SIRT1 and AROS suppress doxorubicin-induced apoptosis via inhibition of GSK3β activity in neuroblastoma cells

ABSTRACT SIRT1, the best-characterized member of the sirtuin family of deacetylases, is involved in cancer, apoptosis, inflammation, and metabolism. Active regulator of SIRT1 (AROS) was the first identified direct regulator of SIRT1. An increasing number of reports have indicated that SIRT1 plays an important role in controlling brain tumors. Here, we demonstrated that depletion of SIRT1 and AROS increases doxorubicin-mediated apoptosis in human neuroblastoma SH-SY5Y cells. Glycogen synthase kinase 3β (GSK3β) promoted doxorubicin-mediated apoptosis, but this effect was abolished by overexpression of SIRT1 and AROS. Interestingly, SIRT1 and AROS interacted with GSK3β and increased inhibitory phosphorylation of GSK3β on Ser9. Finally, we determined that AROS cooperates with SIRT1 to suppress GSK3β acetylation. Taken together, our results suggest that SIRT1 and AROS inhibit GSK3β activity and provide additional insight into drug resistance in the treatment of neuroblastoma.


Introduction
Neuroblastoma is one of the most lethal brain tumor in children, which is largely due to a lack of response to current therapies (Mischel et al. 2004;Löscher and Potschka 2005). Doxorubicin (DOX), the first anthracycline isolated, has been widely used as an effective anticancer drug for many types of cancers including breast cancer, soft tissue sarcomas, and hematological malignancies (Minotti et al. 2004;Lopes et al. 2008). Although DOX has been used as a routine chemotherapeutic agent, it induces drug resistance and exhibits poor penetration across the blood-brain barrier when used in the treatment of brain tumors . However, DOX resistance in neuroblastoma has been poorly studied. SIRT1, the well-characterized mammalian homolog of yeast Sir2, is an NAD + -dependent deacetylase involved in lifespan extension, cancer, inflammation, and metabolism (Herskovits and Guarente 2014;Yang et al. 2015). Active regulator of SIRT1 (AROS) was the first identified direct regulator of SIRT1 (Kim et al. 2007). Recently, an increasing number of reports have shown that SIRT1 plays an important role in brain tumors. SIRT1 is overexpressed in brain metastases of non-small cell lung cancer (NSCLC) and positively regulates the migration of NSCLC cells (Han et al. 2013). In human medulloblastoma cells, SIRT1 expression patterns have also been correlated with their formation and prognosis (Ma et al. 2013). Furthermore, deficiency of miR-34a targeting SIRT1 accelerates medulloblastoma formation (Thor et al. 2015), and its mimics induced cell death in p53-mutated medulloblastoma and glioblastoma cells (Fan et al. 2014).
Glycogen synthase kinase 3β (GSK3β) is a serine/ threonine kinase that was originally identified as a key enzyme in glycogen synthesis (Embi et al. 1980;Kaidanovich-Beilin and Woodgett 2011). GSK3β is also a downstream protein of various signaling pathways, including the Wnt/β-catenin and phosphoinositide 3 kinase (PI3K) pathways, which are involved in cell proliferation and differentiation in many tissues (Tejeda-Muñoz and Robles-Flores 2015; Hermida et al. 2017). Recent studies have shown that GSK3β is overexpressed in several cancer cell types, and inhibition of GSK3β decreased cancer cell proliferation and survival (Luo 2009;Zeng et al. 2014;Domoto et al. 2016). Whereas, several studies have reported that GSK3β promotes apoptosis in various stress conditions (Bijur et al. 2000;Song et al. 2002;Watcharasit et al. 2002). Therefore, further study on the role of GSK3β in cancer is still needed. Furthermore, the functional correlation between SIRT1 and GSK3β in brain tumorigenesis is unknown.
Here, we investigated the role of SIRT1/AROS in DOXmediated cytotoxicity and identified GSK3β as a novel SIRT1/AROS interacting protein. Our results suggest that SIRT1 and AROS suppress GSK3β activity and acetylation, regulating the chemoresistance of doxorubicin in neuroblastoma.

Plasmids and cloning
Full-length GSK3β was PCR amplified, subcloned into Flag-tagged pcDNA3 and pEGFP-C3 vectors (BD Biosciences), and verified by sequencing. Other constructs were generated as previously described (Kim et al. 2007).

Cell culture and transfection
Human neuroblastoma SH-SY5Y cells were maintained in a minimal essential medium. HEK293 cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and an antibioticantimycotic mix (all from Invitrogen). Cells were grown in a humidified incubator at 37°C with 5% CO 2 . Under experimental conditions, plasmids were transfected using Lipofectamine with Plus Reagent (Invitrogen) or Polyethylenimine (PEI, Polysciences Inc.) according to the manufacturer's instructions. To assess the effects of GSK3β, cells were treated with 10 μM SB216763 (Sigma-Aldrich) for 2 h prior to exposure to 0.1 μM DOX for 12 h.

Flow cytometry
Flow cytometry was performed as previously described (Kim et al. 2002). At 12 h after exposure to DOX, adherent and detached cells were collected and fixed in 70% ethanol. Cells were stained with 5 μg/mL propidium iodine-containing 100 μg/mL RNase A for 30 min. A total of 1 × 10 6 cells were analyzed with a flow cytometer (Guava EasyCyte, Cell Signaling Technology).
Hoechst staining DOX-treated cells were fixed in 70% ethanol for 10 min at room temperature and stained with 1 mg/mL Hoechst (Sigma-Aldrich) at a dilution of 1:1000 for 10 min. Apoptotic cells were observed under an immunofluorescence microscope (Carl Zeiss).

Confocal microscopy
Experiments were performed as previously described (Kim et al. 2002). SH-SY5Y cells were seeded in a culture chamber and transfected with GFP-GSK3β, Flag-SIRT1, and Myc-AROS. Cells were washed with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde for 10 min at room temperature. Following permeabilization with 0.3% Triton X-100 for 15 min, cells were blocked in PBS containing 3% bovine serum albumin for 1 h. Cells were then incubated with rabbit polyclonal anti-SIRT1 antibody (sc-15404; Santa Cruz Biotechnology) and mouse monoclonal anti-Myc antibody. Cells were followed by incubation with rhodamine-conjugated anti-rabbit IgG and DyLight 405-conjugated anti-mouse IgG (Jackson ImmunoResearch) for 1 h. Mounted cells were visualized with a confocal microscope (Leica).

Depletion of SIRT1 and AROS increase DOXinduced apoptosis
Based on recent reports on the role of SIRT1 in brain tumors, we first explored the effects of SIRT1 on apoptosis induced by DOX in human neuroblastoma SH-SY5Y cells. As shown in Figure 1(A), the shRNA-mediated knockdown of SIRT1 significantly increased apoptosis compared to sh-Luciferase-transfected cells as indicated by PARP-1 cleavage, a marker of apoptosis. AROS deficiency or depletion of both SIRT1 and AROS also effectively enhanced PARP-1 cleavage (Figure 1(A)). Flow cytometry revealed that the sub-G1 phase population, an indicator of apoptotic cell death, was increased in SIRT1-deficient cells. Similarly, knockdown of AROS or both SIRT1 and AROS also enhanced the percentage of cells in sub-G1 (Figure 1  (B)). Furthermore, Hoechst staining revealed that chromatin condensation and nuclear fragmentation were markedly increased following depletion of SIRT1 and/or AROS (Figure 1(C and D)). Overall, our data suggest that deficiency of SIRT1 and/or AROS increased cellular sensitivity to DOX-induced apoptosis and the effects of AROS may be achieved through activation of SIRT1.

SIRT1 and AROS decrease GSK3β-mediated sensitivity to DOX
A recent study reported that SIRT1 promotes axonogenesis by deacetylating Akt and inactivating GSK3β ). Inhibition of GSK3β attenuates stress-induced apoptosis in SH-SY5Y cells (Bijur et al. 2000;Song et al. 2002). To investigate the functional relevance between GSK3β and SIRT1 in response to DNA damage, we first determined the effects of GSK3β inhibition on apoptosis followed by DOX treatment in SH-SY5Y cells. As expected, treatment with SB216763, a pharmacological inhibitor of GSK3β, greatly attenuated PARP-1 cleavage induced by DOX (Figure 2  (A)). Next, we addressed the effects of GSK3β following DOX treatment. As shown in Figure 2(B), overexpression of GSK3β markedly enhanced DOX-induced PARP-1 cleavage. By contrast, SIRT1 significantly impaired GSK3βmediated sensitivity to DOX (Figure 2(B)). Furthermore, PARP-1 cleavage demonstrated a similar resistance effect of AROS expression on GSK3β-induced apoptosis (Figure

SIRT1 and AROS interact with GSK3β in the nucleus
To examine whether SIRT1 physically interacts with GSK3β, we co-transfected HEK293 cells with empty vector or Flagtagged SIRT1 and GFP-tagged GSK3β. IP using an anti-Flag antibody and subsequent WB with anti-GFP antibody demonstrated that SIRT1 interacts with GSK3β (Figure 3  (A)). Co-transfection followed by IP indicated that AROS also associates with GSK3β (Figure 3(B)). Next, we confirmed the complex formation of these proteins. GFP-SIRT1 and GFP-GSK3β were detected by IP with anti-Flag antibody for AROS, supporting GSK3β forms the complex with SIRT1 and AROS (Figure 3(C)). We previously reported that SIRT1 and AROS primarily colocalize in the nucleus (Kim et al. 2007), whereas GSK3β is predominantly found in the cytoplasm. To further substantiate the interaction, we observed the subcellular distribution of SIRT1, AROS, and GSK3β in SH-SY5Y cells. As shown in Figure 3(D), confocal microscopy revealed that Flag-SIRT1, Myc-AROS, and GFP-GSK3β colocalized in the nucleus. Overall, our data suggest that SIRT1 and AROS physically interact with GSK3β in the nucleus, and may perturb its activity and cytoplasmic function.

SIRT1 and AROS increase inhibitory phosphorylation of GSK3β
To explore the molecular mechanism underlying the regulation of GSK3β activity by SIRT1 and AROS, we analyzed the effects of both SIRT1 and AROS on the inhibitory phosphorylation of GSK3β. GSK3β activity is inhibited by phosphorylation of Ser9 via p70 S6 kinase, p90Rsk, and Akt (Ali et al. 2001;Mulholland et al. 2006). As shown in Figure 4(A), overexpression of SIRT1 or AROS strongly increased phosphorylation of GSK3β Ser9 in HEK293 cells. By contrast, SIRT1 or AROS depletion resulted in markedly decreased phosphorylation of GSK3β Ser9 in SH-SY5Y cells (Figure 4(B)). These results demonstrate that SIRT1 and AROS inhibit DOX-induced apoptosis by suppressing the activity of GSK3β.

SIRT1 deacetylates GSK3β by cooperating with AROS
To investigate the possibility of GSK3β as a SIRT1 substrate, we assessed whether SIRT1 deacetylates GSK3β. HEK293 cells were co-transfected with Flag-tagged GSK3β and GFP-tagged SIRT1 and/or Myc-tagged AROS. Anti-Flag immunoprecipitates probed with antiacetyl lysine antibody revealed that the level of GSK3β acetylation was substantially reduced by SIRT1 and/or AROS expression (Figure 5(A)). By contrast, SIRT1 or AROS depletion resulted in significantly increased the level of Flag-GSK3β acetylation in HEK293 cells ( Figure  5(B)). These results suggest that SIRT1 promotes deacetylation of GSK3β, likely through cooperation with AROS, and may inhibit its role in DOX-induced apoptosis in neuroblastoma cells.

Discussion
Neuroblastoma is the most common type of extracranial solid tumor in children and is responsible for up to 15% of all pediatric oncologic deaths (Whittle et al. 2017;Swift et al. 2018). Neuroblastoma is a heterogeneous tumor with a wide range of clinical behaviors; some tumors exhibit aggressive growth and are more likely to metastasize (Maris 2010). However, the development of therapeutic approaches has been limited despite decades of intensive research and clinical trials. Understanding the molecular mechanisms of chemotherapy resistance in neuroblastoma is critical for improving the poor prognosis.
SIRT1 is an NAD + -dependent deacetylase that plays a crucial role in tumorigenesis through the deacetylation of tumor regulatory proteins . Recent studies have shown that suppression of deacetylase activity or expression of SIRT1 induces apoptosis in human neuroblastoma cells (Tu et al. 2018;Fu et al. 2019). These reports suggest that inhibition of SIRT1 might be a promising target in the treatment of neuroblastoma. GSK3β is also an interesting potential target for neuroblastoma therapy, as it has been implicated in cell growth inhibition and apoptosis in various cancers. GSK3β promotes apoptosis in various damage conditions (Bijur et al. 2000;Song et al. 2002;Watcharasit et al. 2002). Therefore, identifying the functional correlation between SIRT1 and GSK3β is important for the development of novel therapies for neuroblastoma.
Here, we first proposed the reciprocal roles of SIRT1/ AROS and GSK3β in cytotoxicity in response to DOX treatment. Our data demonstrate that DOX-induced apoptosis was enhanced by deficiency of SIRT1 and AROS and increased by GSK3β expression in human neuroblastoma SH-SY5Y cells. We also identified GSK3β as a binding partner of SIRT1 and AROS. The interaction between GSK3β and SIRT1/AROS was determined in the nucleus by IP and confocal microscopy. GSK3β is mainly known as a cytoplasmic protein, but is also found in the nucleus and mitochondria (Bijur and Jope 2003). Recent studies have shown that nuclear localization of GSK3β is regulated by mTOR complex 1 (mTORC1) (Bautista et al. 2018), and nuclear GSK3β facilitates acute myeloid leukemia (AML) cell growth and drug resistance (Ignatz-Hoover et al. 2018). However, the regulation of nuclear GSK3β is not well defined. Our results suggest that SIRT1 might be a potential regulator of nuclear GSK3β. Furthermore, SIRT1 and AROS inhibited GSK3β by increasing inhibitory phosphorylation on Ser9. Finally, we observed that SIRT1 and AROS deacetylated GSK3β. Since GSK3β binds to SIRT1 and AROS in the nucleus, it is expected to be deacetylated in nucleus. SIRT1mediated deacetylation of GSK3β may promote inhibitory phosphorylation of GSK3β on Ser9. Recent evidence shows that high glucose induces HDAC1-mediated deacetylation and enhances phosphorylation of S6 kinase in mesangial cells (Das et al. 2019). In addition, a structural basis study suggests that lysine acetylation disrupts phosphorylation in a kinase motif by inhibiting salt bridge (Parker et al. 2014). Therefore, SIRT1-mediated deacetylation of GSK3β is likely to facilitate its inhibitory phosphorylation and may inhibit GSK3β activity. Overall, our results suggest that SIRT1 and AROS inhibit GSK3β activity and provide a basis for the mechanism of DOX efficacy in brain tumor chemotherapy.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
The present research was conducted by the research fund of Dankook University in 2015.