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. 2020 Nov;13(11):100834.
doi: 10.1016/j.tranon.2020.100834. Epub 2020 Jul 17.

UAE1 inhibition mediates the unfolded protein response, DNA damage and caspase-dependent cell death in pancreatic cancer

Yajing Liu  1 , Sahezeel Awadia  1 , Amy Delaney  1 , Merna Sitto  1 , Carl G Engelke  1 , Heli Patel  1 , Andrew Calcaterra  1 , Sylvia Zelenka-Wang  2 , Hojin Lee  3 , Joseph Contessa  3 , Nouri Neamati  4 , Mats Ljungman  1 , Theodore S Lawrence  1 , Meredith A Morgan  1 , Alnawaz Rehemtulla  5
Affiliations

UAE1 inhibition mediates the unfolded protein response, DNA damage and caspase-dependent cell death in pancreatic cancer

Yajing Liu et al. Transl Oncol. 2020 Nov.

Abstract

The Unfolded Protein Response (UPR) plays a key role in the adaptive response to loss of protein homeostasis within the endoplasmic reticulum (ER). The UPR has an adaptive function in protein homeostasis, however, sustained activation of the UPR due to hypoxia, nutrient deprivation, and increased demand for protein synthesis, alters the UPR program such that additional perturbation of ER homeostasis activates a pro-apoptotic program. Since ubiquitination followed by proteasomal degradation of misfolded proteins within the ER is a central mechanism for restoration of ER homeostasis, inhibitors of this pathway have proven to be valuable anti-cancer therapeutics. Ubiquitin activating enzyme 1(UAE1), activates ubiquitin for transfer to target proteins for proteasomal degradation in conjunction with E2 and E3 enzymes. Inhibition of UAE1 activity in response to TAK-243, leads to an accumulation of misfolded proteins within the ER, thereby aggravating ER stress, leading to DNA damage and arrest of cells in the G2/M phase of the cell cycle. Persistent drug treatment mediates a robust induction of apoptosis following a transient cell cycle arrest. These biological effects of TAK-243 were recapitulated in mouse models of PDAC demonstrating antitumor activity at a dose and schedule that did not exhibit obvious normal tissue toxicity. In vitro as well as studies in mouse models failed to show enhanced efficacy when TAK-243 was combined with ionizing radiation or gemcitabine, providing an impetus for future studies to identify agents that synergize with this class of agents for improved tumor control in PDAC. SIGNIFICANCE: The UAE1 inhibitor TAK-243, mediates activation of the unfolded protein response, accumulation of DNA breaks and apoptosis, providing a rationale for the use as a safe and efficacious anti-cancer therapeutic for PDAC.

Keywords: ER stress; Pancreatic cancer; UAE1 Inhibitor.

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Conflict of interest statement

Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Unlabelled Image
Graphical abstract
Fig. 1
Fig. 1
TAK-243 induces apoptosis in a dose and time dependent manner. (A) A non-invasive reporter for apoptosis. Activated caspase-3 cleaves the DEVD sequence within the reporter to form a functioning luciferase enzyme that enzymatically releases photons of light in the presence of luciferin as substrate. (B) MiaPaCa-2 and Panc-1 cells with apoptosis reporter were dosed with CTEP compounds (1 μM) and luciferase activity was measured every 30 min to up to 19 h. (C) MiaPaCa-2, Panc-1 and KPC2 cells with apoptosis reporter in a 96-well plate were treated with TAK-243 at indicated concentration together with luciferin (150 μg/mL) and the plate was read every 30 min to up to 36 h. (D) MiaPaCa-2 and (E) Panc-1 cells were treated with TAK-243 at 50, 100, 200, 300 and 1000 nM or tunicamycin (5 μg/mL) for 4–24 h and the cells were lysed and assessed by immunoblotting for cleaved PARP.
Fig. 2
Fig. 2
TAK-243 activates the unfolded protein response. (A) MiaPaCa-2 cells were treated with 300 nM TAK-243 for 1, 2, 4 and 6 h and total RNA was extracted for qRT-PCR of GRP78 and spliced XBP-1. Data is presented as mean ± SEM from three experiments, *, p < 0.05; **, p < 0.01; ***, p < 0.001. (B) IRE1 activity sensor expresses mNeonGreen when XBP-1 is spliced. Representative pictures of (C) MiaPaCa-2 and (D) Panc-1 (E) cells with spliced IRE1 reporter after TAK-243 or DMSO treatment at different time point. (E) Quantification of spliced XBP-1 fluorescence signal over surface area in MiaPaCa-2 and Panc-1 cells treated with 300 nM TAK-243, data is presented as mean ± SEM from three technical replicates. Immunoblotting of UPR markers: ATF-4, BIP and CHOP in (F) MiaPaCa-2, (G) Panc-1 and KPC2 (H) cells after TAK-243 or tunicamycin treatment at indicated dose and time. (I) Quantification of spliced XBP-1 fluorescence signal over surface area in MiaPaCa-2 cells treated with 300 nM TAK-243, BAP2, Tunicamycin, NGI-1 and PDI SiRNA. Data is presented as mean ± SEM from three technical replicates.
Fig. 2
Fig. 2
TAK-243 activates the unfolded protein response. (A) MiaPaCa-2 cells were treated with 300 nM TAK-243 for 1, 2, 4 and 6 h and total RNA was extracted for qRT-PCR of GRP78 and spliced XBP-1. Data is presented as mean ± SEM from three experiments, *, p < 0.05; **, p < 0.01; ***, p < 0.001. (B) IRE1 activity sensor expresses mNeonGreen when XBP-1 is spliced. Representative pictures of (C) MiaPaCa-2 and (D) Panc-1 (E) cells with spliced IRE1 reporter after TAK-243 or DMSO treatment at different time point. (E) Quantification of spliced XBP-1 fluorescence signal over surface area in MiaPaCa-2 and Panc-1 cells treated with 300 nM TAK-243, data is presented as mean ± SEM from three technical replicates. Immunoblotting of UPR markers: ATF-4, BIP and CHOP in (F) MiaPaCa-2, (G) Panc-1 and KPC2 (H) cells after TAK-243 or tunicamycin treatment at indicated dose and time. (I) Quantification of spliced XBP-1 fluorescence signal over surface area in MiaPaCa-2 cells treated with 300 nM TAK-243, BAP2, Tunicamycin, NGI-1 and PDI SiRNA. Data is presented as mean ± SEM from three technical replicates.
Fig. 3
Fig. 3
TAK-243 induces cell cycle arrest, DNA damage and leads to a tumor growth delay. (A) FACS analysis of DNA content in MiaPaCa-2 cells at 4, 6, 16, 24 h after DMSO, TAK-243 and tunicamycin treatment. (B) Representative images of comet assay from MiaPaCa-2 cells at indicated time points following 300 nM TAK-243 treatment. (C) Quantification of percentage of tail moment in MiaPaCa-2 cells after TAK-243 treatment, values are expressed as mean ± SEM for at least 100 counts from each condition. (D) MiaPaCa-2 tumor progression in control/DMSO, gemcitabine, TAK-243 and combination group, graph is plotted as mean ± SEM from at least 4 mouse. Representative images of immunohistochemistry on (E) ATF-4 and (F) Caspase-3 in tumors and their quantifications. Error bars represent S.E.M. *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.00005, ns: non-significant. (G) Quantification of ATF-4 and Caspase-3 expression and (H) Hematoxylin and eosin staining.
Fig. 3
Fig. 3
TAK-243 induces cell cycle arrest, DNA damage and leads to a tumor growth delay. (A) FACS analysis of DNA content in MiaPaCa-2 cells at 4, 6, 16, 24 h after DMSO, TAK-243 and tunicamycin treatment. (B) Representative images of comet assay from MiaPaCa-2 cells at indicated time points following 300 nM TAK-243 treatment. (C) Quantification of percentage of tail moment in MiaPaCa-2 cells after TAK-243 treatment, values are expressed as mean ± SEM for at least 100 counts from each condition. (D) MiaPaCa-2 tumor progression in control/DMSO, gemcitabine, TAK-243 and combination group, graph is plotted as mean ± SEM from at least 4 mouse. Representative images of immunohistochemistry on (E) ATF-4 and (F) Caspase-3 in tumors and their quantifications. Error bars represent S.E.M. *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.00005, ns: non-significant. (G) Quantification of ATF-4 and Caspase-3 expression and (H) Hematoxylin and eosin staining.
Fig. 3
Fig. 3
TAK-243 induces cell cycle arrest, DNA damage and leads to a tumor growth delay. (A) FACS analysis of DNA content in MiaPaCa-2 cells at 4, 6, 16, 24 h after DMSO, TAK-243 and tunicamycin treatment. (B) Representative images of comet assay from MiaPaCa-2 cells at indicated time points following 300 nM TAK-243 treatment. (C) Quantification of percentage of tail moment in MiaPaCa-2 cells after TAK-243 treatment, values are expressed as mean ± SEM for at least 100 counts from each condition. (D) MiaPaCa-2 tumor progression in control/DMSO, gemcitabine, TAK-243 and combination group, graph is plotted as mean ± SEM from at least 4 mouse. Representative images of immunohistochemistry on (E) ATF-4 and (F) Caspase-3 in tumors and their quantifications. Error bars represent S.E.M. *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.00005, ns: non-significant. (G) Quantification of ATF-4 and Caspase-3 expression and (H) Hematoxylin and eosin staining.
Fig. 4
Fig. 4
A lack of synergy when TAK-243 is combined with DNA damage inducing therapies. Apoptotic signal in (A) MiaPaCa-2 and (B) Panc-1 cells treated with TAK-243 at indicated concentrations were radiated at 0, 2, 4, 6, 8 Gy and survival was assessed by plating cells 24 h post radiation. The values are expressed as mean ± SEM from three biological replicates. (C) MiaPaCa-2 and (D) Panc-1 cells with 72 h of PDI shRNA induction were radiated at 0, 2, 4, 6, 8 Gy and survival was assessed by plating cells 24 h post radiation, values are expressed as mean ± SEM from three biological replicates.
Fig. 5
Fig. 5
TAK-243 mediated UPR is decoupled from ERAD. MiaPaCa-2 cells were treated with (A) TAK-243 (300 nM), (B) tunicamycin (5 μg/mL), (C) NGI-1 (10 μM) with or without MG132 (10 μM) for 16, 12 and 6 h respectively, the cells were also transfected with (D) PDI siRNA for 48 h and MG132 was added for the last 16 h. Cell lysates were collected for immunoblotting for BIP, ATF4, RAD51, PDI and β-actin, and protein level was quantified and plotted as mean ± SEM from three biological replicates. MiaPaCa-2 cells were treated with (E) tunicamycin (5 μg/mL) with or without MG132 (10 μM) for 8 h and lysates were immunoprecipitated using RAD51 antibody, and the precipitates were immunoblotted with RAD51 and ubiquitin. (F) Cells were transfected with PDI siRNA and treated with or without with MG132 and total lysates were immunoblotted with ubiquitin and RAD51. (G) MiaPaCa-2 cells were treated with TAK-243 (300 nM) with or without MG132 (10 μM) for 6 h and lysates were immunoprecipitated as described above. Error bars represent S.E.M. *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.00005, ns: non-significant.
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