Stress granules affect the dual PI3K/mTOR inhibitor response by regulating the mitochondrial unfolded protein response

doi:10.21203/rs.3.rs-1935001/v2

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Stress granules affect the dual PI3K/mTOR inhibitor response by regulating the mitochondrial unfolded protein response

https://doi.org/10.21203/rs.3.rs-1935001/v2

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Drug resistance remains a challenge in ovarian cancer. In addition to aberrant activation of relevant signaling pathways, the adaptive stress response is emerging as a new spotlight of drug resistance in cancer cells. Stress granules (SGs) are one of the most important features of the adaptive stress response, and there is increasing evidence that SGs promote drug resistance in cancer cells. In the present study, we compared two types of ovarian cancer cells, A2780 and SKOV3, using the dual PI3K/mTOR inhibitor, PKI-402. We found that SGs were formed and SGs could intercept the signaling factor ATF5 and regulate the response of mitochondrial unfolded protein(mtUPR)in A2780 cells. Therefore, exploring the network formed between SGs and membrane-bound organelles, such as mitochondria, may provide new insight into the mechanisms of action of antitumor drugs.

Ovarian cancer is one of the most lethal cancers in women and, in recent years, the development of drug resistance has led to poor treatment outcomes[1]. In addition to cancer cell genetic mutations that lead to drug resistance, research findings suggest that the adaptive stress response induced by chemotherapeutic agents may also be one of the main causes of drug resistance[2]. The integrated stress response maintains the adaptive stress response by phosphorylating eIF2α as a key factor that translates genes containing open upstream reading frames (uORFs), such as ATF4, CHOP, and ATF5, thereby reestablishing proteostasis and leading to reduced sensitivity to chemotherapeutic agents[3, 4]. Furthermore, phosphorylated eIF2α rapidly inhibits mRNA translation by disrupting cap-dependent translation initiation, causing a large number of untranslated mRNAs, RNA-binding proteins, and messenger ribonucleoprotein structures to accumulate in the cytoplasm to form stress granules (SG), believed to play a role in maintaining the adaptive stress response by rapidly regulating changes in intracellular mRNA translation[5, 6]. Further exploration of the role of SGs in drug resistance in cancer cells has received attention[7, 8].

SGs are capable of regulating untranslated mRNAs, selectively suspending, translating, or degrading mRNAs, and acting as a hub for cell signaling during stress[9]. Somasekharan et al. suggested that YB-1 could promote the synthesis of G3BP1 and therefore induce SG formation[10], suggesting that RNA-binding proteins (RBPs), such as YB-1, are involved in the regulation of SG-internal mRNAs[11, 12]. A recent study by Timalsina et al. also reported that SGs are involved in chemoresistance to cisplatin and paclitaxel in human cervical cancer[13].

The PI3K/mTOR pathway is often overactivated and complexly cross-regulated in ovarian cancer, and single-target inhibitors are ineffective, suggesting the existence of more complex network mechanisms in molecular signaling crosstalk[14, 15]. For example, inhibition of the mTOR pathway results in suppressed cap-dependent protein translation[16], and 5' TOP mRNA accumulation in the SGs by the SG-associated protein TIA-1/TIAR, suggesting that the mTOR pathway is involved in SG formation[17]. On the other hand, SGs inhibit the mTOR pathway by sequestering components of the mTORC1 complex [18]. These results suggest that SG formation may be related to targeting mTOR antitumor mechanisms.

Mitochondria, as the main organelles for sensing and alleviating stress, are involved in adaptive stress responses in tumor cells [19]. When oncogenic pathways, such as PI3K/mTOR, are disrupted, the mitochondrial unfolded protein response (mtUPR) is activated to reduce stress caused by protein accumulation in cells[19]. When mitochondrial function is disturbed, ATF5 is selectively translated under stress and enters the nucleus to act as a transcription factor, increasing expressions of mitochondria-associated proteases and molecular chaperones, thus establishing new proteostasis and protecting the cell from death signals[20, 21]. Therefore, the translocation of ATF5 is a key factor in activating mtUPR to induce an adaptive stress response[21]. Although no experimental studies have reported the correlation of ATF5 with SGs, Souquere et al. found that SGs formed near mitochondria when HeLa and H293 cells were in oxidative stress[22]. Based on the above rationale, we speculate that SGs, as a kind of protection mechanism, can intercept ATF5, causing a change in localization, and then an adaptive stress response is activated, increasing the drug resistance. This suggests to us that it is more important to explore the mechanism of molecular targeted drugs for SGs.

In this study, we used two ovarian cancer cell lines to investigate differences in SG formation and the mechanism of SG formation in using the dual PI3K/mTOR inhibitor. We also elucidated the mechanism by which SGs regulate mRNA translation and related signaling molecules to activate mtUPR and regulate mitochondrial function and then reshape cellular proteostasis.

2.1. Cell lines and cell culture

SKOV3 and A2780 ovarian cancer cells were grown in RPMI-1640 (Gibco Life Technologies, USA) supplemented with 10% fetal bovine serum (Invitrogen, USA) at 37°C in a 5% CO2 concentration.

2.2. Reagents and antibodies

Reagents used in this study include the following: 3- (4-dimethylthiazol-2-yl) -2,5-di-phenyltetrazolium bromide (MTT), PKI-402, cycloheximide (CHX) and Thapsigargin (Tg) were commercially sourced from Selleckchem (Houston, TX, USA), anti-G3BP1, anti-eIF2α, anti-4EBP1, anti-caspase-9, anti-Bcl-2, anti-Bax, anti-Hsp60, anti-Lonp, anti-Clpp, anti-Trap1, anti-Actin, anti-vdac (Proteintech, Chicago, IL, USA), anti-YB-1, anti-ATF5, anti-Akt,anti-p-Akt(Ser473) (Santa Cruz, CA, USA), p- eIF2α(Ser51), p-4EBP1(Thr46)( Cell Signaling Technology, USA).

2.3. Cell viability assay

Cells were seeded in 96-well plates overnight at a density of 8000 cells/well. The cells were then treated with drugs for 24 h. Cell viabilities were assessed using an MTT assay and absorbance values were measured at 490 nm using a Vmax Microplate Reader (Molecular Devices, USA).

2.4. Flow Cytometry

Mitochondrial membrane potential (MMP) and ROS production were determined by JC-1 or DCFH-DA staining (Beyotime Biotechnology, Shanghai, China).

2.5. Western blot assay

Cells were lysed in RIPA buffer with protease inhibitors. Lysates were cleared by centrifugation at 10,000 g for 15 min at 4°C, boiled in loading buffer, and resolved using SDS-PAGE. Proteins were transferred to PVDF membranes and membranes were blocked with 5% milk, followed by incubation with primary antibodies overnight at 4°C. The membranes were then incubated with HRP-conjugated secondary antibodies (Proteintech Group, Inc., USA). ECL reagent (Thermo Fisher Scientific, USA) was used for immunodetection and visualization using Syngene Bio Imaging (Synoptics, Cambridge, UK).

2.6. Immunofluorescence assay

Cells were washed with PBS, fixed with 4% paraformaldehyde for 20 min, and permeabilized with 0.1% Triton X-100 for 8 min. After blocking with 5% bovine serum albumin for 30 min, cells were incubated with primary antibody overnight at 4°C. After washing with PBS, cells were incubated at room temperature for 1 h in the dark with secondary antibodies conjugated with FITC / Texas Red ((Proteintech Group, Inc, USA). The images were observed on an Echo-lab Revolve microscope (CA, USA).

2.7. RNA Binding Protein Immunoprecipitation (RIP)

RIP was performed as previously described[23] with some modifications.

2.8. Immunoprecipitation assay

Cells were lysed in NP40 lysis buffer. Equal amounts of lysates were immunoprecipitated with 2µg of p62 antibody overnight at 4°C. Then 25µL of protein A and G agarose (Beyotime, China) was then used for each sample. The beads were washed with PBS three times with 1 ml each. The eluted proteins were examined by western blotting.

2.9. Nuclear and Mitochondrial Isolation

Cells were harvested after treatment with various reagents. Nuclear and mitochondrial fractions were extracted using a nuclear fractionation kit and a mitochondrial fractionation kit, respectively (Beyotime Technology).

2.10. Statistical analysis

All experimental data represent at least 3 independent experiments and were presented as mean ± standard deviation (SD) and were carried out using the Student's t-test. P < 0.05 were considered statistically significant difference. Statistical analysis was performed with GraphPad Prism 7.0 (La Jolla, CA).

3.1. Adaptive Stress Response Was Activated In A2780 Cells

In ovarian cancer cells, the PIK3CA gene that encodes type I PI3Ks P110α is frequently mutated, which is the reason for the reduced sensitivity of tumor cells to chemotherapeutic drugs[24]. We found that two types of ovarian cancer cells, SKOV3 and A2780, showed different sensitivities in the treatment of the dual targeting inhibitor PKI-402. The mutation site in SKOV3 cells has been observed to be H1047R located in the PI3Kα kinase structural domain of the PI3K kinase, while the mutation site in A2780 cells is E365K located in the structural domain C2, which may be the reason for the difference in sensitivity of the two cells to the dual targeting inhibitor PKI-402[25]. We found that under the action of PKI-402, the viability of SKOV3 cells decreased (Fig. 1A), the ratio of Bax/BCL-2 increased, and caspase9 appeared a cleavage band (Fig. 1B, 1C). The above results indicate that SKOV3 cells tend to apoptosis. And compared with A2780, SKOV3 cells were more sensitive to PKI402.

We next found that eIF2α was significantly phosphorylated in A2780 cells when the PI3K/mTOR pathway was completely inhibited; Meanwhile, we also found that

3.2. Stress Granules Form In A2780 Cells Under The Treatment Of Pki-402

Phosphorylated eIF2α leads to a global translational arrest, at which point large amounts of untranslated mRNA, RNA-binding proteins, and messenger ribonucleoprotein complexes undergo liquid phase separation in the cytoplasm to form stress granules[26]. The addition of the dual targeting inhibitor PKI-402 increased the expression level of phosphorylated eIF2α within A2780 (Fig. 1D), and the protein expression levels of phosphorylated eIF2α and ATF5 differed significantly at 12 hours. Immunofluorescence assay staining for G3BP1 and YB-1, marker proteins of SGs[9], revealed that SGs appeared in A2780 cells which treated by PKI-402 for 12 hours (Fig. 2A), while no stress granules formed in SKOV3 cells (Fig. 2B)

3.3 Mtupr Was Activated And Mitochondrial Function Was Enhanced In A2780 Cells

Under PKI-402 treatment, we found an increase in ATF5 expression in A2780 cells (Fig. 1D). ATF5 enters mitochondria to be degraded under normal conditions and enters the nucleus when cells are stressed, where it is involved in transcriptional activation of the mitochondrial unfolded protein response (mtUPR)[27]. Therefore, we isolated mitochondria from two types of ovarian cancer cells and found that mitochondrial protease and molecular chaperone expression were elevated in A2780 cells compared to SKOV3 cells (Fig. 3A), suggesting that mtUPR was activated in A2780 cells. We then examined the mitochondrial membrane potential of both cells and found that the mitochondrial membrane potential was elevated in A2780; we examined intracellular ROS levels and found that ROS was reduced in A2780 cells. Based on these results, we concluded that in A2780 cells mtUPR was activated and mitochondrial function was enhanced. On the contrary, there was no significant change in SKOV3 cells.

3.4 The Sensitivity Of Pki-402 Was Increased And Mtupr Was Inhibited After Inhibiting Sgs

To investigate whether SGs regulate mtUPR, we used CHX to inhibit the formation of SGs. Cycloheximide (CHX), a classic protein translation inhibitor and also a specific inhibitor of stress granules, can inhibit the formation of SG [28]. In immunofluorescence experiments, we observed that CHX treatment for 45 min followed by PKI-402 addition showed that SG formation was inhibited (Fig. 4A) and that the sensitivity of A2780 cells to PKI-402 increased (Fig. 4B). Then we found that inhibition of SGs resulted in reduced protein expression of phosphorylated eIF2α and ATF5 (Fig. 4C), but the overall expression of proteins associated with the mitochondrial unfolded protein response did not change obviously (Fig. 4E). We isolated mitochondria and found a reduction in the expression of associated proteases and molecular chaperones within mitochondria (Fig. 4G), suggesting that only protein expression within mitochondria decreased after inhibiting SGs, indicating that there are some links between SGs and mtUPR. Detecting mitochondrial membrane potential and ROS levels, we found that A2780 cells showed a decrease in mitochondrial membrane potential (Fig. 4I) and an increase in ROS (Fig. 4J) when SGs were inhibited, indicating that mitochondrial function decreased. According to these results, which are SGs formed simultaneously, mtUPR activated and mitochondrial function enhanced under the treatment of PKI-402; however, when the SG inhibitor, CHX, was added, both mtUPR and mitochondrial function were decreased, we can conclude that SGs may have regulated mtUPR.

3.5 SGs regulate mtUPR by affecting the localization of ATF5

Since YB-1 has been reported to regulate G3BP1 and therefore induce SG formation [10], our results showed that after 12 h of treatment with PKI-402, the expression of YB-1 and G3BP1 was reduced in SKOV3 cells compared to A2780 cells (Fig. 5A). We performed RNA immunoprecipitation assays and found (Fig. 5D) that YB-1 interacted with mRNAs of G3BP1 in A2780 cells in the presence of PKI-402. Although there was no significant increase in protein expression levels of YB-1 and G3BP1 in A2780 cells, we can see a significant change in the intracellular distribution of both molecules in Fig. 2A. The above results indicated that YB-1 in A2780 cells induced the production of SG by G3BP1 treated with PKI-402. According to the role of SGs, which can recruit signaling molecules[28]. In the next experiments, immunoprecipitation experiments (Fig. 5E), showed that G3BP1, the signature protein of SGs, interacted with ATF5, suggesting that SG formation protected ATF5 and kept it out of mitochondria after adding dual target inhibitors. Furthermore, we isolated the cytosolic protein and the results showed a clear entry of ATF5 into the nucleus (Fig. 5F). Combining these results, we conclude that A2780's insensitivity to PKI-402 is due to SG formation, which alters the location of ATF5, a key molecule in mtUPR[21]. ATF5 enters the nucleus and initiate mtUPR, followed by increased expressions of protease and molecular chaperones in mitochondria, and improved mitochondrial function, ultimately leading to the resistance of the dual PI3K/mTOR inhibitor (Fig. 6).

Currently, two main causes of high mortality and morbidity in ovarian cancer are late diagnosis and drug resistance[29, 30]. Aberrant activation of oncogenic pathways, such as PI3K/mTOR, in ovarian cancer cells is key to tumor resistance [14]. However, Shen et al. found that translational remodelling of mRNA could cause non-genetic resistance in melanoma, suggesting that selective translation of mRNA caused by adaptive stress responses is emerging as a major cause of drug resistance[31].

The PI3K/mTOR pathway is important for metabolism, growth, and mRNA translation[16]. In cancer cells, this pathway is often over-activated; therefore, inhibiting it can effectively inhibit tumor cell growth. Single PI3K/mTOR target inhibitors of the mTOR pathway activate other loops in the PI3K/mTOR pathway[32]. Dual PI3K/mTOR inhibitors are effective in inhibiting the mTOR signaling pathway, but after using dual PI3K/mTOR inhibitors, we found that A2780 cells developed a more complex adaptive stress response, with phosphorylation of eIF2α in A2780 cells and accumulation of polyribosome catabolism mRNA appearing as a liquid phase separation, leading to SG formation[33, 34]. Langdon et al. showed that mTOR signaling was inhibited at an early stage with a dual PI3K/mTOR inhibitor, indicating that the dual-targeted inhibitor only prevented cancer progression, but tumor cells remained viable and could not be treated[35]. Therefore, it is reasonable to speculate that SG formation may be the main reason for tumor cell insensitivity to the dual PI3K/mTOR inhibitor.

Many studies have examined the relationship between cancer and SGs, with upregulation of SG-related molecules in hepatocellular carcinoma[36], pancreatic cancer,[37], and malignant glioma[7]. Experiments have indicated that the use of sorafenib in human leukemia cells induces the formation of SGs and increases the resistance of chemotherapeutics in cancer cells[36]. Our results showed that A2780 cells were insensitive to the dual PI3K/mTOR inhibitor (Fig. 1A) and intracellularly formed SGs (Fig. 2A). After the addition of the classical SGs inhibitor cycloheximide (CHX) (Fig. 4A)[38], the sensitivity of the dual PI3K/mTOR inhibitor increased in A2780 cells (Fig. 4B), all of which again verified that SGs influence drug resistance.

Figure 5 shows the mechanism of SG formation. After treatment with the dual PI3K/mTOR inhibitor, expressions of SG-related RBP, YB-1, and G3BP1, were elevated in A2780 cells (Fig. 5A), and we also verified that YB-1 promoted G3BP1 synthesis (Fig. 5D) [10]. YB-1 can regulate the stability and translation of mRNAs. In addition, YB-1 is highly expressed in various tumor cells and is involved in tumor drug resistance [39]. Therefore, RBP acts as a scaffold, as well as an important functional protein in SGs[6]. When the mTOR pathway is inhibited, a large number of mRNAs accumulate, and RBPs bind to them to form a dynamic pool of mRNAs[40]. Depending on cellular needs, RBP shuttles between SGs and polyribosomes to selectively translate mRNAs, thus reshaping cellular proteostasis[41, 42]. Thus, the interaction between RBPs not only verifies SG formation but also elucidates the mechanism of adaptive stress response in tumor cells.

When the mTOR pathway was inhibited, mitochondrial-associated protein translation was restricted, leading to the accumulation of large amounts of unfolded or misfolded proteins in mitochondria, which causes stress and initiates mtUPR to prevent mitochondrial dysfunction[27, 43]. In C. elegans, the main factor that activates mtUPR is ATFS-1, which ATFS-1 harbors both a mitochondrial targeting sequence and a nuclear localization sequence, whereas activation of mtUPR is much more complex in mammals[20]. During stress, molecules containing uORFs, such as ATF4, CHOP, and ATF5, can be selectively translated independently of phosphorylated eIF2α[44]. All three molecules are key to the integrated stress response and are involved in mtUPR. However, Fiorese et al. showed that ATF5 is a key factor in the initiation of mtUPR [20]. During the normal mitochondrial function, ATF5 enters the mitochondria for degradation, while during mitochondrial stress, ATF5 enters the nucleus instead, where it functions as a transcription factor. Therefore, ATF5 translocation is critical for initiating mtUPR. Studies indicate that when mitochondrial stress occurs, the efficiency of mitochondrial input is reduced, resulting in the inability of ATF5 to enter the mitochondria[45, 46]. However, there are other effects on the translocation process. According to our results, ATF5 obviously entered the nucleus (Fig. 5F) and it was found that there is an interaction with G3BP1 (Fig. 5E), a core component of SG[47]. SGs can isolate specific signaling factors (such as RACK1[48], HuR, and Lin28[49]) and integrate multiple stress signaling cascades to coordinate some cellular response to counter stress. Therefore, it is likely that ATF5, an important signaling molecule for communication between SGs and mitochondria, is segregated into SGs before entering the nucleus. D'Amico et al. suggest that SGs act as translation hubs to regulate mitochondrial-related translation[19]. This suggests that when stress occurs, cells establish a signaling network with SGs as the centre and mitochondria as effectors to resist stress and reshape cellular proteostasis.

In summary, we explore that during stress, SGs replace the translational function of mTOR and become the center of translational regulation, communicating with mitochondria and other membrane-bound organelles, allowing cells to effectively resist and adapt to stress. Thus, targeting this network for interference provides new ideas for the mechanism of action of antitumor drugs.

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Competinginterests

The authors declare that they have no competing interests.

Authors'contributions

NL performed cell research; HQ and YXZ performed data curation; CJN, MJY, and MHX performed cell research. LKS and XQH designed the research and supervised this study; All authors have read and approved the final manuscript.

Funding

This study was supported by the National Natural Science Foundation of China (82102733), the Jilin Provincial Research Foundation for Innovation in Health Technology (2020Q010, 2021JC005), Health Commission (2021JC008), the Bethune Project of Jilin University (2020B59), the Department of Science and Technology of Jilin Province(20210506024ZP).

Availabilityof data and materials

The datasets in the current study are available from the corresponding authors on reasonable request

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No competing interests reported.

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Stress granules affect the dual PI3K/mTOR inhibitor response by regulating the mitochondrial unfolded protein response

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Abstract

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1. Introduction

2. Methods And Materials

3. Results

3.1. Adaptive Stress Response Was Activated In A2780 Cells

3.2. Stress Granules Form In A2780 Cells Under The Treatment Of Pki-402

3.3 Mtupr Was Activated And Mitochondrial Function Was Enhanced In A2780 Cells

3.4 The Sensitivity Of Pki-402 Was Increased And Mtupr Was Inhibited After Inhibiting Sgs

3.5 SGs regulate mtUPR by affecting the localization of ATF5

4. Discussion

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