HSP90AA1-mediated autophagy promotes drug resistance in osteosarcoma

Abstract

Background: Osteosarcoma is the most common primary bone tumor in children and adolescents. Unfortunately, osteosarcoma treatments often fail due to the development of chemoresistance, of which the underlying molecular mechanisms still remain unclear. In this study, we demonstrated that HSP90AA1 gene is responsible for drug resistance in osteosarcoma through an autophagy-related mechanism.

Methods: shRNAs were transfected into osteosarcoma cells for knockdown of HSP90AA1 gene. Stable HSP90AA1 overexpressing osteosarcoma cell lines were obtained by lentivirus infection. mRNA and protein expressions of HSP90AA1 in osteosarcoma cells were tested by quantitative real-time PCR and western blot, respectively. Autophagy of osteosarcoma cells was detected by western blot of LC3, transmission electron microscopy and fluorescence microscope. mRFP-GFP-LC3 lentiviral transfection was also performed to detect autophagic flux. NOD/SCID mices were inoculated with MG-63 tumor cells transfected with HSP90AA1 specific shRNA. TUNEL and LC3 staining were performed to detect apoptosis and autophagy of resected tumor tissues.

Results: Doxorubicin, cisplatin, and methotrexate, which are commonly used in chemotherapy, each induced HSP90AA1 upregulation in human osteosarcoma cells. Suppression of HSP90AA1 restored the sensitivity of osteosarcoma cells to chemotherapy both in vivo and in vitro. Mechanism study indicated that autophagy is responsible for the chemoresistance in osteosarcoma cells. HSP90AA1 increased drug resistance by inducing autophagy and inhibiting apoptosis. Suppression of HSP90AA1 diminished autophagic protection in response to chemotherapy in osteosarcoma cells. Moreover, HSP90AA1 promotes autophagy through PI3K/Akt/mTOR pathway and inhibits apoptosis through JNK/P38 pathway.

Conclusion: We showed that chemotherapy agents can induce HSP90AA1 expression in osteosarcoma cells. And HSP90AA1, acting as an important regulator of autophagy, is a critical factor in the development of osteosarcoma chemoresistance both in vitro and in vivo. HSP90AA1 provides a novel therapeutic target for improving osteosarcoma treatment.

Keywords: Apoptosis; Autophagy; Chemoresistance; HSP90AA1; Osteosarcoma.

Fig. 1

Chemotherapy agents promote HSP90AA1 expression in osteosarcoma cells. a and b, MG-63, Saos-2 and U-2 OS cells were treated with Cis (20 μmol/L), Dox (0.2 μg/mL), and Mtx (50 μmol/L) for 24 h. Whole cell lysates were subjected to western blot analysis for HSP90AA1 expression (a) and HSP90AA1 mRNA level was assessed by quantitative real-time PCR (b; n = 3; *, p < 0.05 versus untreated group). c MG-63 and U-2 OS cells were treated with Dox (0.2 μg/mL) for 12 to 48 h and then HSP90AA1 protein level was assessed by Western blot

Fig. 2

HSP90AA1 reduces osteosarcoma cells sensitivity to chemotherapy by decreasing apoptosis. a MG-63 and U-2 OS osteosarcoma cells were transfected with control shRNA and HSP90AA1 shRNA for 48 h. Then the expression levels of HSP90AA1 were analyzed by Western blot and quantitative real-time PCR (n = 3; *, p < 0.05 versus control shRNA group). b, c and d, after transfection with control shRNA and HSP90AA1 shRNA, MG-63 and U-2 OS cells were treated with Cis (20 μmol/L), Dox (0.2 μg/mL) and Mtx (50 μmol/L) for 24 h. Then cell viability was determined at 12, 24 and 48 h by CCK-8 assay (b). Apoptosis was analyzed at 24 h by flow cytometric analysis of Annexin V-PE/PI staining (c; n = 3; *, p < 0.05 versus control shRNA group) and both cleaved and total PARP in MG-63 cells were analyzed by Western blot (d). e After transfection with control shRNA and HSP90AA1 shRNA, MG-63 and U-2 OS cells were exposed to Cis (20 μmol/L), Dox (0.2 μg/mL) and Mtx (50 μmol/L) for 24 h in the presence or absence of ZVAD-FMK (20 μmol/L). Then apoptosis was evaluated by Colorimetric Caspase 3 Assay Kit (n = 3; *, p < 0.05 versus control shRNA group). f MG-63 and U-2 OS cells were infected with control (pHBLV control) and HSP90AA1-expressing lentiviruses (pHBLV HSP90AA1). The protein level of HSP90AA1 was assayed by Western blot. G and H, HSP90AA1-overexpressing MG-63 and U-2 OS cells were treated with Cis (20 μmol/L), Dox (0.2 μg/mL) and Mtx (50 μmol/L) for 24 h. Cell viability was determined at 12, 24 and 48 h by CCK-8 assay (g) and apoptosis was analyzed at 24 h by measuring Annexin V-PE positive cells by flowcytometry (h; n = 3; *, p < 0.05 versus control shRNA group)

Fig. 3

Autophagy mediates HSP90AA1-induced sensitivity of osteosarcoma cells to chemotherapy. a and b HSP90AA1-overexpressing MG-63 cells were treated with Cis (20 μmol/L) or Dox (0.2 μg/mL) for 24 h. The protein levels of LC3, p62 and both cleaved and total PARP were assayed by Western blot (a). Autophagic flux were analyzed by mRFP-GFP-LC3 construct (b). c HSP90AA1-overexpressing MG-63 cells were treated with 3-methyladenine (3-MA, 5 mM). Autophagy activity indicated by LC3 levels was analyzed by western blot. d and e HSP90AA1-overexpressing MG-63 cells were treated with Dox (0.2 μg/mL) for 24 h with or without 3-MA (5 mM 2 h). Apoptosis was assessed by analysis of Annexin V-PE/PI staining (d, n = 3; *, p < 0.05 versus pHBLV control group) and proliferation was analyzed by CCK-8 assay (e, n = 3; *, p < 0.05 versus pHBLV control group). NS, not significant

Fig. 4

Knockdown of HSP90AA1 inhibits autophagy during chemotherapy in osteosarcoma cells. a MG-63 osteosarcoma cells were transfected with control shRNA/HSP90AA1 shRNA for 48 h and then were treated with Cis (20 μmol/L) or Dox (0.2 μg/mL) for 24 h. The protein levels of LC3 and p62 were assessed by Western blot. b Control shRNA/HSP90AA1 shRNA transfected MG-63 cells were treated with Dox (0.2 μg/mL) for 24 h with or without bafilomycin A1 (100 nmol/L). LC3 and p62 expression were analyzed by Western blot. c and d control shRNA/HSP90AA1 shRNA transfected MG-63 cells were treated with Cis (20 μmol/L) or Dox (0.2 μg/mL) for 24 h. Then autophagic flux were analyzed by mRFP-GFP-LC3 construct (c) and transmission electron microscopy (d), autophagosome-like structures were indicated (red arrows). e and f Control shRNA/HSP90AA1 shRNA transfected MG-63 cells were pretreated with rapamycin (100 nmol/L) for 6 h and then were exposed to Dox (0.2 μg/mL) for an additional 24 h. Apoptosis was analyzed by measuring Annexin V-PE/PI positive cells by flow cytometric (e, n = 3; *, p < 0.05, versus rapamycin untreated group). Autophagy was determined by measuring LC3 puncta formation (f, n = 3; *, p < 0.05 versus rapamycin untreated group). NS, not significant

Fig. 5

HSP90AA1 promotes autophagy and inhibits apoptosis through PI3K/Akt/mTOR pathway and JNK/P38 pathway respectively. a and b HSP90AA1-overexpressing MG-63 cells and HSP90AA1 shRNA transfected MG-63 cells were treated with Dox (0.2 μg/mL) for 24 h. Total cell lysates were subjected to western blot analysis of key proteins in the PI3K/Akt/mTOR pathway. c Control shRNA/HSP90AA1 shRNA transfected MG-63 cells were treated with Dox (0.2 μg/mL) for 24 h with or without LY294002 (10 μM). Then autophagy was analyzed by measuring LC3 puncta formation (n = 3; *, p < 0.05 versus control shRNA group). NS, not significant. d MG-63 cells transfected with control/HSP90AA1 shRNA for 48 h and were treated with Dox (0.2 μg/mL) for 24 h. Total cell lysates were subjected to western blot analysis of key proteins in JNK/p38 pathway

Fig. 6

Suppression of HSP90AA1 increases sensitivity to chemotherapy in vivo. a NOD/SCID mice were inoculated with control shRNA/HSP90AA1 shRNA transfected MG-63 cells. At the beginning at day 7, docetaxel (5 mg/kg) was administered via intraperitoneal injection. a tumor volumes were calculated for 28 days (n = 3; *p < 0.05). Representative photographs of tumors formed at 4 weeks are shown. b Apoptosis and autophagy in tumor tissues were detected by TUNEL and LC3 stain respectively after insection of tumor samples

Fig. 7

Proposed model of the mechanism of HSP90AA1 gene in osteosarcoma drug resistance. Chemotherapy increases the expression level of HSP90AA1, which leads to disassociation of phosphorylated AKT and JNK/P38. The dephosphorylation of AKT decreases the expressions of phosphorylation of mTOR and promotes autophagy. The dephosphorylation of JNK/P38 results in decreased apoptosis. Moreover, autophagy also can influence HSP90AA1-mediated antiapoptotic effect. Therefore, HSP90AA1 is a critical factor in the development of osteosarcoma chemoresistance