this issue of the JCI, Perego et al. expand that role by showing that Parkin expression stimulated an interferon (IFN) response to modulate CD8+ T cell activity. These findings suggest that, in addition to directly inhibiting tumor progression, Parkin enhances antitumor immune responses, highlighting it as a promising therapeutic target for cancer treatment.

Authors

Hyungsoo Kim, Ze’ev A. Ronai

× Abstract

The activation of innate immunity and associated interferon (IFN) signaling have been implicated in cancer, but the regulators are elusive and links to tumor suppression remain undetermined. Here, we found that Parkin, an E3 ubiquitin ligase altered in Parkinson’s Disease, was epigenetically silenced in cancer and its reexpression by clinically approved demethylating therapy stimulated transcription of a potent IFN response in tumor cells. This pathway required Parkin E3 ubiquitin ligase activity, involved the subcellular trafficking and release of the alarmin High Mobility Group Box 1 (HMGB1) and was associated with inhibition of NF-κB gene expression. In turn, Parkin-expressing cells released an IFN secretome that upregulated effector and cytotoxic CD8+ T cell markers, lowered the expression of immune inhibitory receptors TIM3 and LAG3, and stimulated high content of the self renewal/stem cell factor, TCF1. PRKN-induced CD8+ T cells selectively accumulated in the microenvironment and inhibited transgenic and syngeneic tumor growth in vivo. Therefore, Parkin is an epigenetically regulated activator of innate immunity and dual mode tumor suppressor, inhibiting intrinsic tumor traits of metabolism and cell invasion, while simultaneously reinvigorating CD8 T cell functions in the microenvironment.

Graphical Abstract Introduction

The transcriptional induction of interferons (IFN) and their downstream IFN-stimulated genes (ISG) is a formidable host defense mechanism against pathogenic infections, mostly viruses (1). Identified as innate immunity, this pathway involves the release of damage-associated molecular pattern (DAMP) and pathogen-associated molecular pattern (PAMP) (2) in response to cellular stress (3), mitochondrial damage (4, 5), and exposure to infectious pathogens (6). In turn, DAMP/PAMP signaling activates a multifaceted danger sensing machinery in cytosol, including the cGAS/STING complex (7, 8), as well as mitochondria (9), which leads to the assembly of an IRF3/STAT1 transcriptional complex driving the expression of multiple IFN molecules, ISG, and pleiotropic inflammatory cytokines (10).

In addition to protection against pathogens, there is evidence that innate immunity and IFN signaling have important roles in cancer, modulating a host of tumor responses, including sensitivity to immunotherapy (11, 12). This pathway is complex and highly context dependent (13). As an antitumor mechanism (11, 12), IFN signaling promotes intratumoral recruitment of effector CD8+ T cells (14), activation of MHC class I dendritic cells (15), and improved response to conventional (16), molecular (17), and immune therapy (18). In fact, IFN therapy is feasible, generally well tolerated, and accompanied by positive patient responses alone or in combination with immunotherapy (19, 20). On the other hand, sustained, i.e. chronic, IFN activation can be highly detrimental in cancer, especially contributing to CD8+ T cell exhaustion (21), a process marked by progressive loss of effector functions and insensitivity to therapeutic immune checkpoint inhibitors (ICI) (22). What controls the pro- or antitumorigenic responses to IFN signaling remains elusive, and endogenous regulators of this process potentially linked to tumor suppression have not been identified.

One candidate molecule at the interface between cancer and innate immunity is Parkin (PRKN), an E3 ubiquitin ligase implicated in mitochondrial quality control via mitophagy (23). Defects in this pathway leading to the accumulation of damaged and dysfunctional mitochondria have been linked to neuronal toxicity in patients with Parkinson’s Disease (PD), where the PARK2 gene encoding PRKN can be mono- or biallelically altered (24, 25). In addition, PRKN has been suggested to inhibit multiple mechanisms of innate immunity, including mitochondrial antigen presentation (26), STING signaling (27), and activation of the NLRP3 inflammasome (28). In this scenario, loss of PRKN would contribute to neuroinflammation, another invariable hallmark of PD pathogenesis (29).

On the other hand, it is clear that PRKN has functions beyond the CNS. For instance, PRKN expression is undetectable in virtually all examined human cancers and tumor cell lines (30), suggesting a general role in tumor suppression (31). How this is orchestrated remains to be elucidated, but reintroduction of PRKN in different cancer types is sufficient to inhibit several intrinsic tumor traits, such as mitotic transitions (32), metabolic reprogramming through the pentose phosphate pathway (30), and phosphoglycerate dehydrogenase (33), as well as heightened cell motility and invasion cell motility and invasion (34). While these responses were independent of mitophagy, a role of PRKN innate immunity (26–28) in cancer is unknown and a potential link of this pathway to tumor suppression has not been considered. In this study, we explored a role of PRKN in antitumor immunity.

Results

PRKN activates IFN gene expression in cancer. We began this study by asking whether reexpression of PRKN in tumor cells affected gene expression. By RNA-Seq profiling, we found that transient expression of PRKN in PRKN-negative prostate cancer PC3 cells (30) induced a potent IFN response (Figure 1, A and B) with upregulation of multiple IFNs, ISG, and pleiotropic cytokines (Supplemental Figure 1A; supplemental material available online with this article; https://doi.org/10.1172/JCI180983DS1). Consistent with tumor suppression, effectors of tumorigenesis (Myc, Myb, NKX2-3, and TRIM24), metastasis (NFE2L2), oncogenic transformation (FOXM1), and cell cycle (E2F2 and E2F3) were also inhibited in the presence of PRKN (Supplemental Figure 1B). Further bioinformatics analysis of this dataset showed that PRKN upregulated DNA damage and cell death responses, whereas intrinsic tumor traits of glycolysis, eIF2-α protein translation, and cell cycle were downregulated (Supplemental Figure 1C) (30). The PRKN transcriptome was specific because other pathways associated with inflammation, such as MAPK signaling (Figure 1C) and NF-κB–dependent gene expression (Figure 1D) were unchanged or profoundly suppressed, respectively (Figure 1, C and D). Consistent with this, PRKN expression inhibited NF-κB promoter reporter activity in PC3 cells (Supplemental Figure 1D), whereas several effectors of innate immunity (TLR3, TLR9, LTA, and IL12) were upregulated in the presence of PRKN (Figure 1D).

Figure 1

PRKN IFN response in cancer. (A) PC3 cells were transfected with vector or PRKN and analyzed for an IFN enrichment gene signature by RNA-Seq. (B) Schematic diagram of innate immunity pathways activated by PRKN in PC3 cells by RNA-Seq. Created with BioRender.com. (C) PC3 cells expressing PRKN (as in A) were analyzed in an IFN/MAPK array by RT-qPCR. Heatmap from a representative experiment. (D) The conditions are the same as in A and PRKN-expressing PC3 cells were analyzed in an NF-κB gene array by RT-qPCR. Heatmap from a representative experiment out of 2 independent determinations. (E) The indicated tumor cell types expressing vector or PRKN were analyzed for IFN gene expression by RT-qPCR. Mean ± SD (n = 3). (F) PC3 cells that conditionally express PRKN (TetON system) in response to Doxycycline (Doxy) were analyzed by Western blotting (inset) and RT-qPCR in the presence of vehicle (Veh) or Doxy. Mean ± SD (n = 3). (G) PRKN TetON PC3 cells were analyzed for IFN-β promoter luciferase activity in the presence of vehicle (Veh) or Doxy. RLU, relative luciferase activity. Mean ± SD (n = 4). (H) The conditions are the same as in G except that PRKN TetON PC3 cells were analyzed for WT or mutant (Mut) IFIT1 promoter luciferase activity in the presence of vehicle (Veh) or Doxy. Mean ± SD (n = 3). (I) Normal breast epithelial MCF10A cells expressing endogenous PRKN were transfected with control nontargeted siRNA (siCtrl) or 2 independent siRNA sequences to PRKN (siPRKN #1 and siPRKN #2) and analyzed for IFN gene expression by RT-qPCR. Mean ± SD (n = 3). (J) PC3 cells expressing WT PRKN (WT) or E3-ligase defective PRKN C431S or S65A mutants (inset) were analyzed for IFN gene expression by RT-qPCR. Data are from a representative experiment out of 4 independent determinations. Numbers represent P values by 2-tailed unpaired t test. *P = 0.01; **P = 0.002–0.009; ***P = <0.0001–0.0003.

Next, we asked whether PRKN IFN gene expression was a general property of disparate tumor types. First, and consistent with recent findings (30), endogenous PRKN mRNA levels were mostly undetectable in a large panel of human and murine tumor cell lines (Supplemental Figure 1E). Conversely, normal mammary epithelial MCF10A cells expressed endogenous PRKN (Supplemental Figure 1E), in agreement with previous observations (30). Under these conditions, reintroduction of PRKN by transient transfection upregulated PRKN mRNA (Supplemental Figure 1F) and protein (Supplemental Figure 1G) levels in all human tumor types tested. This was associated with transcriptional induction of an IFN gene signature that comprised multiple IFN molecules, ISG, and inflammatory cytokines (Figure 1E). A similar response was observed in murine prostate cancer cell types MPTEN1 and MP3098, where transient reintroduction of PRKN potently upregulated IFN gene expression (Supplemental Figure 1H). To independently validate these findings, we next conditionally reexpressed PRKN in PC3 cells using a doxycycline-regulated (Doxy-regulated) TetON system (Figure 1F, inset). Here, treatment with Doxy induced a robust IFN response in stably transduced PC3 cells, whereas vehicle had no effect (Figure 1F). Consistent with these data, PRKN expression increased transcription of IFN-β (Figure 1G) and IFIT1 (Figure 1H) promoter reporter activity in PC3 cells, whereas an IFIT1 promoter mutant carrying a double mutation in the ISRE sites was not modulated by PRKN (Figure 1H). Finally, as a complementary approach, we next silenced endogenous PRKN in normal breast epithelial MCF10A cells (Supplemental Figure 1E) (30). In these cells, PRKN silencing using 2 independent siRNA sequences abolished IFN gene expression compared with control cultures (Figure 1I).

In addition to modulation of type I IFNs, IFN-α and IFN-β, RNA-Seq profiling of PRKN-expressing PC3 cells showed a prominent upregulation of IFN-γ (IFNG, Supplemental Figure 1A). Consistent with this, reexpression of PRKN in prostate cancer DU145 or pancreatic adenocarcinoma PANC-1 cells increased IFN-γ mRNA levels (Supplemental Figure 1I), whereas PRKN siRNA silencing in MCF10A cells suppressed IFN-γ expression (Supplemental Figure 1J). Finally, we asked if PRKN E3 ubiquitin ligase activity was required for IFN gene expression. In these experiments, expression of PRKN mutants (Figure 1J, inset) that abolish the E3 ligase catalytic site (Cys431Ser, C431S) or PINK1 phosphorylation site (Ser65Ala, S65A) required for E3 ligase function (23) did not induce an IFN response in PC3 cells (Figure 1J).

PRKN methylation silencing in cancer. In previous studies, PRKN loss in cancer was not accompanied by increased mutagenesis of the PARK2 gene or copy number alterations (30). Instead, analysis of The Cancer Genome Atlas (TCGA) database demonstrated that the PARK2 promoter was hypermethylated in select human tumors, including breast (BRCA) and prostate (PRAD) adenocarcinoma, as well as kidney cancer (KIRC), compared with normal tissues (Figure 2, A and B). Hypermethylation of the PARK2 promoter correlated with shortened patient survival in prostate (PRAD) and pancreatic (PAAD) adenocarcinoma, low-grade glioma (LGG), liver hepatocellular carcinoma (LIHC) and paraganglioma/pheochromocytoma (PCPG) (Figure 2C). Consistent with epigenetic silencing, treatment of PC3 or MDA231 cells with a clinically approved pyrimidine nucleoside analog and DNA hypomethylating agent, decitabine, resulted in near complete demethylation of PARK2 CpG promoter regions located at chr6:162728136 (reference genome hg38), approximately 200 bp upstream of the transcription start site (Figure 2D). Accordingly, decitabine treatment resulted in increased expression of endogenous PRKN mRNA (Figure 2E) and protein (Figure 2F) in multiple human and murine tumor cell types, whereas vehicle had no effect. As a result, decitabine-induced reexpression of endogenous PRKN potently upregulated IFN gene expression in all tumor types tested (Figure 2E).

Figure 2

PRKN epigenetic silencing in human tumors. (A) Heatmap of PARK2 gene methylation in cancer versus normal samples (TCGA). The individual probes are indicated. (B) Hypermethylation of PARK2 promoter in cancer versus normal samples (TCGA). Boxes show the quartiles (0.25 and 0.75) of the data, center lines show the median, and whiskers show the rest of the distribution except for outliers (1-sided paired sample rank-sum test P values are reported). 2 methylation 450 K probes are used. A P value is indicated. KIRC, kidney clear cell carcinoma; BRCA, breast adenocarcinoma; PRAD, prostate adenocarcinoma. (C) Kaplan-Meier curves for PARK2 hyper- or hypomethylation in patient cohorts (TCGA) of PRAD, pancreatic ductal adenocarcinoma (PAAD), liver hepatocellular carcinoma (LIHC), pheochromocytoma and paraganglioma (PCPG), or low-grade glioma (LGG, 2 independent PARK2 methylation probes). A P value per patient cohort is indicated (2-tailed unpaired t test). (D) Methylation-specific PCR amplification of PARK2 promoter region from PC3 or MDA231 cells approximately 200 bp upstream of the transcriptional start site in the presence or absence of the hypomethylating agent, decitabine. Mean ± SD (n = 3). (E) The indicated tumor cell lines were treated with vehicle or decitabine and analyzed for PRKN or IFN gene expression by RT-qPCR. Mean ± SD (n = 4). (F) The indicated human (PC3, DU145, MDA231) or murine (P3098) tumor cell lines were treated with vehicle (Veh) or decitabine (Dec) and analyzed by Western blotting. Numbers represent P values by 2-tailed unpaired t test.

Mechanisms of PRKN activation of IFN gene expression. Next, we studied the mechanism(s) of PRKN induction of IFN signaling in cancer. Consistent with models of innate immunity (7, 8), PRKN expression in PC3 cells activated the cytosolic danger-sensing cGAS pathway with increased production of the second messenger, 2′,3′ cGAMP (8) (Supplemental Figure 2A). This was accompanied by phosphorylation of the cGAMP downstream target, STING as well as IRF3 (Supplemental Figure 2B). In addition, PRKN-expressing PC3 cells exhibited increased phosphorylation of STAT1, a key transcriptional regulator of IFN signaling in a phosphoarray screen (Supplemental Figure 2, C and D). Conversely, PRKN inhibited the expression and phosphorylation of protumorigenic STAT3, as well as other STAT molecules, STAT2 and STAT5, compared with control (Supplemental Figure 2, C and D). Despite the upregulation of several proinflam