CDK12 inhibition enhances oxaliplatin efficacy in gastric cancer by suppressing the MAPK signaling pathway

Abstract

Background: Gastric cancer (GC) is a leading cause of cancer-related mortality worldwide. Oxaliplatin (OXA) based therapy plus/minus targeted therapy and immune check point inhibitors is the standard first-line treatment for advanced GC; however, its clinical efficacy is often hindered by the development of drug resistance. Cyclin-dependent kinase 12 (CDK12), a transcriptional regulator linked to DNA repair, plays a crucial role in transcription, cancer progression as well as drug resistance, where its exact role is unclear. In this study, we will investigate the role of CDK12 in GC progression and its potential as a therapeutic target specifically to enhance the efficacy of OXA.

Methods: CDK12 expression in GC tissues was analyzed by quantitative polymerase chain reaction (qPCR) and tissue microarrays (TMAs). A Kaplan-Meier survival analysis was conducted to assess the relationship between CDK12 levels and clinical outcomes. The effect of the CDK12 inhibitor combined with OXA was evaluated through in vivo and in vitro models. RNA-sequencing and western blots were used to investigate the molecular mechanisms of CDK12 inhibitor sensitizing OXA.

Results: CDK12 exhibited significant amplification frequency in GC. The Mendelian-randomization analysis revealed a positive causal association between elevated CDK12 expression and an increased risk of GC. Additionally, CDK12 was significantly overexpressed in GC tissues compared with adjacent normal tissues, and its high expression was significantly associated with a worse prognosis. The functional assays revealed that combining the CDK12 inhibitor THZ531 with OXA synergistically suppressed GC cell proliferation, induced apoptosis, and reduced colony formation in vitro, while substantially inhibiting tumor growth in xenograft models. Mechanistically, CDK12 inhibition disrupted MAPK signaling, leading to enhanced OXA-induced DNA damage and potentiated anti-tumor effects.

Conclusions: Our findings suggest that CDK12 inhibition may represent a promising strategy for overcoming OXA resistance and improving GC treatment outcomes.

Keywords: Gastric cancer (GC); cell cycle; combination therapy; cyclin-dependent kinase 12 (CDK12); oxaliplatin (OXA).

Figure 1

Cell-cycle pathway dysfunction is a critical characteristic of GC. (A) Functional enrichment: enriched KEGG signaling pathways in GC tissues compared to adjacent non-tumor tissues. (B) Venn diagram analysis illustrating the overlap between the cell cycle-related pathways from the KEGG and CDK family members derived from the GeneCards database. (C) Distribution of genomic alterations in the CDKs in TCGA-GC cohort. (D) Associations between CDK12 expression and GC based on MR analysis. CDK12, cyclin-dependent kinase 12; CI, confidence interval; GC, gastric cancer; IVW, inverse variance weighted; KEGG, Kyoto Encyclopedia of Genes and Genomes; MR, Mendelian randomization; OR, odds ratio; PD-1, programmed cell death 1; PD-L1, programmed death-ligand 1; TCGA, The Cancer Genome Atlas.

Figure 2

CDK12 is increased in tumor tissues, and is associated with the poor prognosis of GC patients. (A) mRNA expression levels of CDK12 in tumor tissues and normal tissues based on TCGA-STAD cohort. (B) The K-M curves of OS in the GC cohort from the K-M plotter database grouped according to high (red, n=460) and low (black, n=171) CDK12 expression. (C) Reverse transcription-qPCR analysis of CDK12 expression in tumor tissues and adjacent normal tissues. (D) Representative IHC images showing CDK12 expression in GC tissues and normal control tissues from the cohort, and the semi-quantitative analysis of CDK12 IHC staining in paired GC tissues (scale bar: 50 and 25 µm). (E) Representative findings of CDK12 protein expression by IHC, which were graded based on staining intensity in 324 GC tissues (scale bar: 50 and 25 µm). (F) OS and DFS according to CDK12 expression. Student’s t-test, ****, P<0.0001. CDK12, cyclin-dependent kinase 12; DFS, disease-free survival; GC, gastric cancer; IHC, immunohistochemical; K-M, Kaplan-Meier; OS, overall survival; qPCR, quantitative polymerase chain reaction; STAD, stomach adenocarcinoma; TCGA, The Cancer Genome Atlas.

Figure 3

OXA efficacy is enhanced by CDK12 inhibition in vitro. (A) CDK12 expression in human gastric cell lines; normal gastric epithelial cells GES-1, and the GC cell lines AGS, NCI-N87, SNU638, SNU668, NUGC3, NUGC4 and HGC27 were evaluated by western blotting. (B) Colony formation assay of high level CDK12 cancer cell lines treated with different dosages of OXA. Colonies were stained with crystal violet. (C) Colony formation assay of cells treated with the CDK12 inhibitor (THZ531), OXA, or a combination of both at 12 days. Colonies were stained with crystal violet. (D) The growth curve of NUGC3 and SNU638 with the CDK12 inhibitor (THZ531, 0.1 µM) combined with OXA (1.0 µM) at the indicated concentration. Data are presented as the mean ± standard deviation. *, P<0.05; **, P<0.01; ***, P<0.001. (E) Representative image of a tumor sphere formation assay of NUGC3 and SNU638 treated with the CDK12 inhibitor (THZ531), OXA, or a combination of these compounds at specified concentrations for a duration of 10 days (scale bar: 200 µm). Tumor spheres were visualized by bright-field microscopy. CDK12, cyclin-dependent kinase 12; DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NC, negative control; OXA, oxaliplatin.

Figure 4

CDK12 inhibition significantly enhances the anti-tumor effect of OXA in vivo. (A) Representative tumor images. (B) Body weight. (C) Growth curve of tumor volume. (D) Tumor weight. (E-G) Representative IHC images showing the expression of Ki-67 (F) and cleaved-caspase 3 (G) in NUGC3 xenograft tumors, visualized using DAB chromogenic staining (scale bar: 50 µm). *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. CDK12, cyclin-dependent kinase 12; DMSO, dimethyl sulfoxide; IHC, immunohistochemical; NC, negative control; OXA, oxaliplatin.

Figure 5

MAPK signaling is involved in GC cell OXA resistance and enhanced DNA damage. (A) Pathway enrichment analysis of RNA-sequencing data revealed the significant activation of various signaling pathways in GC cells after the combined treatment, with the hallmark KRAS signaling downregulation pathway showing the most prominent enrichment (adjusted P value <0.01). (B) GSEA results showing the activation of KRAS signaling downregulation (KRAS signaling DN) in the combined treatment group (OXA + THZ531) compared to the control group as indicated by the elevated running enrichment score (red line) and significant P value (P value <0.001). (C) γH2AX and MAPK signaling pathway analyses by western blotting in the NUGC3 GC cells after treatment with the CDK12 inhibitor THZ531 and OXA. (D) Western blot further confirmed the reduction in MAPK pathway activation (i.e., the phosphorylation of MEK and ERK) following the combined treatment with OXA and THZ531 in the NUGC3 xenografts tumors. CDK12, cyclin-dependent kinase 12; KRAS signaling DN, KRAS signaling downregulation; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GC, gastric cancer; GSEA, gene set enrichment analysis; OXA, oxaliplatin.

Figure 6

Schematic model illustrating the role of CDK12 in mediating OXA resistance in GC via the activation of the MAPK signaling pathway. CDK12 overexpression activates the MAPK signaling pathway, leading to enhanced DNA damage repair and reduced sensitivity to OXA in GC. The pharmacological inhibition of CDK12 significantly suppresses MAPK pathway phosphorylation, thereby impairing DNA repair mechanisms and amplifying OXA-induced DNA damage. Targeting CDK12 may serve as a promising therapeutic strategy for overcoming chemoresistance and improve the clinical outcomes of OXA-based chemotherapy in GC. CDK12, cyclin-dependent kinase 12; GC, gastric cancer; OXA, oxaliplatin.