Stress and interferon signalling-mediated apoptosis contributes to pleiotropic anticancer responses induced by targeting NGLY1

Abstract

Background: Although NGLY1 is known as a pivotal enzyme that catalyses the deglycosylation of denatured glycoproteins, information regarding the responses of human cancer and normal cells to NGLY1 suppression is limited.

Methods: We examined how NGLY1 expression affects viability, tumour growth, and responses to therapeutic agents in melanoma cells and an animal model. Molecular mechanisms contributing to NGLY1 suppression-induced anticancer responses were revealed by systems biology and chemical biology studies. Using computational and medicinal chemistry-assisted approaches, we established novel NGLY1-inhibitory small molecules.

Results: Compared with normal cells, NGLY1 was upregulated in melanoma cell lines and patient tumours. NGLY1 knockdown caused melanoma cell death and tumour growth retardation. Targeting NGLY1 induced pleiotropic responses, predominantly stress signalling-associated apoptosis and cytokine surges, which synergise with the anti-melanoma activity of chemotherapy and targeted therapy agents. Pharmacological and molecular biology tools that inactivate NGLY1 elicited highly similar responses in melanoma cells. Unlike normal cells, melanoma cells presented distinct responses and high vulnerability to NGLY1 suppression.

Conclusion: Our work demonstrated the significance of NGLY1 in melanoma cells, provided mechanistic insights into how NGLY1 inactivation leads to eradication of melanoma with limited impact on normal cells, and suggested that targeting NGLY1 represents a novel anti-melanoma strategy.

Fig. 1

NGLY1 expression in normal and melanoma cells. (a) The expression of the NGLY1 gene in cells at the transcriptional level was measured using qRT-PCR. Orange shading: primary melanocytes (PM). Blue shading: human melanoma cell lines. Pink shading: tumour samples of melanoma patients. Blue dot: undetectable NGLY1 transcript in the sample. Red asterisk: cell samples collected at high passage numbers ( > 16). All data were presented as mean ± standard deviation (n = 3; *P < 0.05, Mann–Whitney U test). The expression level of the ACTB gene in each sample was used as internal control for normalization. Gene expression levels in HEMl cells were used as comparison standards to calculate relative expression values. (b) The protein levels of NGLY1 detected using western blotting in cell samples. Blue shading: human normal cells. Yellow shading: human melanoma cell lines. (c) Representative images for immunohistochemistry staining of NGLY1 in normal skin and melanoma tumour tissues. With the same staining condition, the expression of NGLY1 was positively stained in the melanoma tumour tissue but not detected in the normal skin tissue. (d) WA09 hESC clones with and without the gene editing-mediated ablation of NGLY1 expression. Upper panel: cell morphology. Lower panel: the expression NGLY1 and pluripotency markers NANOG and POU5F1 detected by western blotting in the cells. WA09: parental WA09 hESCs. WA09-C6: a cell clone of WA09 hESCs derived from a gene-editing and selection process without acquiring disruptive mutations in the NGLY1 gene. WA09-C3 and WA09-C4: two NGLY1-deficent cell clones of WA09 hESCs independently derived from a gene-editing and selection process. (e) The positive staining of pluripotency markers in WA09-C6 and WA09-C3 hESCs. (f) NGLY1Pt1i-509 hiPSCs established from cell reprogramming in NGLY1-deficient patient-derived dermal fibroblasts. Upper panel: cell morphology. Lower panel: the expression NGLY1 and pluripotency markers NANOG and POU5F1 detected by western blotting in the cells. (g) The positive staining of pluripotency markers in NGLY1Pt1i-509 hiPSCs

Fig. 2

ER stress-associated apoptosis and synergistic anticancer responses induced by NGLY1 knockdown in melanoma cells. (a) The doxycycline (dox)-inducible pZIP-TRE3GS expression vector of non-targeting shRNA and NGLY1-targeting shRNA sequences. (b) The stable clones of UACC257 cells with dox-inducible shRNA. Cells with induced NGLY1-shRNA645 (green cells) showed morphological features of apoptosis, including shrinkage and fragmentation. Cells with induced non-targeting (NT)-shRNA maintain a morphology similar to the cells before dox induction. Cells were imaged after the treatment of 2 µM dox for 72 h. BF: bright field. ZsGreen: green fluorescence protein. (c) ATF4 and GADD153 signalling was activated by the shRNA-mediated knockdown of NGLY1 in melanoma cells. Tun: 2 µM tunicamycin for 24 h. (D) The accumulation of ubiquitinated proteins was detected using western blotting in MALME3M and SK-MEL-2 cells with NGLY1 knockdown. (e) The representative quadrant plots of flow cytometry analysis to detect apoptosis in normal (HDF51) and melanoma cells with NGLY1 knockdown. The cell samples were collected for analysis after the 72-hour induction of shRNA expression. (f) The quantitative results of flow cytometry analysis to detect apoptosis. All data were presented as mean ± standard deviation (n = 3; *P < 0.05, t-test) in the bar graph. (g) The dose-dependent suppression of viability in MALME3M and SK-MEL-2 cells with the indicated dox-inducible shRNA in response to cisplatin, dacarbazine, vemurafenib and dox treatment. All data were presented as mean ± standard deviation (n = 3, *P < 0.05, logistic regression). (h) The synergistic anticancer responses of NGLY1 knockdown and dacarbazine treatment for 72 h in MALME3M and SK-MEL-2 cells. The cell viability curves of combinatorial treatment were plotted according to the doses of dox used in the treatment. Combination indexes were calculated using Calcusyn software. A combination index value < 1 was considered synergistic. A combination index value < 0.2 was considered highly synergistic. All cell viability data were presented as mean ± standard deviation (n = 3)

Fig. 3

Differential gene expression caused by NGLY1 suppression in melanoma cells, hESCs and the differentiated derivatives of hESCs. SK-MEL-2, COLO829, UACC257 and MALME3M melanoma cells with the expression of the indicated inducible shRNA due to the treatment of 2 µM dox for 48 h were collected for RNA isolation and global gene expression profiling. WA09, WA09-C6, WA09-C3, WA09-C4 hESCs and their differentiated derivatives were also collected for analysis. Samples of two biological replicates for each setting were analysed. (a) A heat map representation of ~750 probes that measured the relative expression levels of differentially expressed genes (P < 0.01, t-test between control and knockdown cells) in melanoma cell samples expressing the indicated shRNA. Red dots: melanoma cells with NGLY1 knockdown. Green dots: control cells. (b) Selected genes that were differentially expressed (P < 0.01 and fold change ≥ 2) in the control and NGLY1-knockdown melanoma cells were annotated in a volcano plot of fold change vs. significance. (c) The qRT-PCR validation of selected genes that were differentially expressed in the control and NGLY1-knockdown melanoma cells (n = 3, *P < 0.05, t-test). The expression level of the ACTB gene in each sample was used as internal control for normalisation. Gene expression levels in SK-MEL-2 cells with NT-shRNA were used as comparison standards to calculate relative expression values. (d) Gene ontology analysis revealed that genes differentially expressed (P < 0.01 and fold change ≥ 2) due to NGLY1 suppression in melanoma cells were highly enriched in multiple biological processes. (e) A volcano plot of fold change vs. significance for selected genes that were differentially expressed (P < 0.01 and fold change ≥ 2) in control and NGLY1-deficient WA09 hESCs. (f) A volcano plot of fold change vs. significance for selected genes that were differentially expressed (P < 0.01 and fold change ≥ 2) in the embryoid bodies of control and NGLY1-deficient WA09 hESCs gone through 6 days of non-directed differentiation

Fig. 4

NGLY1 suppression enhanced the production of IFNβ1 and IL-29 that contributes to viability reduction in melanoma cells. (a) The contents of IFNβ1 and IL-29 in the conditioned media of UACC257 and SK-MEL-2 cell clones with the indicated treatment were measured by cytokine profiling. (b) The NGLY1 knockdown-induced upregulation of IFNβ1 and IL-29 was significantly attenuated by the overexpression of exogenous human NGLY1 in the cells. (c) Left panel: the attenuation of NGLY1 knockdown-induced viability reduction by the treatment of specific IFNβ1 neutralisation antibody in the cells. Right panel: the attenuation of NGLY1 knockdown-induced viability reduction by the treatment of specific IL-29 neutralisation antibody in the cells. NGLY knockdown was induced by the treatment of 2 µM dox for 72 h in the cells. (d) The enhanced expression and activation of IRF3, IRF7 and their upstream kinase TBK1 was detected in SK-MEL-2 and MALME3M cells with NGLY1 knockdown. The serine phosphorylation of IRF3 and TBK1 indicates their activity. NT: non-targeting shRNA. 645: NGLY1-targeting shRNA645. All data were presented as mean ± standard deviation (n = 3; *P < 0.05, t-test)

Fig. 5

The in vivo antitumour activity of targeting NGLY1 in melanoma cells. (a) A schematic illustration of animal study design to test the in vivo antitumour efficacy of NGLY1 suppression in melanoma. (b) The volume changes of xenografted SK-MEL-2 tumours with the induction of NT-shRNA (n = 10) and NGLY1-shRNA645 (n = 8) for 35 days. NGLY1 knockdown was a significant factor that affected the tumour volume (Factorial ANOVA; F = 8.537, P < 0.01). Tumours were harvested at the end of the study for western blotting analysis. Bars: median tumour volumes at the indicated time points. Inset: the volume changes of three tumours with NGLY1-targeting shRNA that initially increased their size but showed regression at the end of the study. (c) The expression of NGLY1, GADD153, IRF3, and GFP (ZsGreen) proteins in selected tumours was analysed by western blotting. (d) The enhanced expression of IL-29 in the tumour tissues with NGLY1 knockdown was detected by immunofluorescence staining

Fig. 6

Anticancer responses induced by novel covalent modifiers that target the catalytic site of human NGLY1 in melanoma cells. The computational homology model of human NGLY1 core domain was generated and used for studying interactions between NGLY1 and novel small molecules that are designed to covalently modified and inactivate the catalytic site of NGLY1. (a) The most favourable binding pose of Z-VAD-fmk, a short peptide with NGLY1 and caspase inhibitory activity, in the human NGLY1 homology model superimposed to the conformation of Z-VAD-fmk bound to mouse NGLY1 in a co-crystalised structure. (b) Novel small molecules (NM-322, NM-348, NM-350, and NM-354) that mimic a GlcNAc-conjugated asparagine in the NGLY1 substrates of NGLY1 and contain strategically positioned electrophilic groups bound to the human NGLY1 homology model in computational docking and showed their high binding affinities with the electrophilic groups pointed towards Cys309 in close proximity at the human NGLY1 catalytic site. (c) Upper panel: the 2-hour reaction of covalent modifiers, including Z-VAD-fmk (20 µM), WRR139 (5 µM), NM-322, NM-348, NM-350 and NM-354, with human NGLY1 suppressed its activity in the deglycosylation of denatured RNase B. Blue arrowhead: recombinant NGLY1-FLAG. RNase B (g): glycosylated RNase B. RNase B (dg): deglycosylated RNase B. Veh: vehicle (DMSO) treatment. M: molecular weight marker. Lower panel: the deglycosylation of NFE2L1 altered by the treatment of 20 µM Z-VAD-fmk and 200 µM NM-350 in bortezomib-treated HEK293T cells. The cells were pretreated with vehicle (DMSO), Z-VAD-fmk and NM-350 for 24 h and subsequently subjected to concomitant treatment with 10 µM bortezomib for an additional 16 h. Cell lysates reacted with and without 500 units of PNGase F for 2 h were analysed using western blotting. Red arrowhead: fully glycosylated NFE2L1. Orange arrowhead: partially glycosylated NFE2L1. Green arrowhead: deglycosylated and truncated NFE2L1. (d) The dose-dependent suppression of cell viability was preferentially induced by the novel NGLY1 inhibitors in melanoma cells compared with normal cells (*P < 0.05, logistic regression). (e) The synergistic effect was observed between NM-322 and dacarbazine in the suppression of melanoma cell viability. The cell viability curve of combinatorial treatment was plotted according to the doses of dacarbazine used in the treatment. (f) The synergistic effect was observed between NM-350 and bortezomib in the suppression of melanoma cell viability. The cell viability curve of combinatorial treatment was plotted according to the doses of bortezomib used in the treatment. All the data of cell viability tests were presented as mean ± standard deviation (n = 3)