Cleavage of CAD by caspase-3 determines the cancer cell fate during chemotherapy

Abstract

Metabolic heterogeneity resulting from the intra-tumoral heterogeneity mediates massive adverse outcomes of tumor therapy, including chemotherapeutic resistance, but the mechanisms inside remain largely unknown. Here, we find that the de novo pyrimidine synthesis pathway determines the chemosensitivity. Chemotherapeutic drugs promote the degradation of cytosolic Carbamoyl-phosphate synthetase II, Aspartate transcarbamylase, and Dihydroorotase (CAD), an enzyme that is rate-limiting for pyrimidine synthesis, leading to apoptosis. We also find that CAD needs to be cleaved by caspase-3 on its Asp1371 residue, before its degradation. Overexpressing CAD or mutating Asp1371 to block caspase-3 cleavage confers chemoresistance in xenograft and Cldn18-ATK gastric cancer models. Importantly, mutations related to Asp1371 of CAD are found in tumor samples that failed neoadjuvant chemotherapy and pharmacological targeting of CAD-Asp1371 mutations using RMY-186 ameliorates chemotherapy efficacy. Our work reveals the vulnerability of de novo pyrimidine synthesis during chemotherapy, highlighting CAD as a promising therapeutic target and biomarker.

Fig. 1. CAD plays a key role in chemotherapy resistance.

a Western blot showing cleaved PARP, P53, CAD, DHODH and UMPS protein levels in HGC27 and MKN45 cells were treated with 5-FU (50 μM), Oxa (20 μM), Dox (20 μM) and PTX (10 nM) for 24 h. b Representative photomicrographs of HGC27 and MKN45 cells expressing empty vector (EV), CAD, or supplemented with uridine (100 μM), dT/dC (20 μM) and DHOA (250 μM), then treated with 5-FU (50 μM). Scale bar, 50 μm. c, d Representative Immunoblot analysis (c) with CAD, cleaved PARP, and p-H2A.X antibody and Immunofluorescence (d) with p-H2A.X antibody of HGC27 and MKN45 cells as described in (b). Scale bar, 20 μm. e Relative quantification of CTP, UTP, dCTP, and dTTP levels in MKN45 cells treated with 5-FU (50 μM) or DMSO for 24 h. n = 6 biologically independent samples per group. f, g Protein expression of CAD, cleaved PARP and P53 were determined after HGC27 and MKN45 cells were treated with different concentrations and time period of 5-FU. h HGC27 and MKN45 cells overexpressing Flag-CAD were treated with different concentrations of 5-FU for 24 h. Flag, cleaved PARP and P53 expression were detected. i Normalized enrichment scores for all gene ontology sets differentially expressed. j Western blot analysis of CAD, cleaved PARP protein expression in 5-FU (100 μM) treatment still attached and suspended cells. k, l Whole-mount images (k) and the statistical analysis (l) of stomach from untreated or 5-FU-treated Cldn18-ATK mice. Tumors were marked with black dotted lines. n = 8 biologically independent samples per group. m Protein expression of CAD, cleaved PARP and P53 were determined in tumor samples. n Representative images of H&E and IHC staining of stomach sections. Scale bar, 50 μm. o Quantification and statistical analysis of Ki67 staining images in (n). n = 8 biologically independent samples per group. Data in (a, c, f, g and j) derive from the same biological experiment but were processed on parallel gels (see Source Data for gel-specific details). Data were expressed as means ± SD, two-tailed Student’s t-test. **p < 0.01, ***p < 0.001.

Fig. 2. CAD is cleaved by caspase-3 during apoptosis.

a Western blot of HGC27 and MKN45 cells transfected with the indicated CAD construct and treated for 24 h with 5-FU. b Immunoblotting analysis of the indicated proteins in HGC27 and MKN45 cells were treated with 5-FU with concentration gradients. c HGC27 and MKN45 cells were treated with 5-FU in the absence and presence of Z-VAD-FMK for 24 h. Total cell lysates were immunoblotted with CAD and cleaved PARP antibody. d, e HEK-293T cells were co-transfected with Flag-CAD and HA-caspase-3 (d) or HA-caspase-6 (e). Flag, cleaved PARP and HA expression were detected by immunoblotting. f Western blot of in vitro cleavage reaction containing purified Flag-CAD by immunoprecipitation and recombinant active caspase-3. g HGC27 and MKN45 cells were treated with 5-FU in the presence of various indicated caspase inhibitors. Cells were collected and lysates were subjected to immunoblot using CAD and cleaved PARP antibody. h casp3-/-, casp6-/- and casp3-/-casp6-/- HGC27 and MKN45 cells were stimulated for 24 h with 5-FU, followed by immunoblot analysis. i casp3-/- HGC27 and MKN45 cells were treated with 5-FU in the presence or absence of recovery of HA-caspase-3. The levels of indicated proteins were determined. j Western blot of in vitro cleavage reaction containing purified Flag-CAD by immunoprecipitation and recombinant inactive mutant caspase-3-C163A. k, l Whole-mount images (k) and the statistical analysis (l) of stomach from untreated or 5-FU-treated Cldn18-ATK casp3-/- mice. Tumors were marked with black dotted lines. n = 8 biologically independent samples per group. m Protein expression of CAD, PARP, and P53 were determined in tumor samples. n Representative images of H&E and IHC staining of stomach sections. Scale bar, 50 μm. o Quantification and statistical analysis of Ki67 staining images in (n). n = 8 biologically independent samples per group. Data in (a, b, f, h–j, m) derive from the same biological experiment but were processed on parallel gels (see Source Data for gel-specific details). Arrowhead highlights band corresponding to cleavage. Data were expressed as means ± SD, two-tailed Student’s t-test. ns not significant.

Fig. 3. Identification of a cleavage site in CAD.

a Schematic representation of the CAD domains. The cleavability of each construct is listed after it. “+” stands for that it is cleavable; “−” stands for that it is uncleavable. b HEK-293T cells were transfected with Flag-tagged CAD mutants and HA-caspase-3 for mapping the CAD cleavage site. 24 h after transfection, total cell lysates were prepared for analysis of Flag, cleaved PARP and HA by Western blot. c Schematic diagram of CAD protein truncation. d HEK-293T cells were transfected with the indicated plasmids and the expression of the three further truncated CAD were detected by anti-Flag antibody. e Schematic diagrams of Flag-CAD-(952-1512) and the different CAD mutants for mapping the cleavage site of CAD. Numbers indicate the position of amino acid in CAD. The pentagrams indicate the putative cleavage sites in CAD. f, g Western blot of in vitro cleavage reaction containing purified Flag-tagged CAD-(952-1512) point mutants of putative cleavage sites (f) or full-length Flag-CAD-D1371A (g) by immunoprecipitation and recombinant active human caspase-3. h Western blot of MKN45 and HCT116 cells transfected with the indicated CAD construct and treated for 24 h with 5-FU. i, j MKN45 cells were co-treated MG-132 and concentration gradient of 5-FU for 24 h. Cell lysates were subjected to immunoblotting using anti-CAD-(1-1371) (i) and anti-CAD-ΔN1371 antibodies (j). Data in (b, d, f–j) derive from the same biological experiment but were processed on parallel gels (see Source Data for gel-specific details). Arrowhead highlights band corresponding to cleavage.

Fig. 4. CAD cleavage by caspase-3 is associated with chemosensitivity.

a Relative quantification of CTP, UTP, dCTP, and dTTP levels in MKN45-EV, MKN45-CAD-WT and MKN45-CAD-D1371A cells treated with 5-FU or DMSO for 24 h. n = 6 biologically independent samples per group. b Cell death assays of HGC27-EV, HGC27-CAD-WT or HGC27-CAD-D1371A cells treated with different concentrations and time period of 5-FU as indicated. Repeat the same treatment and experimental design in MKN45 cells. n = 3 biologically independent samples per group. c Representative photomicrographs of HGC27 and MKN45 cells expressing EV, CAD-WT, or CAD-D1371A, then treated with 5-FU. Scale bar, 50 μm. d–f Representative Immunoblot analysis (d) with Flag, cleaved PARP, and p-H2A.X antibody and Immunofluorescence (e, f) with p-H2A.X antibody of HGC27 and MKN45 cells as described in (c). Scale bar, 20 μm. g, h Size of tumors formed by injected subcutaneously with CAD^WT/WT or homozygous CAD^{D1371A/D1371A} MKN45 cells in xenograft mouse models were treated with DMSO and 5-FU separately and statistical analysis. n = 8 biologically independent samples per group. i Protein expression of CAD, cleaved PARP and P53 were determined by Western blot in tumor samples from untreated or 5-FU-treated Cldn18-ATK Cad^{D1371A/D1371A} mice. j Kaplan–Meier survival analysis for the Cldn18-ATK Cad^+/+ and Cldn18-ATK Cad^{D1371A/D1371A} mice treated with 5-FU (n = 10; Kaplan–Meier). p = 0.0236. Data in (d, i) derive from the same biological experiment but were processed on parallel gels (see Source Data for gel-specific details). Data were expressed as means ± SD, two-tailed Student’s t-test. ns not significant, *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 5. Codon-specific CAD mutations serve as a predictive biomarker for chemotherapy resistance.

a, b Two-dimensional ddPCR amplitude plot showing that the assay detects the wildtype CAD gene (x-axis) and the D1371 mutation (y-axis) in patient FFPE DNA. c Western blot of in vitro cleavage reaction containing purified Flag-CAD, Flag-CAD-D1371E and Flag-CAD-D1371Y by immunoprecipitation and recombinant active human caspase-3. d, e Representative photomicrographs of MKN45 and HCT116 cells expressing EV, CAD-WT, CAD-D1371E (d) or CAD-D1371Y (e), then treated with 5-FU. Scale bar, 50 μm. f, g Representative Immunoblot analysis with Flag, cleaved PARP, and p-H2A.X antibody as described in (d, e). Data in (c, f, g) derive from the same biological experiment but were processed on parallel gels (see Source Data for gel-specific details). Arrowhead highlights band corresponding to cleavage.

Fig. 6. Discovery of a potent CAD inhibitor RMY-186.

a Single-crystal structure of RMY-186. b Flag-CAD-WT or Flag-CAD-D1371A was transfected into MKN45 cells and then treated with RMY-186 (10 μM) for 24 h. c MST assay with RMY-186 and its differential selectivity toward GFP-CAD-D1371A versus GFP. n = 3 biologically independent samples per group. d Flag-CAD-D1371A overexpressed MKN45 cells were pre-treated with MG-132 (5 μM) then treated with RMY-186 (10 μM) for 24 h. Overexpressed protein of Flag-tag and ubiquitination were detected by immunoblotting. e Immunoprecipitation showing ubiquitination of CAD-D1371A protein in HA-Ub and Flag-CAD-D1371A overexpressed MKN45 cells treated with RMY-186. f Immunoprecipitation showing ubiquitination of CAD protein in MKN45 cells transfected with the indicated plasmids. g Computational molecular docking analysis to investigate the interaction of RMY-186 binding to CAD-D1371A (AlphaFold2). CPSase represents the local polypeptides shown. h, i Volcano plots (h) and Heat-map (i) showing the differentially E3 ubiquitination ligases in proteomics MS data. j HEK-293T cells were transfected with Flag-CAD-D1371A and CHX (20 μg/mL) was added to cells 24 h after transfection with and without RMY-186 (10 μM) treatment. Cells were collected after indicated time (0, 3, 6, 9, 12, or 15 h) and cell lystes were immunoblotted with anti-Flag antibody to detect the protein level change. k Scanning densitometry was performed for each Western blot indicated in ( j) using ImageJ. The best-fit exponential decay lines were plotted using GraphPad Prism and the half-life can be read from it (marked by dot-dash line). n = 3 biologically independent samples per group. Data in (d–f, j) derive from the same biological experiment but were processed on parallel gels (see Source Data for gel-specific details). Data were expressed as means ± SD, two-tailed Student’s t-test. ***p < 0.001.

Fig. 7. RMY-186 as a promising therapy for overcoming chemotherapy resistance.

a Protein expression of CAD, cleaved PARP, P53 and p-H2A.X was determined after the MKN45 CAD^{D1371A/D1371A} cells were treated with DMSO, RMY-186 (10 μM), 5-FU (50 μM), or the combination of RMY-186 and 5-FU for 24 h. b p-H2A.X was determined by Immunofluorescence as described in (a). Scale bar, 20 μm. c, d Microscopic images of pyroptotic cells. Arrowheads indicate ballooned cell membrane characteristic of pyroptotic cells. Scale bar, 25 μm. e Western blot was performed to detect the expression levels of GSDME in MKN45 CAD^{D1371A/D1371A} cells with treatment as described in (a). f Western blot was performed to detect the expression levels of GSDME in CAD-KD MKN45 cells with 5-FU treatment. g PI (50 μg/mL) staining of pyroptotic cells in (a). Scale bar, 25 μm. h Culture supernatants in (a) were collected to measure the percentage of LDH release. n = 3 biologically independent samples per group. i PI (50 μg/mL) staining of pyroptotic cells in (f). Scale bar, 25 μm. j Culture supernatants in (f) were collected to measure the percentage of LDH release. n = 3 biologically independent samples per group. k, l Whole-mount images (k) and the statistical analysis (l) of stomach from untreated, RMY-186-treated, 5-FU-treated or the combination of RMY-186 and 5-FU treated Cldn18-ATK Cad^{D1371A/D1371A} mice. Tumors were marked with black dotted lines. n = 8 biologically independent samples per group. m Protein expression of CAD, cleaved PARP, GSDME and P53 were determined in tumor samples. n Representative images of H&E and IHC staining of stomach sections. Scale bar, 50 μm. o Quantification and statistical analysis of Ki67 staining images in (n). n = 8 biologically independent samples per group. p ELISA analyses of the concentrations of LDH in serum. n = 8 biologically independent samples per group. Data in (a, e, f, m) derive from the same biological experiment but were processed on parallel gels (see Source Data for gel-specific details). Data were expressed as means ± SD, two-tailed Student’s t-test. ns not significant, *p < 0.05, ***p < 0.001.