PRIMA-1met Induces Autophagy in Colorectal Cancer Cells With Different p53 Status

doi:10.21203/rs.3.rs-73569/v1

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PRIMA-1met Induces Autophagy in Colorectal Cancer Cells With Different p53 Status

https://doi.org/10.21203/rs.3.rs-73569/v1

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Version 1

posted 16 Sep, 2020

You are reading this latest preprint version

Background: PRIMA-1^met (APR246), a methylated form of PRIMA-1 (p53-reactivation and induction of massive apoptosis-1, APR-017), targets mutant p53 for restoring its wild-type structure and function. We previously demonstrated that PRIMA-1^met was efficient in suppressing the growth of colorectal cancer (CRC) cells in a p53-independent manner, and distinctly induced apoptosis mediated by up-regulation of Noxa in p53-mutant cell lines. Here we aimed to the effect of PRIMA-1^met on autophagy in different CRC cell lines, to further investigate mechanisms underlying the inhibitory effect in cells with different p53 status.

Methods: 3 CRC cell lines with wild-type p53, 5 lines with mutant p53 and 1 line without p53 were obtained for this study. Using western blotting, acridine orange staining, and transmission electron microscopy detection, we assessed autophagy flux in different cells treated with PRIMA-1^met, and detected expression of mTOR/AMPK-ULK1-Vps34 autophagic signaling cascade. We also evaluated cell proliferation of cells with PRIMA-1^met treatment by cell counting Kit-8 proliferation assay, compared to combination of PRIAM-1^met and 3-Methyladenine. Furthermore, we knocked down Noxa gene by siRNA in different CRC cells, to assess LC3 conversion after administration of PRIMA-1^met. Values were expressed as mean + standard error of the mean. Comparison between groups of data was made using one-way analysis of variance.

Results: In this study, we showed that PRIMA-1^met induced autophagy in CRC cells independent on p53 status. PRIMA-1^met not only promoted autophagic vesicles (AVs) formation and AV-lysosome fusion, but also increased lysosomal degradation. Mechanistically, activation of mTOR/AMPK-ULK1-Vps34 autophagic signaling cascade was important for PRIMA-1^met-induced autophagy. Furthermore, autophagy played a crucial role in the inhibitory effect of PRIMA-1^met only in cells harboring wild-type p53, which was closely related to the increased Noxa.

Conclusions: Our results indicated that PRIMA-1^met induced autophagy in CRC cells regardless of p53 status via activating mTOR/AMPK-ULK1-Vps34 signaling cascade. However, induced autophagy was relevant to the cytotoxicity of PRIMA-1^met in cells carrying wild-type p53, along with up-regulation of Noxa. Implying that, PRIMA-1^met-based therapy could be an effective strategy for CRC.

Trail registration: Not applicable.

Cancer Biology

Oncology

colorectal cancer (CRC)

p53

PRIMA-1met

autophagy

oncotherapy

Colorectal cancer (CRC, cancer of colon and rectum) is a worldwide health problem that is the second and the third most common cancer in women and men (www.wcrf.org), respectively. There were over 1.8 million new CRC cases and 0.88 million deaths in 2018[1]. In recent years, the incidence of CRC, especially rectal cancer, showed an increasing trend in youngers [2]. Early diagnosis and radical surgery effectively improve the 5-year survival rate, however, there is still a lack of efficacious measures for advanced patients of stage III and stage IV.

Activation of oncogenes and inactivation/defect of tumor suppressor genes (TSGs) are crucial for the tumorigenesis of CRC. p53, a transcriptional factor, regulates processes of cell cycle arrest, apoptosis, DNA repair, and tumor angiogenesis via mediating numerous targeted genes. Importantly, p53 mutation occurring in about 40–50% CRC patients [3], is associated with resistance to current regimens and poor prognosis. PRIMA-1 (p53-reactivation and induction of massive apoptosis-1, APR-017), a low molecule compound (C9H5NO3), was discovered to restore the sequence-specific DNA binding domain of mutant p53 by forming adducts with thiols for recovering the wild-type structure and function to induce cell apoptosis, thus selectively killing cancer cells with mutant p53 [4, 5]. PRIMA-1^met (APR246) as a methylated analog of PRIMA-1, is more effective on p53-mutant cells, and further certified to inhibit the growth of cells without mutant p53 [5], [6–9]. In our previous study, we have demonstrated that PRIMA-1^met suppressed CRC cell proliferation, migration, invasion, and colony formation independently on p53 status. Nevertheless, PRIMA-1^met induced robust apoptosis in cells carrying mutant p53 via up-regulation of proapoptotic Noxa [10]. However, mechanisms underlying the cytotoxicity of PRIMA-1^met in different CRC cell lines is still not entirely unknown.

Autophagy is a “self-eating” response to intracellular and environmental stimuli, such as starvation, nutrient deprivation, energy exhaustion, through regulatory factors that consist of the products of autophagy-related genes (ATGs) occurring in both normal and cancer cells. Once receiving nutrient or energy signals, regulators of autophagy induce the process, involving the formation of double-membraned autophagosome, fusion with lysosomes with cargoes, degradation, and recycling. By the process, intracellular proteins and organelles are able to be degraded in lysosomes and released into the cytoplasm for biosynthesis and metabolism recycling[11]. Autophagy originally acted as a tumor suppressive process based on the deletion of autophagy gene Beclin-1 (ATG6) in 40–75% human breast, ovarian and prostate cancers [12, 13]. Recent investigation has indicated that autophagy exerted an inhibiting influence on tumor initiation and progression via suppressing chronic inflammation, DNA damage, and genomic instability of tumor environment [14]. Thus, some pivotal points of autophagic pathways, such as mTOR (mammalian target of rapamycin), AMPK (Adenosine monophosphate-activated protein kinase), Akt (protein kinase B), PI3K (phosphoinositide 3-kinases ) class III, Beclin-1, p53, etc, become promising targets for anti-cancer studies.

In this study, we shed new light on the effect of PRIMA-1^met on autophagy in different CRC cell lines, to further investigate mechanisms underlying the inhibitory effect in cells with different types of p53. Our results showed that PRIMA-1^met induced autophagy in different CRC cells irrespectively of p53 status via activating mTOR/AMPK-ULK1-Vps34 signaling cascade. Furthermore, up-regulated autophagy played a crucial role in the suppressed effect of PRIMA-1^met on cells carrying wild-type p53, which was related to up-regulation of Noxa. These findings further indicated deeper mechanisms of PRIMA-1^met for CRC.

Cell lines and drugs

CRC cell lines used in this study with wild-type or mutant p53 were obtained from Dr. Zhang Ting (Cancer Institute, Affiliated Hospital of Jiangnan University, China). The details were as follows: LOVO (wild-type p53), RKO (wild-type p53), HCT116 (wild-type p53), DLD-1 (mutant p53-S241F), SW480 (mutant p53-R237H), SW620 (mutant p53-R237H), HCT15 (mutant p53-P153A), and CaCO2 (mutant p53-E204X). HCT116 null (p53 -/-) cells were provided from Dr. Jimmy Chao (Bioprocessing Technology Institute, A*Star, Singapore). Cells were cultured in DMEM (Dulbecco’s Modified Eagle’s Medium, Hyclone, Logan, USA), except SW620 in RPMI-1640 Medium (Hyclone, Logan, USA), with 10% FBS (fetal bovine serum, Gibco life technology, New York, USA) and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA) in a humidified incubator with 5% CO₂ at 37℃. PRIMA-1^met (APR-246, Santa Cruz Biotechnology, CA) dissolved in DMSO (Dimethyl sulfoxide) at the concentration of 50 mM and 3-Methyladenine (3-MA, Absin Bioscience Inc, Shanghai, China) dissolved in PBS (phosphate buffer saline) as 67.04 mM were both stored at -20℃. Working solutions were diluted to appropriate concentrations with culture medium. Same amount of DMSO as control reagent was added in experiments.

Western blotting analysis

Different CRC cells were seeded at the density of 2 × 10⁶ cells per 100 mm dish overnight, and treated with DMSO, different doses of PRIMA-1^met or 3-MA (800 µM). After incubated for 24 hours, cells were harvested, lysed and analyzed by western blotting. Primary antibodies used in this study were against β-Actin (AC026, ABclonal Technology, Wuhan, China), LC3 (NB100-2220, Novus Biologicals, US), Noxa (OP180, EMD Millipore, Gibbstown, NJ), p62 (#8025), ULK1 (#8054), phospho-ULK1 (Ser757) (#14202), mTOR (#2983), phospho-mTOR (Ser2448) (#5536), AMPK (#2532), phospho-AMPK (Thr172) (#2535) and phospho-PI3K Class III (Ser249) (#13857) (Cell Signaling Technology, Danvers, MA) diluted in blocking solution at the ratio of 1:1000. Second antibodies were goat-anti-rabbit IgG HRP (AS014), and goat-anti-mouse IgG HRP (AS003, ABclonal Technology, Wuhan, China) diluted in blocking solutions at the ratio of 1:5000. Expression level of proteins was estimated with gray value calculated using ImageJ version 1.8.0 (National Institutes of Health, USA) and presented in percentage normalized to β-Actin.

Acridine orange (AO) staining

A panel of CRC cells with different p53 status (HCT116^wt, RKO, CaCO2, SW480 and HCT116^ko) were seeded in 24-well plates containing cell slides at the density of 6 × 10⁴ cells with 1 ml medium per well overnight, and treated with DMSO or 30 µM PRIMA-1^met for 24 hours. AO (Absin Bioscience Inc, Shanghai, China) solution was added to each well at a concentration of 1 µg/ml, followed by incubation at 37℃ for 15 minutes. Rinsed with PBS twice, slides were taken out and inverted onto carry sheet glass. The slides of cells were observed and taken pictures under confocal microscopy with the filter excited at 488 nm and blocked at 515 nm (Hitachi, Japan).

Transmission electron microscopy (TEM) detection

RKO and HCT15 cells were seeded in 100 mm dishes at the density of 2 × 10⁶ cells per dish overnight, and then treated with DMSO, 30 µM PRIMA-1^met or 60 µM PRIMA-1^met. After 24 hours, cell pellets were harvested, and fixed with 4% glutaraldehyde solution at 4℃ for 4 hours, then followed by 2-hour fixation with 1% osmic acid at 20℃. Specimens were dehydrated by 50–100% ethanol and 100% acetone, and embedded with Epon 812. Cut into 60–80 nm, slides were stained with both 2% uranyl acetate and lead citrate to observe AVs with double membrane and autolysosomes under a TEM (Hitachi, Japan).

Cell counting Kit-8 (CCK-8) proliferation assay

After 24 hours of seeding in a 96-well plate at the density of 7000 cells/well with 100 µl medium, cells were treated with DMSO, PRIMA-1^met (45 µM for HCT116^wt, 50 µM for RKO, 55 µM for DLD-1 and HCT15), 3-MA (800 µM) or combination, and incubated for 48 hours. Cell Counting Kit-8 (CCK-8) proliferation assay (Dojindo, Shanghai, China) was used for measuring the cell proliferation by quantitating WST-8 formazan dye which was proportional to living cells according to previous description[15]. The reading value of DMSO control sample was set as 100%, as well as values of other samples were calculated for relative proliferation to DMSO control. Each cell line was obtained three independent experiments.

siRNA transfection

DLD-1 and HCT116^wt cells were seeded in 6-well plates at 6 × 10⁵ cells with 2 ml medium per well and cultured for 24 hours. Mixture containing 110 pmol of Noxa siRNA (sc-37305, Santa Cruz Biotechnology) or control siRNA (sc-36869, Santa Cruz Biotechnology), 200 µl jetPRIME® buffer (Polyplus-Transfection, Illkirch, France), and 4 µl jetPRIME® siRNA transfection reagent (Polyplus-Transfection, Illkirch, France) was vortexed 10 seconds, spun down and incubated for 10 minutes at room temperature. Then the mixture was added to each well. After 24 hours, cells were harvested for further experiments.

Statistical analysis

The statistical analysis was performed by SPSS version 19.0. Values were presented as mean + standard error of the mean (SEM). Comparisons between groups of data were made by one-way analysis of variance (ANOVA) and succedent Tukey’s test. p < 0.05 was considered statistically significant.

PRIAM-1^met induced autophagy flux in different CRC cells

In order to investigate the effects of PRIMA-1^met on autophagy flux, different CRC cells were exposed to PRIMA-1^met for further detection by western blotting. Following 24-hour treatment, increases of LC3-II expression were found in 3 lines carrying wild-type p53, 4 out the 5 p53-mutant lines (exclusive of DLD-1), and 1 p53-deleted cell line (p < 0.01, Fig. 1A, 2A, and 3A). Conversely, reduction of p62 expression was observed in HCT116^ko, RKO, HCT15, DLD-1, SW480, and SW620 cell line (p < 0.01, Fig. 1B, 2B, and 3B). These results suggested that PRIMA-1^met promoted the dissociation of plasma LC3 (LC3-I) to membranous LC3 (LC3-II) for AV-membrane extension as well as degradation of cargoes (p62) in autolysosome. To further determine whether PRIMA-1^met induced autophagy flux in CRC cells, acridine orange (AO) staining was performed on HCT116^wt, RKO, SW480, CaCO2 and HCT116^ko cells with 24-hour treatment, showing that treated cells revealed stronger red fluorescence than DMSO controls under the confocal microscope (Fig. 1C, 2C, and 3C). However, AO had the affinity for acidic vesicular organelles (AVOs) including not only autolysosome but also lysosome. For deeply distinguishing, autolysosomes were observed in PRIMA-1^met-treated RKO, HCT15 cells under a transmission electron microscope (TEM). As shown in Fig. 4, autolysosomes/autophagosomes were increased significantly after PRIMA-1^met administration in both two cell lines compared with DMSO control, and even more prominently at high concentration (p < 0.01). Taken together, PRIMA-1^met promoted the whole process of AVs formation, cargoes removal, and degradation in autophagy flux that was irrespective of p53 status.

PRIMA-1^met regulated mTOR/AMPK-ULK1-Vps34 autophagic signaling cascade in different CRC cells

We next detected several molecules of autophagic signaling cascades using western blotting to dissect distinct mechanisms for the effect of PRIMA-1^met on autophagy in different CRC cells. Decrease in phospho-mTOR (a nutrient sensor) expression was found only in HCT116^wt and RKO cells with wild-type p53 after PRIMA-1^met treatment. On the contrary, increase in phospho-AMPK (an energy sensor) was observed in HCT116^wt, RKO, and HCT15 cells (p < 0.01, Fig. 5A). Furthermore, PRIMA-1^met administration up-regulated expression of phospho-ULK1 (Unc-51-Like Kinase 1) in RKO, DLD-1, and HCT15 cell lines, as well as PI3K Class III (the mammalian homolog of Vps34) in three lines of RKO, HCT116^wt, and HCT15 (p < 0.01, Fig. 5B). These findings indicated that PRIMA-1^met mediated mTOR/AMPK-ULK1-Vps34 signaling cascade for autophagy in CRC cells independent on p53 status.

PI3K inhibitor suppressed inhibitory effect of PRIMA-1^met in CRC cells with wild-type p53

To determine if the effects of PRIMA-1^met on autophagy made an impact on growth inhibition in CRC cell lines, PI3K inhibitor 3-methyladenine (3-MA) was used for further detection. CRC cells of four lines, including HCT116^wt, RKO, DLD-1, and HCT15, were exposed to PRIMA-1^met, 3-MA either alone or in combination for 48 hours, following by Cell Counting Kit-8 (CCK-8) proliferation assay. As expected, cell proliferation decreased in all cell lines with PRIMA-1^met alone (p < 0.01, Fig. 6A) while 3-MA exposure resulted in a rare suppression (p > 0.05, Fig. 6A). However, in the presence of combination, inhibition of cell growth was obviously weakened compared to PRIMA-1^met single in both HCT116^wt and RKO lines expressing wild-type p53 (p < 0.01, Fig. 6A). In contrast, there were no significant differences in cell proliferation between PRIMA-1^met and combination regimens in DLD-1 and HCT15 cell lines carrying mutant p53 (p > 0.05, Fig. 6A). Decreased expression of LC3-II was confirmed by western blotting after 3-MA treatment in both HCT116^wt and RKO cells (Fig. 6B).

PRIMA-1^met-induced LC3 conversion in CRC cells with wild-type p53 was positively correlated to Noxa

We previously reported that up-regulation of Noxa, a pro-apoptotic molecule, was crucial for PRIMA-1^met to induce robust apoptosis in CRC cell lines with mutant p53 [10]. To investigate the role of Noxa in PRIMA-1^met- induced autophagy, we knocked down Noxa gene by siRNA in DLD-1 and HCT116^wt cell lines, and confirmed decreased expression by western blotting (Fig. 7). Notably, LC3-II expression was reduced in HCT116^wt cells after knocking down Noxa (p < 0.01), whereas knocked-down Noxa in p53-mutant DLD-1 cell line failed to produce significant impacts on LC3 conversion (p > 0.05) (Fig. 7).

Autophagy proceeds through several phases, including initiation (prepare membrane to form AVs), membrane curative, LC3 family conjugation cascade, cargo loading, AV maturation, AV-lysosome fusion, lysosomal degradation and recycling[16]. This dynamic process is also called autophagy flux. In this study, several methods, such as western blotting, AO staining, and TEM observation, provided sufficient evidence for PRIMA-1^met-induced autophagy in CRC cell lines with different p53 status. PRIMA-1^met treatment not only up-regulated LC3 conversion and resulted in the increase of AVs, but also promoted degradation of cargoes combining with receptor p62. This effect was regardless of p53 status, but relative to different cell types. Furthermore, our results suggested a molecule mechanism underlying PRIMA-1^met-induced autophagy in CRC cells via mediating mTOR/AMPK-ULK1-Vps34 signaling cascade. ULK1, the mammalian homolog of ATG1, plays a convergent role in multiple signals that regulate autophagy, receiving nutrient and energy signals from the two upstream factors of AMPK and mTOR. AMPK activated under low energy condition with an elevated AMP/ATP ratio phosphorylates ULK1 [17]. On the other hand, mTOR as an autophagy inhibitor reversely regulates the phosphorylation of ULK1 by nutrient stress and even disrupts the interaction of ULK1, and AMPK [18]. Once activated, ULK1 complex that consisting of ULK1, ATG13, FIP200, and ATG101 promotes the initiation of AV formation [19], then further leads to activation of Vps34 complex, which including Vps34 (PI3K Class III), Beclin-1, Vps15, ATG14, for AV membrane curvature [20]. In this study, we found PRIMA-1^met up-regulated phosphorylation of AMPK, ULK1, and PI3K Class III simultaneously in CRC cells carrying both wild-type p53 and mutant p53. However, decrease in the phosphorylation of mTOR was only observed in cells with wild-type p53. It was worth nothing that PRIMA-1^met increased the phosphorylation of PI3K Class III but without ULK1 in the the HCT116^wtcell line, supposed due to activation by AMPK directly. These findings provided a pronounced link between PRIMA-1^met and autophagy in different CRC cell lines mediated by mTOR/AMPK-ULK1-Vps34 signaling cascade. 3-MA, targeting at Vps34 and PI3Kγ for inhibiting PI3K Class I permanently as well as PI3K Class III temporally, is a useful reagent to block the formation of the autophagosome. Combined with 3-MA in PRIMA-1^met treatment, we suppressed inducible autophagy and further increased cell proliferation effectively in CRC cells expressing wild-type p53 compared to PRIMA-1^met alone. This result supported our hypothesis that the mechanism underlying the cytotoxicity of PRIMA-1^met in cells with wild-type p53 was relative to the induction of autophagy. Previous studies demonstrated the crosstalk between autophagy and apoptosis was required for regulating cell growth and survival especially the Bcl-2 family [21, 22]. Our study showed that reduced expression of Noxa by siRNA suppressed LC3 conversion in the HCT116^wt cell line, but not in the DLD-1 cell line. In other words, up-regulation of Noxa was supposed to influence cell autophagy after PRIMA-1^met treatment in CRC cells carrying wild-type p53. These findings confirmed that different mechanisms were involved in the inhibitory effect of PRIMA-1^met in CRC cells with different types of p53. PRIMA-1^met mainly promoted apoptosis in cells harboring mutant p53, whereas PRIMA-1^met induced autophagy in cells carrying wild-type p53 or null p53. Inducible autophagy including autophagic proteins was supposed to participate in apoptosis regulation through the complex network of molecular interactions between them in p53-mutant cells after PRIMA-1^met treatment.

In conclusion, our study elucidated that PRIMA-1^met induced autophagy in CRC cells via activating mTOR/AMPK-ULK1-Vps34 signaling cascade in a p53-independent manner (Fig. 8). Nevertheless, induced autophagy was closely related to the cytotoxicity of PRIMA-1^met in cells carrying wild-type p53, along with up-regulation of Noxa. According to the complex interaction between autophagy and apoptosis, a deeper insight into the correlation between them induced by PRIMA-1^met especially in p53-mutant cells is an attractive avenue for further investigation. Our new findings further elaborate a deeper understanding of mechanisms for the anti-tumor activity of PRIMA-1^met in CRC, that will support to PRIMA-1^met-based therapy as a strategy for improving advanced CRC patients’ outcome.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and materials

The datasets during and/or analysed during the current study available from the corresponding author upon reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

This study was supported by grants from National Natural Science Foundation of China (NSFC, 81603128) *, Gusu Health Layer Training Program for Young Top Talent (GSWS2019059), and Science and Technology Project of Suzhou (SYSD2018136).

Authors’ contributions

Li XL, Zhou J, and Chen ZR were responsible for the experimental design; Li XL and Zhou J wrote the manuscript; Chen ZR revised the manuscript; Li XL, Xia CJ and Lu ZK performed experiments. All authors read and approved the final manuscript.

Acknowledgements

We gratefully acknowledge to Dr Jun-dong Zhou (The Affiliated Suzhou Hospital of Nanjing Medical University, China) and Dr Fang Lin (Soochow University, China) for their grateful help to this study.

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PDF

Version 1

posted 16 Sep, 2020

You are reading this latest preprint version

PRIMA-1met Induces Autophagy in Colorectal Cancer Cells With Different p53 Status

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Cell lines and drugs

Western blotting analysis

Acridine orange (AO) staining

Transmission electron microscopy (TEM) detection

Cell counting Kit-8 (CCK-8) proliferation assay

siRNA transfection

Statistical analysis

Results

PRIAM-1^met induced autophagy flux in different CRC cells

PRIMA-1^met regulated mTOR/AMPK-ULK1-Vps34 autophagic signaling cascade in different CRC cells

PI3K inhibitor suppressed inhibitory effect of PRIMA-1^met in CRC cells with wild-type p53

PRIMA-1^met-induced LC3 conversion in CRC cells with wild-type p53 was positively correlated to Noxa

Discussion

Declarations

References

Status:

Version 1

PRIMA-1met Induces Autophagy in Colorectal Cancer Cells With Different p53 Status

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Cell lines and drugs

Western blotting analysis

Acridine orange (AO) staining

Transmission electron microscopy (TEM) detection

Cell counting Kit-8 (CCK-8) proliferation assay

siRNA transfection

Statistical analysis

Results

PRIAM-1met induced autophagy flux in different CRC cells

PRIMA-1met regulated mTOR/AMPK-ULK1-Vps34 autophagic signaling cascade in different CRC cells

PI3K inhibitor suppressed inhibitory effect of PRIMA-1met in CRC cells with wild-type p53

PRIMA-1met-induced LC3 conversion in CRC cells with wild-type p53 was positively correlated to Noxa

Discussion

Declarations

References

Status:

Version 1

PRIAM-1^met induced autophagy flux in different CRC cells

PRIMA-1^met regulated mTOR/AMPK-ULK1-Vps34 autophagic signaling cascade in different CRC cells

PI3K inhibitor suppressed inhibitory effect of PRIMA-1^met in CRC cells with wild-type p53

PRIMA-1^met-induced LC3 conversion in CRC cells with wild-type p53 was positively correlated to Noxa