CRISPR-mediated WNK4 point mutation aggravates tumor progression and weakens chemotherapy sensitivity in gastric
Xiaojun Ying1, Zhen Ying2, Xiaobing Gao1
1Department of General Surgery, Yongkang First People's Hospital, PR China.
Objective:
Gastric cancer (GC) is the fifth most common malignancy, the molecular targets of which have been increasingly explored in recent years. As a serine/threonine protein kinase, the role of WNK lysine deficient protein kinase 4 (WNK4) in GC was clarified in this study.
Methods:
Human GC lines AGS and MKN45 were stably transfected with a WNK4 mutant constructed by the CRISPR/Cas9 method and treated with cis-dichlorodiammine platinum (CDDP, 2 μg/mL) and 5-fluorouracil (5-FU, 5 μg/mL) for 48h. Tumor-bearing mice were established with 5×106 mutant-type AGS cells, and injected with 40 mg/kg WP1066, the inhibitor of signal transducer and activator of transcription 3 (STAT3), for 21 days. Cell malignant potential and tumor growth were assessed. STAT3 activation was identified by western blot and immunohistochemistry. The interaction between WNK4 and STAT3 was determined using co-immunoprecipitation and immunofluorescence co-localization.
Results:
WNK4 mutation promoted proliferation and invasion, and upregulated the p-STAT3/STAT3 value in GC cells with/without 5-FU and CDDP treatments, while inhibiting apoptosis of GC cells without drug treatment. In tumor-bearing mice, WNK4 mutation accelerated tumor growth, increased levels of p-STAT3, STAT3, and p-STAT3/STAT3, and strengthened the co-immunoprecipitation and co-localizing with STAT3; however, these effects were reversed by WP1066 treatment.
Conclusion:
Through activating STAT3, WNK4 mutation impacts both the natural and drug-treated growth of GC cells or tumors, suggesting a new avenue for preclinical research.
Insights
WNK4 mutation promotes gastric cancer (GC) growth and invasion by activating STAT3. This pathway is implicated in both natural and drug-treated GC progression, offering a potential target for new therapies.
Area of Science:
- Oncology
- Molecular Biology
- Biochemistry
Background:
- Gastric cancer (GC) is a significant global health concern, ranking as the fifth most common malignancy.
- Recent research has focused on identifying novel molecular targets for GC treatment.
- WNK4, a serine/threonine protein kinase, has emerged as a potential player in GC pathogenesis.
Purpose of the Study:
- To elucidate the role of WNK4 in gastric cancer.
- To investigate the molecular mechanisms by which WNK4 influences GC progression.
- To explore WNK4 as a potential therapeutic target in GC.
Main Methods:
- CRISPR/Cas9 was used to create WNK4 mutants in human GC cell lines (AGS, MKN45).
- Cells and tumor-bearing mice models were treated with chemotherapy agents (CDDP, 5-FU) and a STAT3 inhibitor (WP1066).
- Cell proliferation, invasion, apoptosis, tumor growth, STAT3 activation, and WNK4-STAT3 interactions were assessed using various molecular and cellular assays.
Main Results:
- WNK4 mutation enhanced GC cell proliferation and invasion, and increased p-STAT3/STAT3 levels, with or without chemotherapy.
- WNK4 mutation inhibited apoptosis in GC cells without drug treatment.
- In vivo, WNK4 mutation accelerated tumor growth and increased STAT3 activation, effects reversed by WP1066.
Conclusions:
- WNK4 mutation promotes gastric cancer growth and invasion through STAT3 activation.
- This mechanism affects both spontaneous and drug-induced GC progression.
- Targeting the WNK4-STAT3 pathway presents a promising strategy for preclinical GC research.
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