Sulindac (K-80003) with nab-paclitaxel and gemcitabine overcomes drug-resistant pancreatic cancer

Abstract

The Nab-paclitaxel combined with gemcitabine (AG) regimen is the main chemotherapy regimen for pancreatic cancer, but drug resistance often occurs. Currently, the ability to promote sensitization in drug-resistant cases is an important clinical issue, and the strategy of repurposing conventional drugs is a promising strategy. This study aimed to identify a classic drug that targets chemotherapy resistance's core signaling pathways and combine it with the AG regimen to enhance chemosensitivity. We also aimed to find reliable predictive biomarkers of drug combination sensitivity. Using RNA sequencing, we found that abnormal PI3K/Akt pathway activation plays a central role in mediating resistance to the AG regimen. Subsequently, through internal and external verification of randomly selected AG-resistant patient-derived organoid (PDO) and PDO xenograft models, we discovered for the first time that the classic anti-inflammatory drug sulindac K-80003, an inhibitor of the PI3K/Akt pathway that we focused on, promoted sensitization in half (14/28) of AG-resistant pancreatic ductal adenocarcinoma cases. Through RNA-sequencing, multiplex immunofluorescent staining, and immunohistochemistry experiments, we identified cFAM124A as a novel biomarker through which sulindac K-80003 promotes AG sensitization. Its role as a sensitization marker is explained via the following mechanism: cFAM124A enhances both the mRNA expression of cathepsin L and the activity of the cathepsin L enzyme. This dual effect stimulates the cleavage of RXRα, leading to large amounts of truncated RXRα, which serves as a direct target of K-80003. Consequently, this process results in the pathological activation of the PI3K/Akt pathway. In summary, our study provides a new treatment strategy and novel biological target for patients with drug-resistant pancreatic cancer.

Fig. 1

PI3K/Akt pathway plays a vital role in the resistance of PDAC to GEM-based chemotherapy. A, Overall experimental scheme for this study. B, Complex heatmap showing differentially expressed genes (DEGs) and distribution of clinical parameters between AG-sensitive and AG-resistant PDAC. C, KEGG pathway enrichment analysis for the upregulated DEGs in the RNA-seq comparing the AG-resistant group and AG-sensitive group. D, KEGG pathway enrichment analysis of external RNA-seq data, SRP303224. E, Representative hematoxylin and eosin (H&E) staining images of AG-sensitive and AG-resistant PDAC and patient-derived organoids (PDOs). Scale bar, 100 μm. F, IC₅₀ values for 10 PDOs after GEM treatment. G-H, Representative images of PDOs from PDO-AGS and PDO-AGR groups. PDOs treated for 48 h with nab-paclitaxel (10nM) and gemcitabine (30nM). Live/Dead cell viability staining showing live cells stained with calcein-AM (green) and dead cells with Ethidium-1 (red). Scale bar, 50 μm. I, RNA-seq results for PDO-AGS and PDO-AGR groups from PDOs. J, Heatmap showing DEGs between PDO-AGS and PDO-AGR groups. K, KEGG pathway enrichment analysis for upregulated DEGs in the RNA-seq comparing the PDO-AGS group and PDO-AGR groups. L, Representative images of H&E and IHC staining for p-Akt (Thr308) in PDOs from PDO-AGS and PDO-AGR groups

Fig. 2

K-80003 can enhance the sensitivity of some AG-resistant PDAC patients to AG chemotherapy. A, Method used to apply different treatments to PDO-AGR in vitro and quantitation of PDO fluorescence. Method used for corresponding PDOX treatments in vivo. B-F, Representative images of different PDO-AGRs that received different treatments. PDOs in each group treated for 48 h with saline or AG (Nab-paclitaxel 10nM plus GEM 30nM) or K-80003 (5 nM) or AG (Nab-paclitaxel 10nM plus GEM 30nM plus K-80003 5 nM). Live cells stained with calcein-AM (green), and dead cells with ethidium-1 (red). Quantitative data are shown on the right. Tumors from different groups of PDOXs received different treatments from weeks 2 after orthotopic xenograft injection of 1 million corresponding PDOs. Mice were treated with saline, K-80003 (20 mg/kg, twice weekly; bule arrows), the dual combination of gemcitabine (25 mg/kg, weekly), and nab-paclitaxel (15 mg/kg, weekly; red arrows), or the triple combination of gemcitabine and nab-paclitaxel (as dosed for the monotherapies) plus K-80003 for 3 weeks. Tumors were harvest at 3 weeks after injection. Fluorescence of tumors from mice in the different groups (5 mice per group). Survival curves for mice that received different treatments (5 mice per group). Not significant (ns), *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 3

Response status of AG-resistant PDAC with K-80003 treatment from in-house and external cohorts. A, Flow chart for screening PDAC patients for K80003 sensitization AG regimen. B, Complex heatmap showing distribution of clinical parameters between in-house cohort and external cohort of AG-resistant PDAC. C, Kaplan–Meier overall survival (PFS) curves between in-house cohort and external cohort of AG-resistant PDAC. D, Representative HE staining images of 12 AG-resistant PDAC samples and PODs from in-house cohort, and ability of K-80003 to sensitize samples to AG chemotherapy. E, Weight of tumors from mice in the different groups (5 mice per group), and survival curves for mice that received different treatments (5 mice per group). Tumors from different groups of PDOXs received different treatments from weeks 2 after orthotopic xenograft injection of 1 million corresponding PDOs. Mice were treated with saline, the dual combination of gemcitabine (25 mg/kg, weekly), and nab-paclitaxel (15 mg/kg, weekly; red arrows), or the triple combination of gemcitabine and nab-paclitaxel (as dosed for the monotherapies) plus K-80003 (20 mg/kg, twice weekly; blue arrows) for 3 weeks. Tumors were harvest at 3 weeks after treatment. Survival curves for mice that received different treatments (5 mice per group). F, Representative HE staining images of 11 AG-resistant PDAC-derived PDOs in external cohort, and ability of K-80003 to sensitize them to AG chemotherapy. G, Weight of tumors from mice in the different groups (5 mice per group), and survival curves for mice that received different treatments (5 mice per group). Not significant (ns), *P <0.05; **P < 0.01; ***P < 0.001

Fig. 4

cFAM124A is a potential biomarker for the ability of K-80003 to increase AG sensitivity of PDAC. A, Flowchart for screening for K-80003 response markers. B, Volcano plot showing upregulated and downregulated circRNAs between PDO-AGS and PDO-AGR groups. C, RT-qPCR detection of the expression of 10 relevant circRNAs in PDAC PDOs and cells. D, GSEA of 10 circRNAs demonstrating enrichment of DEGs in the PI3K/Akt pathway. E, Pearson correlation analysis between PI3K/Akt signaling pathway marker expression and the 10 circRNAs and number of related genes. F, Necrosis in 3D tumor microspheres based on propidium iodide (PI) staining (red) and its quantification after treatment with Gem (1 µM) and/or K-80003 (5 nM). Scale bar, 100 μm. G, Representative H&E and IHC staining for p-Akt (Thr308) and FISH staining for cFAM124A in samples from GEM-sensitive (Gem-S) and GEM-resistant (Gem-R) patients. Quantification of p-Akt (Thr308) staining in PDAC tissues collected from Gem-S and Gem-R groups. Quantification of cFAM124A staining in PDAC tissues collected from Gem-S and Gem-R groups. Pearson correlation analysis between p-Akt (Thr308) expression and cFAM124A expression. H, Kaplan–Meier OS and PFS curves according to cFAM124A expression for PDAC patients in Table S3 (n = 132). Not significant (ns), *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 5

cFAM124A is the best marker to predict the ability of K-80003 to promote AG sensitivity in PDAC. A-B, Representative multiplex IHC staining for cFAM124A, p-Akt (Thr308), and p-Akt (Ser473) expression in PDAC tissues collected from K-80003 responsive and K-80003 non-responsive groups. Quantification of cFAM124A, p-Akt (Thr308), and p-Akt (Ser473) staining in PDAC tissues collected from K-80003 responsive and K-80003 non-responsive groups. Scale bar, 100 μm. C-D, Representative H&E, FISH staining for cFAM124A, and IHC staining for p-Akt (Thr308) and p-Akt (Ser473) in PDAC tissues collected from K-80003 responsive and K-80003 non-responsive groups. Quantification of cFAM124A, p-Akt (Thr308), and p-Akt (Ser473) staining in PDAC tissues collected from K-80003 responsive and K-80003 non-responsive groups. Scale bar, 100 μm. E-F, Representative H&E, FISH staining for cFAM124A and IHC staining for p-Akt (Thr308) and p-Akt (Ser473) in paracancer tissues collected from K-80003 responsive and K-80003 non-responsive groups. Quantification of cFAM124A, p-Akt (Thr308), and p-Akt (Ser473) staining in paracancer tissues collected from K-80003 responsive and K-80003 non-responsive groups. Scale bar, 100 μm. G, Fold changes in cFAM124A, p-Akt (Thr308) and p-Akt (Ser473) expression levels in PDAC tissues collected from K-80003 responsive and K-80003 non-responsive groups. H, Peak fold changes in cFAM124A, p-Akt (Thr308), and p-Akt (Ser473) expression in different groups. Not significant (ns), *P < 0.05; **P < 0.01; ***P < 0.001)

Fig. 6

cFAM124A activates PI3K/Akt pathway through tRXRα to cause GEM resistance in PDAC, and K-80003 can reverse this effect. A, Western blot analysis of Akt, p-Akt (Ser473), p-Akt (Thr308), and cleaved caspase 3/8 expression after 6 h of treatment with different inhibitors and GEM treatment. B, IC₅₀ values for GEM in PDAC cells overexpressing cFAM124A and control cells after 6 h of treatment with different inhibitors and GEM treatment. C, Necrosis in 3D tumor microspheres based on PI staining (red) and after 6 h of treatment with different inhibitors and GEM treatment. Scale bar, 100 μm. D, Colony formation by cells overexpressing cFAM124A after 6 h of treatment with different inhibitors and GEM treatment in 6-well dishes (800 cells/well) for 2 weeks. Each inhibitor, copanlisib (2 nM, pan-PI3K inhibitor) or K-80003 (5 nM, tRXRα-dependent Akt activation inhibitor). Quantitative data are shown on the right. E, Subcutaneous xenograft model of mice in the different groups treated with GEM (40 mg/kg i.p. 2×/week for 4 weeks), copanlisib (1 mg/kg, iv. 2×/week for 4 weeks), or K-80003 (20 mg/ kg i.p. 2×/week for 4 weeks) at 2 weeks after subcutaneous injection of 5 × 10⁶ cells overexpressing cFAM124A and control cells. Representative images of tumors are shown (n = 5). F, Body weights of subcutaneous tumor-bearing mice in the indicated groups (n = 5). G, Volcano plot showing upregulated and downregulated protein between EV-PATU8988T and cFAM124A-PATU8988T cells. H, Western blot analysis of RXRα, Akt, p-Akt (Ser473), and p-Akt (Thr308) expression in PDAC cells overexpressing cFAM124A or with cFAM124A knockout and control cells. I, cFAM124A increased RXRα protein degradation: Indicated PDAC cell lines were incubated with CHX for indicated time periods before western blot analysis of RXRα and GAPDH expression. Representative images are shown (left). J, Indicated PDAC cell lines were incubated with MG132 and then with CHX for indicated time periods before western blot analysis of RXRα and GAPDH expression. Representative images are shown (left)

Fig. 7

cFAM124A upregulates CTSL enzyme expression through bridging effect and promotes an increase in the tRXRα protein level. A, Western blot analysis of RXRα expression after 24 h of treatment with PD150606 (200 nM, m-calpain inhibitor) or ZFY-CHO (10 µM, CTSL inhibitor). Representative images are shown (left). B, Indicated PDAC cell lines were incubated with ZFY-CHO and then with CHX for indicated time periods before western blot analysis of RXRα and GAPDH expression. Representative images are shown (left). C, CTSL enzyme‑linked immunosorbent assay (ELISA) in PDAC cells with cFAM124A overexpression or knockout. D, Potential cFAM120A-binding proteins were pulled down in cell lysate by RAP assay; these were incubated with the cFAM124A probe and subsequently visualized by MS and silver staining. E, Venn diagram showing the overlap of potential binding proteins of cFAM124A. F, IGF2BP2 was pulled down by the LacZ probe (control) or cFAM124A probe and cFAM124A mutant. G, IGF2BP2 was pulled down by the CTSL 3ʹ-UTR probe after overexpression of CTSL 3ʹ-UTR. H, Association of cFAM124A with CTSL mRNA on RAP assay. I, IGF2BP2 was pulled down by the CTSL 3ʹ-UTR probe on western blotting in the indicated groups. J, Upper: Schematic of the RNA-binding domain within IGF2BP2 and list of different IGF2BP2 truncation mutants. Lower: Immunoblotting with anti-Flag antibody after RNA pulldown assay in PATU8988T cells using RNA cFAM124A or CTSL mRNA probes. K, Pattern diagram of binding among cFAM124A, CTSL mRNA, and IGF2BP2. L, CTSL mRNA expression in the indicated groups. M, CTSL, Akt, RXRα, p-Akt (Thr308), and p-Akt (Ser473) protein expression in the indicated groups. N, CTSL protein concentrations detected by ELISA in the indicated groups

Fig. 8

cFAM124A competes with CSTB through bait effect and enhances CTSL enzyme activity. A, MS of CSTB protein. B, CSTB was pulled down by the cFAM124A probe or LacZ probe (control). C, RIP assay showing the association of CSTB with cFAM124A. D-E, Co-IP assay of CSTB and CTSL in PDAC cells with cFAM124A overexpression or knockout. F, Predicted binding regions in cFAM124A for CSTB by CatRAPID. G, After PATU8988T cells were transfected with plasmids for wild-type or truncated cFAM124A overexpression, RNA pulldown assay was performed with cFAM124A-specific probes. H, Co-IP assay of CSTB and CTSL in PDAC cells in different treatment groups. I, Western blot analysis of CTSL, RXRα, Akt, p-Akt (Ser473), and p-Akt (Thr308) protein expression in the indicated groups. J, CTSL concentrations by ELISA in the indicated groups. K, Proposed mechanism by which cFAM124A promotes PDAC chemoresistance via “scaffolding” and “decoys-like” effects. Not significant (ns), *P < 0.05; **P < 0.01; ***P < 0.001