Interplay between Endoplasmic Reticulum (ER) Stress and Autophagy Induces Mutant p53H273 Degradation.

The unfolded protein response (UPR) is an adaptive response to intrinsic and external stressors, and it is mainly activated by the accumulation of misfolded proteins at the endoplasmic reticulum (ER) lumen producing ER stress. The UPR signaling network is interconnected with autophagy, the proteolytic machinery specifically devoted to clearing misfolded proteins in order to survive bioenergetic stress and/or induce cell death. Oncosuppressor TP53 may undergo inactivation following missense mutations within the DNA-binding domain (DBD), and mutant p53 (mutp53) proteins may acquire a misfolded conformation, often due to the loss of the DBD-bound zinc ion, leading to accumulation of hyperstable mutp53 proteins that correlates with more aggressive tumors, resistance to therapies, and poorer outcomes. We previously showed that zinc supplementation induces mutp53 protein degradation by autophagy. Here, we show that mutp53 (i.e., Arg273) degradation following zinc supplementation is correlated with activation of ER stress and of the IRE1α/XBPI arm of the UPR. ER stress inhibition with chemical chaperone 4-phenyl butyrate (PBA) impaired mutp53 downregulation, which is similar to IRE1α/XBPI specific inhibition, reducing cancer cell death. Knockdown of mutp53 failed to induce UPR/autophagy activation indicating that the effect of zinc on mutp53 folding was likely the key event occurring in ER stress activation. Recently discovered small molecules targeting components of the UPR show promise as a novel anticancer therapeutic intervention. However, our findings showing UPR activation during mutp53 degradation indicate that caution is necessary in the design of therapies that inhibit UPR components.


Introduction
Tumor suppressor p53 plays a central role in tumor prevention and response to therapies. The presence of a functional p53 pathway is incompatible with neoplastic growth and, for this reason, p53 is the most frequently mutated gene in tumors [1]. The majority of p53 mutations are missense mutations (i.e., R175H, R248Q, and R273H) that are mainly within the DNA-binding domain (DBD), leading to the synthesis of p53 proteins that are unable to bind target gene promoters; however, some mutant (mut) p53 proteins can physically bind other transcription factors, profoundly remodeling the

Viability Assay and Cell Death/PI Staining
Subconfluent cells were plated in triplicate in six-well plates and, the day after, were treated with Zn (II)-curc (100 µg/mL) for 24 h. Both adherent and floating cells were collected and cell viability was assessed by Trypan blue (Sigma-Aldrich, #72571) exclusion counting blue (dead)/total cells with a Neubauer hemocytometer using light microscopy.
Cell death was detected by cytofluorimetric analysis of propidium iodide (PI)-stained cells, as previously reported [12]. Briefly, both floating and adherent cells were fixed in 80% ethanol and stained in a PBS solution containing PI (62.5 mg/mL; Sigma-Aldrich, #P4864) and RNase A (1.125 mg/mL; Sigma-Aldrich, #R6148). Samples were acquired with a FACScan instrument (Becton Dickinson) and the percentage of cells in the sub-G1 compartment was calculated using ModFit LT software (Becton Dickinson).

Endoplasmic Reticulum (ER) Staining
Subconfluent cells were seeded on coverslips in 35 mm Petri dishes and, the day after, were treated with Zn (II)-curc (100 µg/mL) for 16 h. After treatment, cells were fixed with 3.7% paraformaldehyde (Thermo Fisher Scientific, #50-980-487) for 10 min at room temperature (RT) and stained with ER Staining Kit-Red Fluorescence (Abcam, #ab139482) following the manufacturer's instructions. Immunofluorescence was visualized by an Olympus BX53 microscope equipped with epifluorescence, and photographs were taken (×40 objective) using a cooled camera device (ProgRes MF). ImageJ (NIH) software [33] (http://imagej.nih.gov) was used to calculate the relative fluorescence from 40× magnification images and normalized to cell size from phase-contrast images. At least 25 cells were analyzed in duplicate for each group at the same exposure time.

RNA Extraction and Semi-Quantitative Reverse Transcription (RT)-PCR Analysis
Cells were harvested in TRIzol Reagent (Invitrogen, #15596026) and total RNA was isolated following the manufacturer's instructions. The first strand cDNA was synthesized from 2 µg of total RNA with a MuLV reverse transcriptase kit (TermoFisher Scientific, #28025013). Semi-quantitative Reverse-Transcribed (RT)-PCR was carried out by using Hot-Master Taq polymerase (Geneaid Biotech Ltd., New Taipei City, Taiwan, #TQ050) with 2 µL cDNA reaction and genes specific oligonucleotides under the conditions of linear amplification. The primers used for amplification of Xbp1s were as follows: Xbp1s-for GGAGTTAAGACAGCGCT; Xbp1s-rev TGTTCTGGAGGGGTGAC. PCR products were run on a 2% agarose gel and visualized with ethidium bromide. The housekeeping 28S gene, used as the internal standard, was amplified from the same cDNA reaction mixture. Densitometric analysis was applied to quantify mRNA levels compared to control gene expression.

siRNA Interference
Subconfluent cells were plated in 35 mm Petri dishes and, the day after, were transfected with control pSuper and pSuper-p53 (for p53 interference, si-p53) vectors [34] using the LipofectaminePlus reagent (Invitrogen, #11514-015), according to the manufacturer's instructions. Twenty-four hours after transfection, cells were trypsinized and replated for the indicated experiments.

Statistical Analysis
Each experiment was performed at least three times. Results are expressed as values of mean ± standard deviation (S.D.). Statistical significance was determined using Student's t-tests for two sample comparison. Difference was considered statistically significant when the p-value was at least <0.05.

ER Stress Sis Activated by Zn(II)-Curc in mutp53H273 Cancer Cells
As Zn (II)-curc may induce mutp53 protein degradation through autophagy [14,15], and autophagy can be induced by UPR activation [27], we investigated the effect of Zn(II)-curc on ER stress/UPR activation. To address this issue, we used several approaches. U373 cells were treated with 100 µM Zn (II)-curc, the dose that induces mutp53 degradation and autophagy [14,15]. ER analysis by specific fluorescent red labelling shows that Zn (II)-curc caused a marked increase in ER size (Figure 1a,b). This increase correlated with the augmented expression of BiP/Grp78 as well as with the IRE1α and p-IRE1α protein levels in both U373 and HT29 cells (Figure 1c,d), while the other arms of the UPR were not affected in this experimental condition (not shown). Western blot and semiquantitative RT-PCR evidenced spliced (s) Xbp1 mRNA (Figure 1c-e), in agreement with the notion that activated IRE1α functions as an endoribonuclease, splicing a 26 base pair intron from Xbp1 mRNA [35]. The Zn (II)-curc-induced activation of IRE1α correlated with the reduction of mutp53 expression levels (Figure 1c), while the p53 gene expression was not affected (Figure 1f). Interestingly, the Zn (II)-curc treatment of T98 cells (expressing M237I p53 mutation), which we previously reported not been affected by Zn (II)-curc at a biological level [12], did not increase BiP levels or reduced p53 protein levels (Supplementary Material Figure S1). * p ≤ 0.05. (c) Western blot analysis of p53, BiP, total (tot) IRE1α, phosphorylated (p) IRE1α, and XBP1 spliced (s) protein levels evaluated in U373 and HT29 cells untreated or treated with Zn (II)-curc (100 µg/mL) for 24 h. (d) Densitometric analysis was performed using Image J software to calculate the ratio of the protein levels, as detected in (c), vs. β-actin. Histograms represent the mean ± SD of three independent experiments. * p ≤ 0.05. (e) Total mRNA was extracted from U373 and HT29 cells untreated or treated with Zn (II)-curc (100 µg/mL) for 24 h. Spliced (s) Xbp1 gene expression was assayed by the polymerase chain reaction (PCR) of reverse-transcribed cDNA. Densitometric analysis was performed using Image J software to calculate the Xbp1s/28S ratio. Histograms represent the mean ± SD of three independent experiments. * p ≤ 0.05. (f) p53 gene expression was assayed by PCR as in (e). The p53/28S ratio is indicated.
To further address the correlation between mutp53H273 degradation and ER stress activation, wtp53-expressing cells were treated with Zn (II)-curc. As shown in Figure 2a, Zn(II)-curc did not increase BiP expression levels or induce Xbp1 splicing (not shown) in wtp53-expressing cells, while it stabilized endogenous wtp53 protein levels in accordance with previous studies where zinc supplementation induced wtp53 oncosuppressor activities and the transcription of target genes such as p21, Puma, and Bax [14,36]. On the contrary, both BiP ( Figure 2b) and Xbp1 splicing (Figure 2c) were efficiently induced in wtp53-expressing cells by Tunicamycin (Tn), a drug causing ER stress by inhibiting N-linked glycosylation [37]. Altogether, these results indicate that the IRE1α/XBP1 arm of UPR was activated in response to Zn (II)-curc only in mutp53H273 cells.

ER Stress/IRE1α Inhibition Impairs Zn(II)-Curc-Induced Autophagy and Mutp53 Degradation
To evaluate the role of ER stress/UPR activation in Zn (II)-curc-induced autophagy, we analyzed the LC3I/II level (a cellular readout of autophagy) [18] by Western blot in U373 cells treated with Zn (II)-curc with or without pre-treatment with 4-Phenyl butyric acid (PBA), a molecular chaperone that dampens the ER stress [30]. The results show reduction of LC3II levels in the presence of 4-BPA, concomitantly to the reduction of Zn (II)-curc-induced BiP protein levels (Figure 3a,b) and Xbp1 splicing (Figure 3c), suggesting that a possible autophagy inhibition was occurring following ER stress inhibition, which is in agreement with the known role of Xbp1 splicing in triggering autophagy [38].
Concomitantly, we verified the effect of ER stress inhibition on mutp53 levels and found that mutp53 protein was no longer downregulated in the presence of 4-BPA (Figure 3a,b). (b) Densitometric analysis was performed using Image J software to calculate the ratio of the protein levels, as detected in (a), vs. β-actin. Histograms represent the mean ± SD of three independent experiments. * p ≤ 0.05. (c) Total mRNA was extracted from U373 cells untreated or treated as in (a). Spliced (s) and unspliced (u) Xbp1 gene expression were assayed by the PCR of reverse-transcribed cDNA. Densitometric analysis was performed using Image J software to calculate the Xbp1s/28S ratio. Histograms represent the mean ± SD of three independent experiments. * p ≤ 0.05.

ER Stress or IRE1α Inhibition Reduces Zn(II)-Curc-Induced Cell Death
Next, we evaluated the role of ER stress/IRE1α activation on Zn (II)-curc-induced cell death. To this aim, we combined Zn (II)-curc with either ER stress or IRE1α inhibitors. The results show that both 4-BPA and STF efficiently reduced the Zn (II)-curc-induced cell death (Figure 5a,b), suggesting that the activation of ER stress balanced the cell death/survival equilibrium towards cell death. Similarly, the inhibition of wtp53 transcriptional activity with PFT-α (Figure 5a,b), as well as p53 knockdown (Figure 5c), reduced the Zn (II)-curc-triggered cell death, in agreement with the effect of Zn(II)-curc on the reactivation of wtp53 oncosuppressor functions, as previously reported [12,14,15]. Altogether, these results suggest that both ER stress/UPR activation and wtp53 reactivation by Zn (II)-curc promoted cell death in mutp53 cells. (c) U373 and HT29 cells were transfected with control pSuper (si-ctr) and pSuper-p53 (si-p53) vectors for p53 knockdown and 36 h after transfection, cells were treated with Zn(II)-curc (100 µg/mL) for 24 h. Cell viability was measured by a trypan blue exclusion assay and expressed as percentage ± SD of three independent experiments. * p ≤ 0.05.

Mutp53 Knockdown Abrogates the Zn(II)-Curc-Induced ER Stress
Finally, we asked whether Zn (II)-curc-induced mutp53 downregulation was upstream of the ER stress or rather the consequence of UPR/autophagy activation. To address this issue, p53 was knocked-down with siRNA, and ER stress was analyzed by several approaches. Analysis of ER by immunofluorescence shows that the increase in ER size by Zn (II)-curc was abrogated in the U373 cells undergoing p53 knockdown ( Figure 6a); in agreement, the Zn (II)-curc-induced BiP protein levels ( Figure 6b) and Xbp1 splicing (Figure 6c) were strongly reduced by p53 knockdown in both U373 and HT29 cells, suggesting that mutp53H273 degradation by Zn (II)-curc was likely upstream of the ER stress/UPR activation.

Discussion
Cancer cells are often exposed to intrinsic and external factors that alter protein homeostasis. The consequence is the activation of ER stress and UPR, which is the adaptive mechanism used to cope with ER stress and to restore ER proteostasis [21]. Activation of the UPR through three different but interconnected signaling pathways may favor pro-death or prosurvival signaling and is strictly linked to autophagy. Autophagy is usually a prosurvival mechanism, playing a crucial role in drug resistance, although it may also induce cell death [39,40]. The interplay between autophagy and UPR and their abnormal activation may pave the way to degenerative and chronic diseases including cancer [28,41]. Therefore, understanding the crosstalk between UPR activities and autophagy should help in developing new treatment options for various pathologies, including cancer. Here, we found that autophagy-mediated mutp53H273 degradation following zinc supplementation correlated with activation of ER stress and of the IRE1α/XBPI arm of the UPR. ER stress or IRE1α/XBPI inhibition impaired mutp53 degradation and reduced cell death, suggesting that both ER stress/UPR activation and the clearance of mutp53 by zinc played a pro-death role in mutp53-carrying cells, as summarized in Figure 7. . Schematic representation of the interplay between ER stress/unfolded protein response (UPR) activation and autophagy in response to Zn (II)-curc in mutp53H273-carrying cancer cells. Diagram illustration describes the pathways contributing to mutp53H273 degradation by autophagy and the role of ER stress/UPR activation. The arrows indicate activation, and the red lines indicate blocking. The possible starting even could be the Zn-induced mutp53 folding/unfolding balance, as previously reported [8,11,36,37].
Mutp53 proteins may acquire a misfolded conformation forming hyperstable micro-and macro-aggregates that cannot undergo degradation, with consequent accumulation in tumors [5]. This aggregation may result from the loss or alteration of DBD-bound zinc, which is necessary for the thermodynamic stability of the DBD, and is needed for wtp53 oncosuppressor function [6]. Our previous studies demonstrated that zinc supplementation was able to modify the equilibrium between the p53 mutant and wild-type conformation, positively reactivating wtp53 functions of some of the most frequently p53 mutated residues, such as Arg175 and Arg273 [9][10][11][12]14,42,43], which is in line with the findings that zinc metallochaperone rescues mutant p53 conformation [44]. Based on these findings, here we can speculate that the effect of zinc on mutp53 folding/unfolding unbalance could act as a cellular stress to trigger UPR activation. Thus, the depletion of mutp53 failed to recapitulate the effect of zinc on UPR activation, and zinc did not activate UPR in wtp53-carrying cells. In addition, zinc treatment of cells carrying a different mutant p53 protein (i.e, M237I) did not induce mutp53 degradation or trigger UPR activation. Therefore, we can reasonably affirm that the UPR activation by zinc is linked to the degradation of the mutp53 protein and, in particular, of mutp53H273. However, further studies are needed to unveil the mechanisms that are involved in UPR activation by degradation of some mutp53 proteins by zinc.
UPR activation is commonly observed in various tumor specimens and correlates with drug resistance [45]. UPR is orchestrated by three main sensors, and our data show that the IRE1α-XBP1 arm of UPR was mainly activated during mutp53 degradation through autophagy, likely in an autoregulatory loop, although the exact mechanism by which mutp53 degradation increases ER stress-induced activation of IRE1α/XBPI remains to be defined. The IRE1α-XBP1 pathway is the most conserved signaling branch of the UPR and plays important roles in both physiological and pathological settings, with its activity having profound effects on disease progression and prognosis [35]. The IRE1α signaling pathway induces the expression of the transcription factor Xbp1s, which increases the expression of ER chaperones and ER mass, stimulates lipid biogenesis, and degrades unfolded proteins to enhance the secretory function of ER and triggers autophagy initiation, mainly serving as a pro-survival pathway in multiple human cancers [35]. XBP1 pathway activation has been shown to induce triple-negative breast cancer progression and is correlated with poor patient survival, suggesting that UPR inhibitors in combination with chemotherapy may improve tumor regression [46]. In addition, unspliced XBP1 is associated with longer survival of breast cancer patients treated with tamoxifen, which is opposite to XBP1 splicing that is associated with shorter survival [47]. Therefore, developing combination therapies that target specific UPR signaling pathways will hopefully bypass anticancer drug resistance. On the other hand, we found here that XBP1 inhibition impaired zinc-induced autophagy and mutp53 degradation, indicating that UPR modulation may achieve different effects depending on cell context. To make the story more complex, mup53 has been shown to favor cancer cell survival and promote cancer progression by its crosstalk with cellular stress pathways [48]. For instance, the downregulation of mutp53 interferes with the downstream activation of the cross-talk between NRF2 and p62, restoring the cytotoxic effect of chemotherapies [49], and mutp53 inhibits the pro-apoptotic and pro-survival UPR effectors PERK and ATF6 in cancer cell lines [50], suggesting that UPR inhibitors combined with mutp53 inhibitors may increase cell death.

Conclusions
In conclusion, this study demonstrates for the first time that a curcumin-based zinc complex was able to induce UPR activation to trigger autophagy and mutp53H273 degradation, highlighting the interplay between ER stress and autophagy. As UPR activation has both pro-survival and pro-death effects, caution is necessary in the design of therapies that target UPR components in combination with chemotherapies to increase the antitumor response, as they could hamper mutp53 degradation.