DNAJB8 in small extracellular vesicles promotes Oxaliplatin resistance through TP53/MDR1 pathway in colon cancer

Abstract

Chemotherapy is one of the most frequently used therapies for the treatment of colon cancer (COAD). However, Oxaliplatin (L-OHP) resistance is a major obstacle to the effective treatment of COAD. Here, we investigated whether DNAJB8, a heat shock protein 40 (HSP40) family protein, could be used for the prognosis and therapy of L-OHP resistance in COAD. Treatment with small interfering RNA targeting DNAJB8 could restore the response to L-OHP in vitro and in vivo. On the mechanism, we demonstrated that DNAJB8 could interact with TP53 and inhibit the ubiquitination degradation of TP53, leading to MDR1 upregulation which promotes colon cancer L-OHP resistance. We found that small extracellular vesicle (sEV)-mediated transfer of DNAJB8 from L-OHP-resistant COAD cells to sensitive cells contributed to L-OHP resistance. A prognostic signature based on the DNAJB8 levels in both tissue and serum showed that COAD patients with high-risk scores exhibited significantly worse overall survival and disease-free survival than patients with low-risk scores. These results indicate that DNAJB8 levels in serum sEVs may serve as a biomarker for COAD. DNAJB8 from sEVs might be a promising therapeutic target for L-OHP resistance and a prognostic predictor of clinical response.

Fig. 1. Overexpression of DNAJB8 in L-OHP-resistant COAD cells.

A IC₅₀ Values of L-OHP in COAD cells treated with L-OHP. B Expression levels of MDR1(P-glycoprotein) were determined by qRT-PCR in L-OHP-resistant COAD cells. Expression levels of DNAJBs were determined by qRT-PCR in L-OHP-resistant SW480 C and SW620 D cells. Experiments were repeated three times and representative results are shown. E Expression analysis of DNAJB8 protein in tissues from COAD patients treated with L-OHP therapy by immunohistochemistry. F DNAJB8 expression was analyzed in responding and nonresponding groups of patients. Results shown are mean ± s.d. from a representative experiment. *p < 0.05; **p < 0.01; Student’s t test. Similar results were obtained in three independent experiments.

Fig. 2. DNAJB8 silencing sensitizes COAD cells to L-OHP chemotherapy.

A DNAJB8 expression was detected by western blot assay in L-OHP-resistant COAD cells following siRNA transfection. SW620/L-OHP and SW480/L-OHP cells with DNAJB8 knockdown were seeded for cell proliferation B, C mammosphere formation D and flow cytometry assay E Scale bars = 40 μm. Luminescence imaged tumor-bearing nude mice were injected with DNAJB8 knockdown L-OHP resistant cells and control cells F, and transplanted tumor volume was detected G. Results shown are mean ± s.d. from a representative experiment. *p < 0.05; **p < 0.01; Student’s t test. Similar results were obtained in three independent experiments.

Fig. 3. DNAJB8 interacts with TP53 in L-OHP-resistant COAD cells.

A Expression of TP53 was analyzed by western blot assay in L-OHP-resistant COAD cells transfected with DNAJB8 specific siRNA. MDR1 promoter binding activity by TP53 was detected in L-OHP-resistant colon cancer cells B and parental colon cancer cells C using a luciferase reporter gene assay. D Restoration of DNAJB8 knockdown L-OHP-resistant colon cancer cells using a TP53 (R273H) overexpression vector. Indicated proteins were detected by western blot assay. E Restoration of DNAJB8 overexpression L-OHP-resistant colon cancer cells using a TP53 (R273H) specific siRNA. Indicated proteins were detected by western blot assay. L-OHP-resistant SW480 F and SW620 G cells transfected with DNAJB8-specific siRNA were treated with cycloheximide (CHX) (20 μg/ml) for the indicated time. H L-OHP-resistant colon cancer cells transfected with DNAJB8-specific siRNA were treated with MG132. I TP53 ubiquitylation expression was detected in DNAJB8 knockdown L-OHP-resistant colon cancer cells by western blot assay. Results shown are mean ± s.d. from a representative experiment. *p < 0.05; **p < 0.01; Student’s t test. Similar results were obtained in three independent experiments.

Fig. 4. DNAJB8 directly interacts with TP53 in vivo and in vitro.

A HEK293T cells transfected with the indicated constructs were collected 24 h later. Cells were lysed with NETN buffer. C Immunoprecipitation (IP) using S-protein agarose was performed and the western blotted was done with the indicated antibodies. B Endogenous DNAJB8 associated with TP53 was analyzed using SW480/L-OHP cells. D Beads coated with GST or GST-TP53 fusion proteins were incubated with SFB-DNAJB8 protein overnight. GST pulldown was immunoblotted with the indicated antibodies. E The design of the AlphaLISA assay for DNAJB8–TP53 interactions. The binding signal expressed in binding relative luminescence units (RLUs) between DNAJB8 and TP53 in SW480/L-OHP cells F and SW620/L-OHP cells G. Results shown are mean ± s.d. from a representative experiment. *p < 0.05; **p < 0.01; Student’s t test. Similar results were obtained in three independent experiments.

Fig. 5. DNAJB8 positively correlates with TP53/P-gP pathway expression in COAD.

A DNAJB8 and TP53 expressions were tested using IHC in the COAD tissues. B The results of multivariate COX regression analysis were shown in a tree diagram. C The calibration curve for predicting patient survival at 5 years; the nomogram-predicted probability of overall survival is plotted on the x-axis; and the actual overall survival is plotted on the y-axis. D The results of the receiver operating characteristic (ROC). E Prognostic nomogram for COAD. F Kaplan–Meier curve analyses. G Decision curve analysis for the prognostic nomogram, where the y-axis measures the net benefit. Results shown are mean ± s.d. from a representative experiment. *p < 0.05; **p < 0.01; Student’s t test. Similar results were obtained in three independent experiments.

Fig. 6. DNAJB8 transfer by small extracellular vesicles confirmed in colon cancer.

A The apoptosis rate was detected in parental cells cocultured with culture medium (CM) from L-OHP resistant cells and DNAJB8-knockdown-resistant cells. B The apoptosis rate was detected in parental cells cocultured with parental cells and L-OHP resistant cells. C DNAJB8 expression was analyzed using ELISA assay in the CM of L-OHP cells treated with Proteinase K (PK) alone or combined with Triton X-100 for 20 min. D DNAJB8 expression was analyzed using ELISA assay in the CM of DNAJB8 knockdown L-OHP cells treated with GW4869. E DNAJB8 and sEVs biomarker expression in sEVs from colon cancer cells were detected using by western blot assay. F The sizes of sEVs from colon cancer cells were verified using the NTA method. G CD63, CD81, and TSG101 expression was detected in fractions collected from OptiPrepTM density gradient centrifugation by western blot assay. (H) sEVs obtained from fraction 7 (density 1.10 g/mL) were verified using transmission electron microscopy. Scale bar = 200 nm. Results shown are mean ± s.d. from a representative experiment. *p < 0.05; **p < 0.01; Student’s t test. Similar results were obtained in three independent experiments.

Fig. 7. Intercellular Transfer of DNAJB8 by sEVs increases L-OHP Resistance.

A DNAJB8 expression was detected by western blot assay in parental cells incubated with sEVs from resistant cells. B DNAJB8 expression was detected by western blot assay in parental cells incubation with sEVs from resistant cells treated with actinomycin D (ActD). C Parental cells were incubated directly with sEVs from L-OHP resistant cells. Scale bar = 40 µm. D The apoptosis and proliferation E were evaluated in parental cells incubated with sEVs from L-OHP resistant cells. F Subcutaneous xenograft assay of SW480 and SW620 cells in nude mice with intratumoral injection of indicated exosomes upon L-OHP treatment. G Volumes of tumors are shown (n = 5 per group). H TP53 and P-gP expression were detected in parental COAD cells incubated with sEVs from L-OHP resistant cells. Results shown are mean ± s.d. from a representative experiment. *p < 0.05; **p < 0.01; Student’s t test. Similar results were obtained in three independent experiments.

Fig. 8. A schematic diagram of sEVs DNAJB8 based signaling pathway in CRC L-OHP resistance.

In parental cells, sEV-mediated transfer of DNAJB8 derived from L-OHP resistant COAD cells promoted L-OHP resistance by binding and inhibiting ubiquitination of TP53, leading to the increased expression of P-gP and upregulation of the L-OHP efflux.