SAP30, a novel autophagy regulatory gene in neuroblastoma

Abstract

Neuroblastoma (NB), a devastating pediatric cancer originating from neural crest cells crucial for nervous system development, poses a significant therapeutic challenge. Despite chemotherapy being the primary treatment, approximately 70% of high-risk NB cases develop resistance. Autophagy is vital for neuronal development, balance, and differentiation of neural stem cells into mature neurons. However, the intricate mechanisms governing autophagy and the pivotal genes orchestrating its regulation in NB remain largely elusive. In this study, we first identified Sin3A Associated Protein 30 (SAP30) as a novel regulator of autophagy in NB. Silencing SAP30 inhibits autophagy and disrupts starvation-induced physiological autophagy in NB cells. Conversely, ectopic expression of SAP30 induces autophagy in NB cells under normal or starvation conditions. Mechanistically, SAP30 transcriptionally regulates STX17, a crucial protein involved in autophagosome-lysosome fusion during autophagy. Reduction of SAP30 decreases STX17 expression, hindering its translocation to the autophagic membrane and inhibiting autophagosome-lysosome fusion. SAP30-mediated autophagy enhances cell growth and provides protection in NB cells treated with chemotherapy drugs. Notably, suppressing SAP30 in vivo increases LC3B accumulation, an autophagy marker, along with reduced proliferation markers, both in vivo and in PDX tumors. Therefore, SAP30 emerges as a potential target to enhance NB responsiveness to chemotherapy drugs.

Keywords: MT: Novel therapeutic targets and biomarker development special issue; SAP30; autophagosome; autophagy; chemotherapy response; neuroblastoma.

Graphical abstract

Figure 1

Silencing of SAP30 inhibits autophagy in NB cells (A and B) Western blot analysis illustrates the protein levels of SAP30, p62, LC3B-I, and LC3B-II in SAP30-silenced stable SK-N-AS, NB-19, and SK-N-BE(2) cells (A) or transfection with SAP30-specific siRNAs in SK-N-AS, and NB-19 cells (B) for 48 h. (C) Immunofluorescence images show the expression levels of LC3B and p62 in SAP30-silenced stable SK-N-AS cells for 48 h. LC3B was visualized using an anti-LC3B-AF488 (green) antibody, while p62 was detected using an anti-p62-AF647 (red) antibody. (D) Immunofluorescence images depict the expression levels of GFP-LC3B in stable SK-N-AS cells with silenced SAP30. The cells were transfected with GFP-LC3B or GFP-empty vector (EV) for 48 h, followed by immunofluorescence analysis using a GFP antibody.

Figure 2

Silencing of SAP30 disrupts starvation-induced physiological autophagy in NB cells (A and B) Western blot analysis of SAP30, p62, LC3B-I, and LC3B-II protein levels in SAP30-silenced stable NB-19 (A) and SK-N-AS (B) cell lines following 12 and 24 h of serum starvation. SFM, serum-free medium; SRM, serum-rich medium. (C) Flow cytometry plots illustrate the degradation of GFP-LC3B in SAP30-silenced stable SK-N-AS cell lines after 4 h of amino acid and serum starvation using HBSS. Stable NB cell lines were transiently transfected with the GFP-LC3B-RFP-LC3BΔG plasmid before starvation, and the normalized GFP/RFP ratio, an indicator of LC3B degradation, is provided. (D and E) Western blot analysis of p62 and LC3B-II protein levels in SAP30-silenced stable SK-N-B(E)2 (D), SK-N-AS, and NB-19 (E) cell lines after treatment with the lysosome-targeting autophagy inhibitor bafilomycin (5 nM) for 24 h. (F) Immunofluorescence images depict the co-localization of LC3B and LAMP-1 in SAP30-silenced stable SK-N-AS cells following 4 h of HBSS starvation. LC3B was detected with an anti-LC3B-Alexa Fluor 488 antibody (green), while LAMP-1 was visualized using an anti-LAMP-1-Alexa Fluor 568 antibody (red). DAPI staining in blue was used to mark the nucleus. White arrows indicate inhibited colocalization.

Figure 3

SAP30 ectopic expression induces physiological autophagy in NB cells under starvation conditions (A and B) Western blot analysis of p62, LC3B-I, and LC3B-II protein levels in SAP30-overexpressing stable SK-N-AS (A and C) and NB-19 (B and D) cell lines after 2 and 4 h of amino acid starvation using HBSS (A and B) or cultured in medium containing different percentages of FBS (C and D) and treatment with or without the autophagy inhibitor bafilomycin (5 nM) for 24 h. The fold change in the expression levels of p62 and LC3B-II proteins normalized to GAPDH is provided below the respective western blot images. (E–G) Immunofluorescence images depict the expression of LC3B in SAP30-overexpressing stable NB-19 (E and F) and SK-N-AS (G) cell lines under different starvation conditions. Cells were starved with HBSS (E and G) or SFM (F) and treated with bafilomycin or hydroxychloroquine (HCQ). LC3B was detected using an anti-LC3B-Alexa Fluor 568 antibody (red), and DAPI staining (blue) was used to label the nucleus. The fold change in expression levels of p62 and LC3B-II proteins, normalized to GAPDH, is provided below the respective western blot images. HE, higher exposure; LE, lower exposure.

Figure 4

The stability of SAP30 and its degradation mechanisms independent of autophagy during starvation (A and B) Western blot analysis of p62, SAP30, LC3B-I, and LC3B-II protein levels in SK-N-AS (A) and NB-19 (B) cells following 4 h of amino acid starvation using HBSS, and treatment with bafilomycin (50 nM), the proteasomal inhibitor MG132 (1 μM), and HCQ (50 μM). (C) Immunofluorescence analysis depicts the localization of p62 (red) in the cytosol and SAP30 (green) in both the nucleus and cytosol in SK-N-AS cells after 4 h of HBSS starvation, with or without treatment with bafilomycin (50 nM). (D and E) Western blot analysis of p62, SAP30, LC3B-I, and LC3B-II protein levels in SK-N-AS (D) and NB-19 (E) cells after 24 or 9 h of starvation, respectively, using SFM (0% FBS) in the presence of bafilomycin or HCQ. (F and G) Western blot analysis of p62, SAP30, LC3B-I, and LC3B-II protein levels in SK-N-AS (F) and NB-19 (G) cells after 48 h of starvation using reduced serum (1% FBS) medium in the presence of bafilomycin (2.5 nM), HCQ (25 μM), or MG132 (0.5 μM). The fold change in expression levels of p62 and SAP30 proteins, normalized to GAPDH, is provided below the respective western blot images.

Figure 5

Silencing of SAP30 reduces STX17 expression and its availability for translocation to the autophagic membrane (A) Real-time qPCR (left) and western blot (center) analyses display the mRNA and protein levels of SAP30, STX17, and ATG14 in stable NB-19 and SK-N-AS cells with silenced SAP30. A western blotting densitometric quantification graph shows the fold change in expression levels of STX17, normalized to GAPDH protein (right). (B and C) Immunofluorescence images depict the expression levels of STX17 in stable NB-19 (B) and SK-N-AS (C) cells with silenced SAP30 following 4 h of starvation using HBSS in the presence of bafilomycin (50 nM). Polyclonal rabbit antibody against STX17 was utilized, followed by immunofluorescence analysis using secondary antibody labeled with Alexa Fluor 488 (green). (D and E) Confocal microscopy images display the co-localization of STX17 and LC3B in stable SK-N-AS cells with silenced SAP30 following 4 h of HBSS incubation along with bafilomycin (50 nM) (D) and HCQ (50 μM) (E). Antibodies against STX17 and LC3B-II were employed, followed by immunofluorescence analysis using secondary antibodies labeled with Alexa Fluor 488 (green) and AF568 (red), respectively. The magnified confocal microscopy images illustrate the orange-yellow colocalized dots of STX17 (green) and LC3B-II (red) resulting from the combination of the two channels in cells. (F) Western blot analysis of p62, LC3B-I, and LC3B-II protein levels in stable SK-N-AS cells with silenced SAP30 following 48 h of transfection with FLAG-EV or FLAG-STX17 overexpression plasmids and with or without 4 h of serum and amino acid starvation using HBSS. Bafilomycin and HCQ were added with HBSS. Densitometric quantification numbers show the fold change in expression levels of p62 and LC3B-II, normalized to GAPDH, at the bottom of the western blot images.

Figure 6

SAP30 promotes cell growth and autophagy protection under therapeutic stress in NB cells (A and B) Quantification graphs depict live cell counts analyzed using the trypan blue exclusion method in stable SK-N-AS (A) and NB-19 (B) cells with silenced SAP30, cultured in either 10% FBS medium (FM) or 1% FBS medium (1% FBS M) for 4 days. (C and D) Flow cytometry plots illustrate the distribution of NB cells (%) in the G1-, S-, and G2-phases upon SAP30 silencing. Stable SK-N-AS (C) and NB-19 (D) cells with silenced SAP30 were cultured in either 10% FM or 1% FBS M for 2 days, followed by apoBrdU-allophycocyanin (APC) and 7-amino-actinomycin D (7-AAD) staining. Quantification graphs display the cells (%) in the S-phase on the right side of the flow cytometry graphs. (E and F) Flow cytometry plots depict the intracellular expression of LC3B in SK-N-AS (E) and NB-19 (F) cells with stable expression of either FLAG-EV or FLAG-SAP30, treated with doxorubicin (0.5 μg/mL) alone or in combination with bafilomycin (2.5 nM) and HCQ (25 μM) for 48 h. (G) Cell viability quantification graphs, analyzed by MTT assay in SK-N-AS, NB-19 cells with stable expression of either FLAG-EV or FLAG-SAP30, treated with doxorubicin (0.5 μg/mL) or cisplatin (2.5 μg/mL) alone or in combination with HCQ (25 μM) for 48 h. These experiments were independently replicated three times, with error bars representing the SE. Statistical comparisons were conducted using a two-tailed unpaired Student’s t test, and significance levels were indicated as ∗∗∗∗p < 0.001, ∗∗p < 0.01, and ∗p < 0.05, respectively.

Figure 7

Silencing SAP30 results in increased accumulation of LC3B and reduced levels of SAP30-responsive STX17 in NB, both in vivo and in PDX tumors The IHC images depict the expression levels of (A) LC3B (top), STX17 (center), and Ki67 (bottom), in tumors from NSG mice injected with stable SK-N-B(E)2 cells expressing either control shRNA or two different SAP30-specific shRNAs. (B) Additionally, LC3B expression in PDX tumors from NB patients at diagnosis and relapse stages is shown. The intensity of LC3B staining in the cytoplasm of each cell/frame was quantified using HALO software to compute an H-score. Protein expression is indicated by brown staining, while blue staining represents the nucleus. Images were captured at 20× magnification, with enlarged images provided at 40× magnification in the lower right corner. Scale bar: 75 μm. A two-way ANOVA denotes statistical significance levels as ∗∗∗∗p < 0.001.