Intracellular AFP Blocks All-trans retinoic acid induced ATG7 expression and Autophagy in Hepatocellular carcinoma cells

Background: Retinoic acid and retinoid acid receptor (RA-RAR) signaling exhibits suppressive functions in the progression of hepatocellular carcinoma (HCC) through multiple mechanisms. However, whether RA-RAR signaling induces autophagy that contributes its anti-tumor activity in HCC remains elusive. Methods: The effects of RA-RAR pathway were investigated in two HCC cell lines: AFP positive PLC/PRF/5 cells and AFP negative HLE cells. Cell autophagy, apoptosis and proliferation were analyzed by Western blotting, co-immunoprecipitation (CoIP), Immunofluorescence staining, chromatin immunoprecipitation (ChIP), Caspase-3 activity and Cell Counting Kit (CCK)-8. Results: ATRA dosage-dependently induced high levels of cell autophagy through its specific nuclear receptor RAR in both the PLC/PRF/5 and HLE cells. ChIP assay showed that RAR bind to response elements of key autophagy-initiated gene autophagy-relevant protein (ATG) 7 gene in the 5’-flanking region. Analyses based on CoIP further revealed that AFP formed complex with RAR in PLC/PRF/5 cells. Knockdown of AFP reduced the AFP and RAR combination, and thus up-regulated the expression of ATG7 gene and cell autophagy. Interestingly, in the HLE cells, AFP overexpressed and combination with RAR resulted in down-regulated ATG7 gene expression and reduction of cell autophagy. In both cell lines, ATRA inhibited cell proliferation and induced cell apoptosis, which was impacted by AFP action. Conclusion: The current study indicated that autophagy participated in the functionality of ATRA on HCC cells and AFP is a key regulator of ATRA induced autophagy through forming complexes with RAR in HCC cells.

work by us showed that AFP could perturb RA-RAR signaling through interaction with RARα, resulting in transcriptional dysregulation of RAR targets, like survivin [3], Fn14 [4], GADD45 [5], GADD153 [6] etc. in HCC cells. ATRA or its chemical derivatives have long been tested as candidates for treatment of HCC as single reagent or in combination with other clinically used drugs [7,8], the results was, however, far to be satisfactory, which could be partially attributed to the perturbation of AFP and the resultant dysregulation of RAR target genes. Given the profound effect of ATRA on HCC cells and the broad distribution of retinoic acid response elements (RAREs) in human genome, whether other target genes of RA-RAR signaling and related biological process is regulated by AFP in HCC cells is tempting to investigation.
Macroautophagy (referred to as autophagy hereafter) is a conserved degradation system for damaged, misfolded, or senescent cellular components, like organelles or certain proteins to maintain cellular homeostasis [9]. About 40 autophagy related genes (ATGs) have been identified to date and participate in the whole process of autophagy that mainly composed of initiation and elongation of the phagophore, autophagosome formation, autophagosome fusion with lysosomes and final degradation of the intracapsular products, in a highly ordered manner [10]. Important signaling molecules like AMPK, mTOR, PI3K/Akt etc. showed potent regulation on autophagy [11]. For example, mTORC1 inhibited autophagosome formation elicited by ULK1 (ATG1) while activated AMPK was able to inhibit mTORC1and directly phosphorylates ULK1, leading to autophagy initiation [12]. RA-RAR signaling has also been implicated in the modulation of autophagy through multiple mechanisms in different cell types. In acute promyelocytic leukemia (APL) cells, ATRA was able to induce autophagy through inhibition of mTOR pathway, which contributed to the degradation of the fusion oncoprotein PML/RARα, resulting in cell differentiation and the remission of the tumor [13,14]. In breast cancer cells, ATRA was reported to induce autophagy dependent on RARα, and ablation of autophagy promoted ATRA induced apoptosis of the cancer cells [15]. Fang et al. also suggested induction of autophagy and expression of a panel of ATGs by ATRA in Hepa1-6 mouse hepatoma cells [16], however, the generality of autophagy induction by ATRA in HCC and the underlying mechanism remains to be further addressed.
Conventional chemotherapeutic drugs for HCC like doxorubicin, oxaliplatin, cisplatin have all been reported to induce autophagy in vitro and in vivo that seemed be protective for the cells under treatment, for inhibition of autophagy was able to enhance the anti-tumor activity of these drugs [17].
Multiple ATGs and related signaling pathways were shown to regulate sensitivity of HCC cells to chemo-or targeted reagents, which might hold potential therapeutic potentials [18]. We recently provided intriguing evidence that AFP played a suppressive role in the maintenance of the basal level of autophagy in HCC cells through interaction with PTEN, which led to inhibition of the phosphatase activity of PTEN and subsequent over-activation of PI3K/Akt/mTOR, and finally promoted cell survival [19]. As AFP also interacted with RARα and perturbed RA-RAR signaling as well as the anti-tumor effect of ATRA in HCC cells, whether this perturbation also participates in regulation of cell autophagy is of great interest to be investigated.
In the present study, we found that ATRA robustly induced autophagy in human HCC cells through t RAR mediated transcriptional up-regulation of ATG7, an essential ATG for autophagosome initiation, which played a protective role for ATRA treated cells. Furthermore, AFP interacted with RARα and attenuated its regulation on ATG7 expression and autophagy. Our results was supposed to be helpful for developing novel therapeutics for HCC composed of ATRA and autophagy inhibition reagents, where the level of AFP needs to be taken into consideration.

Materials And Methods
Cell lines AFP-producing hepatocellular carcinoma cell line PLC/PRF/5 cells and AFP-non producing cell line HLE were both maintained in a 5% CO 2 incubator and cultured in DMEM medium supplemented with 10% FCS.

Western blotting
For western blotting, total cell proteins from each sample were extracted with radioimmunoprecipitation (RIPA, Thermofisher, Waltham, MA, USA) cell lysis buffer containing protease inhibitor cocktail (CST, Beverly, MA, United States), 15µ g of which were then subjected to 12% SDS-PAGE. Electrophoretic transfer of proteins from gels onto nitrocellulose membrane was carried out in a transblotting cell. Membranes were blocked by immersing in 5% nonfat milk (w/v) /PBS for 1 hour, and then incubated with primary antibodies at 4℃ for overnight. After rinsing with PBS/0.1% Tween-20, membranes were incubated with horseradish peroxidase-conjugated secondary Ab. The signals were visualized by incubation with the Enhanced Chemiluninescence kit and exposure on an X-ray film. Primary and secondary antibodies used in this study are listed in Table 1.

Transient transfection
The AFP-expressing plasmid (pcDNA3.1-afp) was used to overexpress AFP in HLE cells, and the AFP interference siRNA (AFP-siRNA923) was employed to knockdown endogenous AFP in PLC/PRF/5 cells.

Chromatin immunoprecipitation and PCR (ChIP-PCR)
ChIP was performed to verify the capacity of RAR for binding to the 5'regulatory region of ATG7 gene.
Briefly, HCC cells were cross-linked in 1% formaldehyde/PBS for 10 min at 37℃and were then washed twice with ice-cold PBS prior to be lysed. Chromatin fragments ranging from 200 to 1000 bp were obtained by sonication (SCIENTZ, China). The solution containing chromatin fragments was then incubated overnight at 4 °C with anti-RAR or anti-IgG. The chromatin-antibody complexes were then washed, eluted and reverse cross-linked at 65 °C for 6 h. The eluted DNA was purified with the phenol-chloroform. Immunoprecipitated chromatin was analyzed with PCR using primers targeting the human ATG7 promoter containing RAR binding motif. The primers used for ChIP-PCR are listed in Table 1.

Evaluation of fluorescent LC3 puncta
The tandem mRFP-GFP-LC3 adenoviruses construct was obtained from Hanbio Inc (Shanghai, China) and was used to evaluate autophagy induction. Briefly Caspase-3 activation assay PLC/PRF/5 and HLE cells were homogenized in lysis buffer. Thereafter, 30 µL lysates were added to a white 96-well plate, and then mixed with 60 µL assay buffer. 90 µL assay buffer was added into the blank well. After incubation for 10minutes at 37℃, each well was added 10 µL AC-DEVD-AFC at final concentration of 10 µg/mL, followed with further incubation for 1 h at 37℃ in the dark. The luminescence was measured using a Imaging Multi-Mode Reader (BioTek

Statistical Analysis
The results of at least three separate experiments are presented as the mean ± s.d. Statistical significance was determined using the one-way and two-way ANOVA tests (SPSS 16 software). Immunofluorescence analyses further confirmed that ATRA significantly reduced the expression of P62/SQSTM1 in both PLC/PRF/5 and HLE cells (Fig. 1C). All these results indicated that ATRA induced autophagy in HCC cells. The activation of RA-RAR signaling in HCC cells was further verified with nuclear accumulation of RAR as demonstrated with western blot analyses for nuclear proteins (supplementary Fig. 1A and 1B) and cellular immunofluorescence (supplementary Fig. 1C and 1D).

Results
The ATRA-RAR signaling regulated transcription of ATG7 To further reveal the potential molecular mechanism underlying ATRA induced autophagy in HCC cells. Expressions of ATG5, Beclin and ATG7 were evaluated with RT-qPCR in PLC/PRF/5 and HepG2 cells. According to preliminary experimental results (supplementary Fig. 2), expression of ATG7, an E1-like activating enzyme that are indispensable for autophagosome formation [20], was most significantly up-regulated under ATRA treatment in both HCC cell lines. We thus focused on potential regulation of ATG7 by ATRA-RAR in the following studies. Western blotting showed that ATRA induced robust increment of ATG7 in both PLC/PRF/5 ( Fig. 2A) and HLE ( Fig. 2A') cells, in a dose-dependent, reaching maximum at 40 µM. Similar results were observed at the mRNA level with the qRT-qPCR assay ( Fig. 2B and 2B'). Ethyl Alcohol, solvent for ATRA, did not influence the expression of the ATG7 gene ( Fig. 2A, B, 2A' and 2B'). The alteration of ATG7 at the mRNA level prompted us to investigate if ATG7 was transcriptionally regulated by RAR. Two adjacent binding sequence for RAR was discovered at the proximal promoter of ATG7 (Fig. 2C). To validate RAR was able to binding to the regeion, ChIP assays were performed. As shown in Fig. 2D and 2D', RAR was able to bind to the 5'-flanking regions containing its responsive elements at the ATG7 promoter in both PLC/PRF/5 and HLE cells, indicating a direct transcriptional regulation of ATRA-RAR signaling on ATG7 via RAR.
ATG7 played a protective role in HCC during ATRA treatment.
We next investigated whether the induction of ATG7 expression and autophagy were functional in ATRA treated HCC, or merely indicators for the activity of ATRA. CCK-8 and caspase-3 activity assays were carried out. The CCK-8 results shown that the cell viability was further decreased with knockdown of ATG7 in response to ATRA in both PLC/PRF/5 and HLE cells ( Fig. 3A and 3A´), which was accompanied with increment of caspase-3 activity (Fig. 3B and B´). These results indicated that the induced ATG7, and quite probable the autophagy, played a protective role in ATRA induced apoptosis in HCC cells.

AFP interacted with RAR in hepatoma cells
To investigate whether AFP could possibly regulate ATRA-RAR mediated autophagy, western blotting analyses were first employed to detect the endogenous expression of AFP in PLC/PRF/5 and HLE cells.
As previously reported, AFP protein was undetectable in HLE cells, but robustly expressed in PLC/PRF/5 cells (Fig. 4A). Further analyses with confocal microscopy showed that AFP and RAR colocalized in cytoplasm in PLC/PRF/5 cell (Fig. 4B), but not in HLE cells (Fig. 4C), which were further confirmed by Co-IP analysis ( Fig. 4D and 4E).
AFP perturbed ATRA-RAR signal and reduced ATG7 expression in hepatoma cells.
To further investigate if AFP was involved in ATRA-RAR signal transduction as well as ATG7 transcription by interacting with RAR, expression of AFP was first knockdown by specific siRNA in PLC/PRF/5 cell. Immunofluorescence and confocal microscopy assays showed that AFP expression was obviously depleted upon specific siRNA transfection compared with scramble siRNA (Fig. 5A).
Following AFP depletion, binding of AFP with RAR was significantly decreased as demonstrated by Co-IP assay in PLC/PRF/5 cells (Fig. 5B). On the contrary, when AFP was introduced into HLE cells with pcDNA3.1-afp vectors (Fig. 5A'), notable interaction between AFP and RAR was observed as shown by Co-IP results (Fig. 5B'), accompanied with co-localization of AFP and RAR in the cytoplasm (Fig. 5A´).
One intriguing phenomena was observed that alteration of the alteration intracellular AFP level not only changed its interaction with RAR, but also exhibit a negative regulation on the protein level of RAR itself ( Fig. 5A and 5A'), which needs further investigation.
As we previously demonstrated that interaction between AFP and RAR was able to disrupt the transcriptional regulation of RAR on its targets, we wonder whether it was also the case in ATG7. Not surprisingly, when AFP was down regulated by siRNA in PLC/PRF/5 cells, the ATG7 protein level was remarkably increased as compared with the control (Fig. 5C). On the other hand, AFP expression in HLE cells showed an apparent reduction of ATG7 protein (Fig. 5C´).

Discussion
In in ATRA induced autophagy [14,[21][22][23][24]. In other cell types, including several other solid tumor types, ATRA was also able to induce autophagy [15,25]. In these studies, expression alterations of certain ATGs or signaling molecules were always displayed as the underlying mechanisms, which seemingly was not powerful enough to establish direct links between ATRA and autophagy, as the involvement and the function of RAR always lacked. For example, ATRA induced autophagy in human B cells through mTOR inhibition [26], and induced autophagy in APL cells via potent up-regulation of TFEB [23], how the inhibition or promotion occurred, directly through RAR or by other alternative pathways?? The present study directly linked ATRA and autophagy in HCC cells with RAR mediated transcriptional activation of ATG7. Of course, as ATRA was able to elicit a number of other downstream signaling pathways [27], it still cannot rule out the possibility that other regulators were also involved in ATRA induced autophagy in HCC cells.
Blockade of autophagy by knockdown Atg1, Atg5 and PI3KC3 etc. or by specific autophagy inhibitors like 3-methyladenine (3-MA) impaired ATRA induced differentiation of APL cells [14], suggesting the necessity of autophagy for the primary function of ATRA. In breast cancer cells, autophagy was reported to be cell protective and inhibition of autophagy genetically or pharmacologically resulted in robust apoptosis [15]. In the current study, as in most cases of chemotherapeutic drug treatment, ATRA induced autophagy also played a protective role for the cancer cells though overwhelmed by the potency of ATRA. Thus autophagy constituted a resistance mechanism for HCC cells to the stimuli.
ATRA has been shown to induce differentiation of tumor initiating cells in HCC, and potentiated the cytotoxicity of chemotherapeutic drugs like cisplatin [28]. It has also been shown to enhance the antitumor activity of sorafenib through activation of AMPK [29], a potent regulator for autophagy induction. Together with the reports that most reagents for HCC treatment induced autophagy, and autophagy was cell protective in most cases, it is plausible to consider combinational use of chemotherapeutic drugs with ATRA and autophagy inhibitors like chloroquine to improve the efficacy of chemotherapy for HCC. Further in vitro and in vivo experiments are needed to address these issues.
We recently reported that AFP was able to block basal level of autophagy in HCC cells through direct sequestration of PTEN, which leads to overactivation of PI3K-Akt-mTOR cascade [19]. Together with current results, AFP was thus able to disrupt both the basal and ATRA induced autophagy through interaction of different partners (PTEN or RAR) and modulation of different autophagy regulators (PI3K/Akt/mTOR or ATG7). However, whether autophagy and AFP played identical roles in these conditions still needs further illustration. Under basal conditions, increased level of autophagy with AFP knockdown was accompanied with PTEN overactivation and increased cell apoptosis [19].
However, to what extent did autophagy contribute to increased apoptosis could not be figured out

Conclusion:
In this study, we found that RA-RAR pathway upregulated HCC cells autophagy levels by directly regulated ATG7 gene express, and this function was inhibited by AFP, which could block RA-RAR pathway by combining with RAR. And further RA-RAR pathway influence HCC cells apoptosis and proliferation, which was impacted by AFP action too.           and HLE (C') cells were analyzed by Western blotting.

Supplementary Files
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