Reciprocal Negative Feedback Regulation of ATF6α and PTEN Promotes Prostate Cancer Progression

doi:10.21203/rs.3.rs-2622377/v1

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Reciprocal Negative Feedback Regulation of ATF6α and PTEN Promotes Prostate Cancer Progression

https://doi.org/10.21203/rs.3.rs-2622377/v1

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published 16 Sep, 2023

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Phosphatase and tensin homolog (PTEN) loss tightly correlates with prostate cancer (PCa) progression and metastasis. Inactivation of PTEN leads to abnormal activation of PI3K/AKT pathway. However, results from clinical trials with AKT inhibitors in PCa have been largely disappointing. Identification of novel regulators of PTEN in PTEN-dysfunctional PCa is urgently needed. Here we demonstrated that the expression level of PTEN was inversely correlated with the signature score of unfolded protein response (UPR) in PCa. Importantly, PTEN suppressed the activity of ATF6α, via interacting to de-phosphorylate ATF6α and consequently inhibiting its nuclear translocation. Conversely, ATF6α promoted the ubiquitination and degradation of PTEN by inducing CHIP expression. Thus, ATF6α and PTEN formed a negative feedback loop during PCa progression. Combination of ATF6α inhibitor with AKT inhibitor suppressed tumor cell proliferation and xenograft growth. Importantly, this study highlighted ATF6α as a therapeutic vulnerability in PTEN dysfunctional PCa.

PTEN

UPR

ATF6α

prostate cancer

Prostate cancer (PCa) is characterized as being histologically and molecularly heterogeneous, with low-risk localized PCa having an indolent natural history, while others have aggressive and lethal disease [1, 2]. Phosphatase and tensin homolog (PTEN) is one of the most commonly inactivated tumor suppressor gene in PCa and its loss is associated with poor clinical outcome [2, 3]. PTEN deficiency, resulting mainly from PTEN deletion or mutation, could be detected in tissues representing all stages of PCa progression, and especially at much higher frequency in metastatic PCa and castration resistant prostate cancer (CRPC)[4–6]. Inactivation of PTEN leads to abnormal activation of PI3K/AKT pathway[7]. Until recently, however, results from clinical trials with AKT inhibitors in PCa have been largely disappointing[8–10]. Therefore, an appropriate design of rational combinations of PI3K/AKT inhibitors with other novel agents is urgently needed. Most recently, Sweeney et al. reported that co-targeting AKT and androgen-receptor signaling pathway was a potential treatment strategy for men with PTEN-loss mCRPC[11]. Of note, PTEN function is also regulated at the transcriptional, post-transcriptional and translational levels[12–14]. Thus far, it remains largely unknown as to what extent dysregulation of these processes could contribute to PCa progression.

The unfolded protein response (UPR) is a homeostatic mechanism to maintain endoplasmic reticulum (ER) function[15]. Previous studies have demonstrated that aberrant UPR signaling pathway plays an important role in PCa progression[16, 17]. The UPR is mediated by at least three well-conserved stress sensors that are ER-localized transmembrane receptors: pancreatic ER kinase-like ER kinase (PERK), inositol-requiring kinase 1 (IRE1), and activating transcription factor 6 (ATF6)[15]. ATF6 consists of two isoform ATF6α and ATF6β[18], both of which can be activated by ER stress[19]. Upon activation, the N-terminal fragment of ATF6α and ATF6β, designated as ATF6α(N) and ATF6β(N), is cleaved from their full-length in the Golgi apparatus and transported into the nucleus to induce gene expression[20]. ATF6α plays a dominant role in inducing the expression of ATF6 target genes. Clinically, Liu et al suggested that ATF6α is expressed at a higher level in PCa tissues than in benign prostatic hyperplasia (BPH)[16]. In the present study, we found that PTEN expression is adversely related to UPR signature in human PCa tissues. Reintroduction of PTEN into PTEN-deficient LNCaP cells leads to ATF6α dephosphorylation, inhibits its nuclear translocation and transcriptional regulatory activity. In turn, ATF6α could promote PCa progression by destabilizing PTEN protein in CHIP-dependent way. Importantly, co-targeting ATF6α and PI3K/AKT pathways may be a promising therapeutic approach for PCa, especially the ones with PTEN dysfunction.

Cell culture and reagents

LNCaP, VCaP, DU145, PC3 and HEK293T cell were purchased from American Type Culture Collection (ATCC). All cell lines were authenticated by short tandem repeat analysis and were tested for mycoplasma contamination. The information of reagents is shown in Supplementary Table 1.

Animals

All experimental protocols for animal studies were approved by the Institutional Animal Care and Use Committee of School of Basic Medical Sciences, Shandong University (Document No. ECSBMSSDU2021-2-126) and conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Different groups were allocated in a randomized manner and investigators were blinded to the allocation of different groups when doing drug treatment. More detailed information on animals is included in Supplementary Materials and Methods.

Patients And Tissue Specimens

A total of three tissue microarrays were constructed representing 15 patients with BPH and 130 clinically localized PCa patients who underwent radical prostatectomy between 2012 and 2015 at Qilu Hospital of Shandong University (Jinan, China). Three cores (0.6 mm in diameter) were taken from each representative tumor focus and the morphology was confirmed by two pathologists (J.H. and M.Q). The localized tissues consisted of Gleason score 3 + 3 = 6 (n = 17), Gleason score 3 + 4 = 7 (n = 20), Gleason score 4 + 3 = 7 (n = 24), Gleason score 4 + 4 = 8 (n = 30), and Gleason score ≥ 9 (n = 39) tumors. This study was conducted in accordance with the International Ethical Guidelines for Biomedical Research Involving Human Subjects. This study protocol was approved by Shandong University Medical Research Ethics Committee according to the Declaration of Helsinki (Document No. ECSBMSSDU2021-1-61).

Cell Viability

Cell viability was performed as previous described[21]. Cell proliferation rates were measured by MTT assays and all values were normalized to the value of day 0.

Immunohistochemistry And Immunofluorescence Staining

Immunohistochemistry was carried out as described previous[22]. More detail information on the Immunohistochemistry procedure and tissues microarray evaluation are included in Supplementary Materials and Methods. For immunofluorescence, proteins were detected using goat anti-mouse Alexa Fluor 594. For quantitation of ATF6α subcellular distribution, at least 150 cells were scored for each experiment. Cells were rated as showing nuclear staining (N), cytoplasmic staining (C), or staining within both the nucleus and the cytoplasm (N + C). The ATF6α nuclear location (%) = N + (N + C) / total cells. Nuclei were identified by DAPI staining. The information of antibodies is shown in Supplementary Table 2.

Western Blot And Co-immunoprecipitation

Western blot and co-immunoprecipitation

Preparation of cell lysates and performance of western blot and immunoprecipitation were carried out according to our published procedure[21]. Cytoplasmic and nuclear proteins were extracted using the ProteoExtract subcellular proteome extraction kit (#539791, Millipore). The information of antibodies is summarized in Supplementary Table 2.

Rna Isolation And Real-time Qpcr Analysis

Total RNA was extracted using TRIzol reagent according to the manufacturer’s protocol (Vazyme Biotech, R401-01 regent kit) and 1 µg of RNA was reverse-transcribed with ReverTra Ace qPCR RT kit (PCR-311, Toyobo). Real-time qPCR was performed using the SYBR Green mix (QPK-201, Toyobo). The primer sequences used are listed in Supplementary Table 3.

Transient Transfection And Viral Transduction

Premade siRNA constructs were purchased from RiboBio (Guangzhou, China) and all the siRNA constructs sequences were list in Supplementary Table 4. For DNA transfections, the plasmid constructs were purchased from Biosun Inc (Jinan, China) and WZ Biosciences Inc (Jinan, China). The transfection procedure was performed as previously described[23]. Human Lenti-ATF6α-GFP and its control Lenti-GFP were obtained from Genecopoeia. To acquire a stable cell line, single-cell clonal isolates were selected by puromycin.

Luciferase Reporter Gene Assay And Chromatin Immunoprecipitation (Chip)

Luciferase activity was determined using the Promega luciferase assay kit (E1910, Promega) following the manufacturer’s protocol. Reporter activity was detected using the Molecular devices spectraMzx iD3. ChIP experiments were performed with ChIP assay kit (#17–295, Millipore) according to the manufacturer’s protocol. Briefly, Cells were sonicated, pre-cleaned, mixed with magnetic beads and incubated with indicated antibodies. After DNA purification, the enrichment of the DNA template was analyzed by PCR and real-time qPCR using primers. The primers were list in Supplementary Table 5.

Gene Set Enrichment Analysis

We used the java GSEA v4.0 program. GSEA were performed according to the instructions. Gene sets of Hallmark Collection, Canonical Pathway were used. All gene sets are from the Molecular Signatures Database v7.5.1.

Quantification And Statistical Analysis

All of the statistical details including the statistical tests used, exact number of animals, definition of center, and dispersion and precision measures for the experiments can be found in the figures, figure legends or in the results. All statistical analyses were carried out using GraphPad Prism 8 software (GraphPad software, CA, US). The two-tailed unpaired t-test was used to calculate statistical significance between the two groups. Survival information was verified by Kaplan-Meier analysis and compared using the log-rank test. All results were presented as the mean and the standard error of the mean. P values considered to be significant as follows: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

PTEN expression is adversely related to UPR signature and PTEN deficiency synergizes with ATF6α to activate UPR signaling in PCa cells

To determine the correlation between PTEN expression and UPR signaling pathway in PCa, we have analyzed various PCa public datasets. As shown in Fig. 1a, 1b, gene set enrichment analysis (GSEA) was performed using microarrays from Gene Expression Omnibus (GEO) datasets (GSE3325 and GSE6919). The UPR gene signature was significantly enriched in PTEN-low tumors compared with PTEN-high tumors. Of note, ATF6α target genes (HSPA5 and PDIA6) were up-regulated in PTEN-low samples (Supplementary Fig. 1a, 1b).

To further confirm the relation between PTEN function and UPR activation, prostate-specific Pten knockout (Pten^flox/flox: Pb-Cre⁺, referred to herein as Pten^PC–/– mice) mice were used. As shown in Fig. 1c, PTEN protein levels were significantly decreased in wild type (WT) mice under ER stress inducer tunicamycin (Tm) treatment. In addition, spliced ATF6α (ATF6α-N), and its target gene BiP were increased significantly after Tm treatment in Pten^PC–/– prostate tissues than those in WT mice. To further verify the effects of PTEN on UPR activity, we next silenced PTEN with siRNA in DU145 and VCaP (PTEN-WT) cells and overexpressed PTEN in LNCaP and PC3 (PTEN-deficient) cells. Western blot showed that the protein levels of ATF6α and its target gene were increased after silencing PTEN expression, especially in the presence of Tm treatment in DU145 and VCaP cells (Fig. 1d and Supplementary Fig. 1d). In contrast, PTEN overexpression inhibited protein expression of ATF6α and its target gene induced by Tm in LNCaP and PC3 cells (Fig. 1e and Supplementary Fig. 1e). Similarly, real-time qPCR revealed that mRNA levels of the ATF6α target genes were suppressed by overexpression PTEN in LNCaP cells and activated by silence PTEN in DU145 cells (Fig. 1f, 1g and Supplementary Fig. 1f). Additionally, as shown in Fig. 1h, 1i, the BiP luciferase activity was highly induced by Tm. However, these activity effects of Tm were further enhanced by PTEN silencing in DU145 cells but decreased by PTEN overexpression in LNCaP cells. Collectively, these results demonstrated that ATF6α signaling was adversely related to PTEN expression.

Further analyses on The Cancer Genome Atlas (TCGA) and GSE29010 showed that UPR gene signature was enriched in PTEN-loss prostate tumors when compared with PTEN-WT prostate tumors (Supplementary Fig. 1g, 1h). We next analyzed the relationship between PTEN loss and expression of UPR target genes. As shown in Supplementary Fig. 1i, compared with target genes (ASNS, ATF3, ATF5) of PERK and target genes (HSPA13, SERP1) of IRE1α, the mRNA levels of ATF6α target genes (HSPA5, PDIA4)[24] were significantly increased in PCa tissues with PTEN loss. Collectively, these results supported that PTEN dysfunction can activate the ATF6α signaling.

Since ATF6α could be activated by PTEN knockout or knockdown, we aimed to confirm whether PTEN dysfunction participated in the activity of ATF6α signaling. As shown in Fig. 1j, the combination of PTEN silencing and ATF6α overexpression significantly increased mRNA levels of HERPUD1, SEL1L, and BiP in DU145 cells. However, the increase induced by PTEN silencing could be attenuated when ATF6α was silenced (Fig. 1k). Likewise, we identified that the increase of the luciferase activity of BiP promoter caused by PTEN knockdown could be further strengthened by introducing ATF6α (Fig. 1l) but abolished by silencing ATF6α in DU145 cells (Fig. 1m).

ATF6α protects against ER stress-induced cytotoxicity and promotes PCa progression in vivo

To define the function of ATF6α in PCa progression, we performed IHC in a cohort of 145 Chinese PCa cases from Qilu hospital with three tissue microarrays (TMAs). We found that ATF6α was expressed in both nucleus and cytoplasm. Of note, nuclear staining of ATF6α was frequently seen in PCa cases with high Gleason score. Representative image of IHC staining for nuclear ATF6α expression of PCa cases were shown in Fig. 2a. We showed a significantly positive correlation between nuclear ATF6α expression with Gleason score (Fig. 2b). Furthermore, high expression of ATF6α was significantly associated with poor clinical outcome (Fig. 2c). We also observed that the expression levels of ATF6α target genes were significantly higher in localized PCa than those in BPH tissues as shown in GSE46602 and GSE70768 (Supplementary Fig. 2a). And the Kaplan-Meier curve showed that patients with high levels of ATF6α target genes had a shorter biochemical relapse free survival rate than those with low expression (Supplementary Fig. 2b). In all, these results suggested that upregulation of ATF6α target gene was associated with PCa progression to advanced clinical states. To elucidate the biological functions of ATF6α in PCa cells, we designed siRNA against ATF6α. As shown in Supplementary Fig. 2c, 2d, transfection of siRNA constructs can significantly deplete ATF6α protein and mRNA levels in DU145 and LNCaP cells. We showed that Tm treatment resulted in decreased cell viability in DU145 and VCaP cells (Fig. 2d, 2e and Supplementary Fig. 2e, 2f) and the inhibitory effects of Tm on cell viability were further enhanced by treatment with ATF6α siRNA or its inhibitor (Fig. 2e and Supplementary Fig. 2f) but compromised by ATF6α overexpression (Fig. 2d and Supplementary Fig. 2e). Next, in vivo experiments demonstrated that, introduction of ATF6α into DU145 cells showed an increased tumor growth in the xenograft models when compared to its parental controls (998 ± 145.6mm³vs. 456 ± 93 mm³) (Fig. 2f, 2h). Moreover, the protein levels of ATF6α and its target genes in DU145-ATF6α group were relatively increased compared with that in DU145-vector group (Fig. 2i, 2j). Collectively, our data suggested that ATF6α contributes to PCa progression.

PTEN interacts with ATF6α and inhibits ATF6α activity

To elaborate on the effect of PTEN on ATF6α, we sought to determine whether ATF6α physically associates with PTEN. As shown in Fig. 3a, HA-PTEN co-precipitated with the full-length Flag-ATF6α (short for ATF6α(P)), but not the N-terminal of ATF6α (short for ATF6α(N)) in LNCaP cells. Interestingly, Tm diminished the interaction between HA-PTEN and ATF6α(P). Additionally, the endogenous interaction between ATF6α(P) and PTEN in DU145 cells dramatically decreased with the presence of Tm (Fig. 3b). Finally, to determine whether PTEN directly interacts with ATF6α, GST, and recombinant GST-ATF6α were used to pull down the lysates of HEK293T cells with HA-PTEN overexpression. As shown in Fig. 3c, GST-ATF6α, but not GST protein, interacted with PTEN. Together, these results demonstrated the direct interaction between ATF6α and PTEN.

Immunofluorescent staining analysis showed that the nuclear staining of ATF6α markedly increased when LNCaP cells were treated with Tm, whereas the staining of ATF6α in nucleus significantly decreased when PTEN was overexpressed (Fig. 3d, 3e). We then investigated whether PTEN could inhibit the transcriptional activity of ATF6α using a BiP promoter-luciferase system. As shown in Fig. 3f, introducing PTEN profoundly diminished the transcriptional activity of BiP promoter in LNCaP cells with overexpression ATF6α. Likewise, PTEN can significantly suppress the mRNA levels of HERPUD1, SEL1L, and BiP induced by ATF6α overexpression in LNCaP cells (Fig. 3g).

We then performed rescue experiments and the data showed that introduction of PTEN could decrease the cellular viability of LNCaP, PC3, VCaP and DU145. However, such decrease could be restored partly by ectopic overexpression of ATF6α, suggesting that ATF6α could promotes cell growth, at least in part, by deregulating PTEN (Fig. 3h and Supplementary Fig. 3a, 3b). Furthermore, overexpression of ATF6α could increase the cell viability of PCa cells with PTEN deficiency, which indicates that ATF6α may enhance cell growth independently of PTEN. Since BiP has been shown to control ATF6α export from ER through a dissociation mechanism[25], we next determined whether PTEN plays a role in their dissociation. The interaction between ATF6α and BiP was determined by immunoprecipitation. In consistent with the previous study, ATF6α dissociated from BiP when PCa cells were challenged with Tm. However, there was no visible change in dissociation when PTEN was overexpressed in LNCaP cells (Supplementary Fig. 3c) or knocked down in DU145 cells (Supplementary Fig. 3d). This suggested that PTEN cannot affect the dissociation between BiP and ATF6α.

ATF6 α activity was repressed by PTEN mediated-phosphatase activity

We subsequently explored the molecular basis by which PTEN inactivates ATF6α. Since PTEN is a protein phosphatase that inactivates protein function by dephosphorylation[26, 27], we first determined whether ATF6α could be phosphorylated or not in PCa cells. As shown in Fig. 4a, 4b, the tyrosine phosphorylation levels of ATF6α were dramatically increased when DU145 cells were stimulated by Tm. To define the exact phosphorylation site on ATF6α, we constructed several plasmids targeting the tyrosine sites of ATF6α. The results showed that compared with ATF6α^WT, the expression levels of phosphorylated and spliced ATF6α were decreased more significantly in ATF6α^Y111A in the presence of Tm (Fig. 4c). Concordantly, real-time qPCR analysis demonstrated that the expression level of HERPUD1 was significantly decreased in ATF6α^Y111A group than ATF6α^WT (Fig. 4d). In addition, PTEN only pulled down ATF6α^WT but not ATF6α^Y111A, which suggests that Y111 is required for its interaction with PTEN (Fig. 4e). To examine whether PTEN dephosphorylates ATF6α or not, PTEN was silenced in DU145 (Fig. 4f) or overexpressed in LNCaP cells (Fig. 4g). The levels of phosphorylated ATF6α(P) were significantly increased in DU145 cells but decreased in LNCaP cells compared with their control. However, no observable change was detected in DU145 or LNCaP cells with mutant ATF6α^Y111A in response to PTEN knockdown or overexpression (Fig. 4f, 4g), indicating tyrosine residue Y111 is important for the phosphorylation of ATF6α.

To further confirm the specific phosphatase sites of PTEN for ATF6α inactivating dephosphorylation, we transfected LNCaP cells with a variety of PTEN expression constructs with mutations diagramed in Fig. 4h. These mutations include the protein phosphatase dead PTEN^Y138L, the lipid phosphatase-dead PTEN^G129E, dual phosphatase-deficient PTEN^C124S, and PTEN^G129R constructs. After transfection with PTEN^G129E, the levels of phosphorylated ATF6α were reduced in LNCaP cells (Fig. 4i). In addition, none of the PTEN constructs affected ATF6α^Y111A phosphorylation levels (Fig. 4j). Collectively, these data indicated that PTEN inhibits ATF6α activation by dephosphorylation. To examine the functional consequence of PTEN-mediated dephosphorylation of ATF6α, we analyzed its nuclear translocation in DU145 cells. Our data showed that ATF6α^WT, but not ATF6α^Y111A was transported into the nucleus in the presence of Tm (Fig. 4k). Furthermore, as shown in Fig. 4l, ATF6α^Y111A exhibited low transcriptional activity in both unstressed and stressed cells when compared with ATF6α^WT, which was consistent with the results obtained from real-time qPCR of HERPUD1, SEL1L, and BiP (Fig. 4m). These results highlighted that PTEN-inactivity-mediated phosphorylation is required for the protein cleavage, nuclear translocation, and the subsequent transcription modulation of ATF6α.

ER stress induces PTEN protein instability through ATF6α and ATF6 α destabilizes PTEN protein via CHIP-mediated polyubiquitin

Since a significant decrease in PTEN protein levels were observed in mice prostate tissues in the presence of Tm (Fig. 1c), we then treated DU145 and VCaP cells with ER stress inducers in vitro. We found that there was a progressive reduction in PTEN expression in a time- and dose-dependent manner at the protein levels (Fig. 5a and Supplementary Fig. 4b) but not at the mRNA levels (Supplementary Fig. 4c), suggesting the potential of posttranslational modification. We then aimed to determine of which the UPR signaling pathways (ATF6α, PERK, and IRE1) was required for the decrease of PTEN expression by using MEFs from Atf6α, Perk, and Ire1 knockout mice. Our data showed that the reduction of PTEN protein levels caused by Tm challenge could be partly reversed in Atf6α^−/− MEFs compared with those in Perk^−/− and Ire1^−/− MEFs (Fig. 5b). Similar effects were also observed when ATF6α, PERK or IRE1 was silenced in DU145 cells (Fig. 5c). However, PTEN protein levels could be rescued after introducing Atf6α into Atf6α^−/− MEFs (Fig. 5d). Collectively, these results suggest that Tm-induced decrease of PTEN protein expression was mediated by ATF6α.

To characterize the inhibitory effect of Tm on PTEN expression, we treated DU145 cells with MG132 (a proteasome inhibitor) or leupeptin (a protease inhibitor). Our data showed that MG132, but not leupeptin, could rescue Tm-induced PTEN protein levels downregulation (Fig. 5e and Supplementary Fig. 4d). This suggested that PTEN protein undergoes degradation in a proteasome-dependent manner in the presence of Tm. Our results were consistent with previous report, which demonstrated that PTEN protein levels were regulated by both polyubiquitylation and monoubiquitylation[28]. Further analysis showed that more polyubiquitination in PTEN protein occurred in the presence of Tm stimulation or ATF6α overexpression in DU145 cells (Fig. 5f, 5g). Moreover, polyubiquitination of PTEN proteins could hardly be detected in Atf6α^−/− MEFs when compared to WT (Fig. 5h). However, such decrease in polyubiquitination could be restored by introducing ATF6α into Atf6α^−/− MEFs (Fig. 5h). These results confirmed that ATF6α plays an important role in PTEN degradation induced by Tm.

We further investigated the molecular mechanism by which ATF6α mediated PTEN suppression. It was previously reported that CHIP, NEDD4-1, XIAP, and WWP-2 [29–32] regulated PTEN by promoting protein degradation. To search for genes that participate in PTEN degradation induced by ATF6α, we performed real-time qPCR assay in DU145 cells treated with Tm. The data showed that the increase of CHIP mRNA levels in DU145 cells induced by Tm could be reduced by silencing ATF6α (Supplementary Fig. 4e). This result was also confirmed by western blot (Fig. 5i, 5j and Supplementary Fig. 4f). Furthermore, CHIP protein levels were decreased more significantly in Atf6α^−/− MEFs, compared with those of Perk^−/− and Ire1^−/− MEFs (Supplementary Fig. 4g). However, such inability in Atf6α^−/− MEFs could be rescued by introducing ATF6α (Fig. 5k). These data supported that ATF6α was important for protecting CHIP protein stability. We next aimed to assess whether ATF6α destabilizes PTEN protein via CHIP-mediated polyubiquitination. As shown in Fig. 5l, the knockdown of CHIP expression inhibited PTEN polyubiquitination induced by ATF6α in DU145 cells. Similarly, the decreased PTEN polyubiquitination caused by ATF6α knockdown could be reversed by introducing CHIP expression in DU145 cells (Fig. 5m).

To determine whether ATF6α directly regulates CHIP expression, we conducted a luciferase activity assay with CHIP promoter containing reporter plasmid. Our data showed that Tm treatment could significantly increase the transcriptional activity of CHIP promoter, which also could be enhanced by overexpressing ATF6α (Fig. 5n). It was previously reported that TGAC is the core sequence critical for ATF6α binding[33]. We aligned this sequence with CHIP promoter from − 2000 ~ + 300bp and found two perfectly matched sequences. We constructed two TGAC-mutant plasmids and performed transient transfection assays with Tm treatment. As shown in Fig. 5o, mutant construct failed to affect CHIP activity in the presence of Tm.

Combined inhibition of ATF6α and AKT synergistically delays PCa progression

In the context of PTEN inactivity, the most pronounced signaling event is the constitutive activation of AKT which associates with the UPR pathway[34]. Indeed, we identified that silencing ATF6α could attenuate the activation of PI3K/AKT pathway in DU145 cells (Fig. 6a). Similar effects were also observed in Atf6α^−/− MEFs when compared with WT MEFs (Fig. 6b). Furthermore, the decreased activity of AKT signaling elicited by ATF6α deficiency was rescued by re-introducing ATF6α in Atf6α^−/− knockout MEFs (Fig. 6c). Either PTEN silencing or ATF6α overexpression enhanced AKT activity in DU145 cells, and the combination of them was more effective in increasing AKT activity (Fig. 6d).

We then evaluated the potential benefit of co-targeting ATF6α and AKT in PCa cells. As shown in Fig. 6e and Supplementary Fig. 5a, combination of AKT inhibitor (AZD5363) with ATF6α inhibitor (Ceapin-A7) was more effective in inhibiting cell proliferation in DU145 and VCaP cells. Similar to the pharmacologic inhibition, siATF6α combined with AKT inhibitor treatment significantly reduced cell viability of PCa cells (Fig. 6f and Supplementary Fig. 5b).

To determine the impacts of AZD5363 and Ceapin-A7 on tumor growth, we have established DU145 xenograft model. As shown in Fig. 6g-6i, either AZD5363 or Ceapin-A7 treatment reduced the tumor volume, and a combination of AZD5363 and Ceapin-A7 induced further inhibition in tumor growth. The expression level of activated ATF6α was detected by western blot and real-time qPCR assay in these tumors (Fig. 6j, 6k). These results suggested that ATF6α inhibitor combined with AKT inhibitor would be useful for the treatment of PCa.

This study discovered a novel reciprocal negative feedback regulation of ATF6α and PTEN that promotes PCa progression. We demonstrated that 1) ATF6α is a novel negative regulator of PTEN by promoting PTEN protein degradation. 2) PTEN can shift the UPR pathway by selective inactivation of ATF6α. 3) Co-targeting ATF6α and AKT suppresses tumor cell proliferation and xenograft growth in PCa. These results highlighted co-targeting the AKT pathway and ATF6α oncogenic signaling as potential future therapies for PCa cases with PTEN dysfunction.

Inactivation of PTEN leads to abnormal activation of PI3K/AKT pathway. Multiple studies have demonstrated a key role of the PI3K/AKT signaling axis in the development of PCa and maintenance of CRPC[35, 36]. Until recently, however, results from clinical trials with AKT inhibitors in PCa have been largely disappointing. Crabb et al. showed that pan-AKT inhibitor capivasertib with docetaxel and prednisolone failed to improve progression-free survival in patients with mCRPC irrespective of PI3K/AKT/PTEN pathway activation status[10]. Therefore, an appropriate design of rational combinations of PI3K/AKT inhibitors with other novel agents is urgently needed. Recently, Cen et al. reported that the antitumor activity of AKT inhibitor is attenuated by upregulation of receptor tyrosine kinase which is controlled by Pim-1 in a cap independent manner[8]. Conversely, drugs targeting both Pim-1 and AKT results in a synergistic decrease in tumor growth in vitro and in vivo[8]. Carver et al. showed that PI3K pathway inhibition activates AR signaling by relieving feedback inhibition of HER kinase and co-targeting AR and AKT signaling may be beneficial in mCRPC with PTEN loss[37]. In this study, we demonstrated reciprocal negative feedback regulation of ATF6α and PTEN in PCa (Fig. 7). Combined inhibition of ATF6α and AKT synergistically delays PCa progression in vitro and in vivo. Our data may provide a potential new strategy for the treatment of PCa cases.

UPR signaling can affect various aspects of tumor cell biology, including angiogenesis, invasion, and therapy resistance [17, 38–40]. Recent works have established that steroid hormone pathway, in particular androgens and estrogens related signaling, can regulate the UPR and enhance survival benefits for breast cancer and PCa cells[41]. Sheng et al. reported that AR activated the IRE1 branch and promoted PCa cell growth[17]. In addition, androgens can inhibit the activity of PERK and the phosphorylation of eIF2α. Although it has been documented that ATF6 is a major mammalian UPR sensor, there is limited data on the potential role of ATF6α in cancer progression thus far. In gastric cancer models, LncRNA MIR503HG suppressed gastric cancer progression in an ATF6α-mediated UPR dependent manner[42]. Zhang et al. elaborated that high OTUB1 expression promotes bladder cancer progression by stabilizing ATF6α[43]. Most recently, Pachikov et al reported that GCC185 played an important role in translocation of S1P and S2P proteases to the ER, which subsequent promoted ATF6 activity, and ATF6 is associated with PCa progression[44], however, the exact role of ATF6α in PCa merit further investigation. Here, we found that knockdown of ATF6α in PCa cells inhibits tumor cell proliferation, whereas overexpression of ATF6α can promote it, the latter of which became decreased when PTEN was overexpressed simultaneously. These data suggest that active ATF6α might play a significant role in promoting PCa progression, especially under PTEN dysfunction conditions.

PTEN is progressively inactivated in PCa and its loss is associated with aggressive disease, especially at much higher frequency in PCa and mCRPC. PTEN deficiency is resulting mainly from PTEN deletion or mutation. However, PTEN can also be regulated at transcriptional, post-transcriptional and translation levels. Post-translational modifications, such as phosphorylation and ubiquitylation, have been shown to decrease PTEN protein levels[45]. Polyubiquitylation and monoubiquitylation play important roles in the regulation of PTEN degradation, in which NEDD4-1, WWP2, XIAP4, and CHIP [29–32] are involved. Of them, CHIP is a highly conserved ubiquitin E3 ligase that can control the ubiquitination of multiple substrates, including p53, c-Myc, ErbB2, EGFR and PTEN[46]. In this study, our results demonstrated that ATF6α is a PTEN negative regulator and thereby facilitates prostate progression via degradation of PTEN protein, and ATF6α may serve as a key mediator linking a probable bidirectional regulatory pathway between PTEN and ER stress. Although this study identified PTEN as an important target for ATF6α, we cannot exclude the possibility that ATF6α may also modulate cell growth independently of PTEN. This based on our observations that overexpression ATF6α also promote cell growth in PTEN-deficiency cells. More works are still being needed to clarify the exact role of ATF6α in PTEN-deficient PCa cells.

In summary, our study suggested that ER stress suppressed PTEN expression and enhanced ATF6α activity to promote PCa cell proliferation. Furthermore, ATF6α could protect PCa cells against ER stress-induced apoptosis by directly inducing CHIP and subsequently destabilizing PTEN. Overall, our study provides evidence that there is a link between PTEN and UPR in the pathogenesis of PCa. PCa patients with PTEN dysfunction may benefit significantly from combined therapies that target both ATF6α and AKT.

Funding

This work was supported by the National Natural Science Foundation of China (Grants No. 82172818, 81972416), (Grant No. 81772760, 82072850), Joint Research Fund of Natural Science of Shandong Province (ZR2019LZL014), Academic promotion program of Shandong First Medical University (LJ001), China Postdoctoral Science Foundation (2022M711900) and Natural Science Foundation of Shandong Province (ZR202111150013).

Competing interests

The authors declare no competing interests.

Author contributions

HB, LW, TF, and RZ wrote the manuscript, performed, designed, and analyzed experiments, FS, HZ, and LG accomplished some of in vitro experiments, WC and MW analyzed experiments, TF, and YX accomplished some of in vivo studies, JH, MQ, and LL analyzed IHC assay. All authors read and approved the final manuscript.

Data Availability

All data and material during the current study are available from the corresponding author on reasonable request.

Ethics approval

This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of Shandong University (Document No. ECSBMSSDU2021-1-61). All animal experimental protocols were performed following the Ethical Animal Care and Use Committee of Shandong University (Document No. ECSBMSSDU2021-2-126).

Consent to participate

Informed consent was obtained from all individual participants included in the study.

Consent to publish

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Acknowledgements

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Reciprocal Negative Feedback Regulation of ATF6α and PTEN Promotes Prostate Cancer Progression

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Methods

Cell culture and reagents

Animals

Patients And Tissue Specimens

Cell Viability

Immunohistochemistry And Immunofluorescence Staining

Western Blot And Co-immunoprecipitation

Rna Isolation And Real-time Qpcr Analysis

Transient Transfection And Viral Transduction

Luciferase Reporter Gene Assay And Chromatin Immunoprecipitation (Chip)

Gene Set Enrichment Analysis

Quantification And Statistical Analysis

Results

ATF6α protects against ER stress-induced cytotoxicity and promotes PCa progression in vivo

PTEN interacts with ATF6α and inhibits ATF6α activity

Combined inhibition of ATF6α and AKT synergistically delays PCa progression

Discussion

Conclusions

Declarations

References

Supplementary Files

Status:

Journal Publication

Version 1