CDK5RAP3 as tumour suppressor negatively regulates self-renewal and invasion and is regulated by ERK1/2 signalling in human gastric cancer

Abstract

Background: Toward identifying new strategies to target gastric cancer stem-like cells (CSCs), we evaluated the function of the tumour suppressor CDK5 regulatory subunit-associated protein 3 (CDK5RAP3) in gastric CSC maintenance.

Methods: We examined the expression of CDK5RAP3 and CD44 in gastric cancer patients. The function and mechanisms of CDK5RAP3 were checked in human and mouse gastric cancer cell lines and in mouse xenograft.

Results: We show that CDK5RAP3 is weakly expressed in gastric CSCs and is negatively correlated with the gastric CSC marker CD44. CDK5RAP3 overexpression decreased expression of CSC markers, spheroid formation, invasion and migration, and reversed chemoresistance in gastric CSCs in vitro and vivo. CDK5RAP3 expression was found to be regulated by extracellular-related kinase (ERK) signalling. ERK inhibitors decreased spheroid formation, migration and invasion, and the expression of epithelial-to-mesenchymal transition (EMT)-related proteins in both GA cells and organoids derived from a genetically engineered mouse model of GA. Finally, CDK5RAP3 expression was associated with reduced lymph-node metastasis and better prognosis, even in the presence of high expression of the EMT transcription factor Snail, among patients with CD44-positive GA.

Conclusions: Our results demonstrate that CDK5RAP3 is suppressed by ERK signalling and negatively regulates the self-renewal and EMT of gastric CSCs.

Fig. 1. The relation of CDK5RAP3 and CD44 expression in tumours of GA patients and gastric cell lines.

a The expression of CDK5RAP3 and CD44 in the same patients by IHC and immunofluorescence. Scale bar 50 μm. b Proportion of tumours with low vs. high staining of CDK5RAP3 and CD44. c Kaplan–Meier survival curve comparing overall survival between patients with low or high CDK5RAP3 expression in gastric cancer patients with low CD44 expression. d Kaplan–Meier survival curve comparing overall survival between patients with low or high CDK5RAP3 expression in gastric cancer patients with high CD44 expression. e Western blot demonstrating levels of CDK5RAP3, CD44, Sox2, Oct4, Nanog and c-Myc in monolayers and spheroids conditions of AGS, KATOIII, NCI-N87 and SNU668. f Photos of monolayers and spheroids conditions of AGS and KATOIII cells following immunofluorescence staining for DAPI (blue), CDK5RAP3 (red) and CD44 (green). Scale bar 20 μm. β-actin was used as a loading control.

Fig. 2. Effect of CDK5RAP3 on EMT and acquisition of CSC phenotypes in gastric epithelial cells.

a Western blot demonstrating levels of CDK5RAP3, CD44, Sox2, Oct4, Nanog and c-Myc in HFE-145 spheroid cells transduced with sh. CDK5RAP3 or sh. Scr. b Photos and graph of HFE-145 cells following transduction sh.CDK5RAP3 or sh.Scr and grown in spheroid formation conditions. c Immunofluorescence of HFE-145 spheroids for DAPI (blue), CD44 (green) and Sox2 (red) following transduction with sh.CDK5RAP3 or sh. Scr. Scale bar 50 μm. d Photos and graphs of migration and invasion assays for HFE-145 cells in monolayer cells and in spheroids transduced with sh.CDK5RAP3 or sh.Scr. e Western blot demonstrating levels of CDK5RAP3, E-cadherin, N-cadherin, Vimentin, Snail, Slug and Zeb1 in HFE-145 spheroid cells transduced with sh.CDK5RAP3 or sh.Scr. f Photos following immunofluorescence staining HFE-145 spheroid cells transduced with sh.CDK5RAP3 or sh.Scr for DAPI (blue), CD44 (Green) and Snail (Red). Scale bar, 50 μm. β-actin was used as a loading control.

Fig. 3. Effect of CDK5RAP3 in gastric CSCs.

a Photos and graph of AGS and KATOIII cells transduced with CDK5RAP3 or Vector and grew in spheroid formation conditions. Scale bar, 50 μm. b Western blot analysis and immunofluorescence staining demonstrating levels of CDK5RAP3, CD44, Sox2, Oct4, Nanog and c-Myc in spheroid cells of AGS and KATOIII transduced with CDK5RAP3 or Vector. c Graphs and photos of migration and invasion assays for AGS and KATOIII spheroid cells transduced with CDK5RAP3 or Vector. d Photos and graph of NCI-N87 and SNU668 cells transduced with sh.CDK5RAP3 or sh.Scr and grown in spheroid formation conditions. Scale bar, 50 μm. e Photos and graphs of migration and invasion assays for AGS and KATOIII in spheroids transduced with CDK5RAP3 or Vector. f Photos and graphs of migration and invasion assays for NCI-N87 an dSNU668 in spheroids transduced with sh.CDK5RAP3 or sh.Scr. β-actin was used as a loading control. *p < 0.05 compared to control.

Fig. 4. Effect of CDK5RAP3 on chemotherapy resistance in gastric CSCs and gastric cancer xenografts.

a Proliferation assays for AGS spheroid cells transduced with CDK5RAP3 or Vector following treatment whit 5-fluorouracil (5-FU) or cisplatin chemotherapy. b Western blot analysis of tumour lysates for CDK5RAP3 and cleaved caspase-3 treated with 5-FU or cisplatin. c Tumour growth curves for AGS xenografts transduced with CDK5RAP3 or Vector and treatment with PBS or cisplatin. d Photos and graph of representative tumours from Vector plus PBS or CDK5RAP3 plus cisplatin. e Photos and graphs following Immunofluorescence analysis of tumours for proliferation using Ki-67 (green), apoptosis using cleaved caspase-3 (red), CD44 (green) and Sox2 (red). Scale bar, 20 μm. Bars represent standard deviation. β-actin was used as a loading control. *p < 0.05 compared to control.

Fig. 5. ERK signalling promotes CSC phenotypes and inhibits expression of CDK5RAP3 in gastric cancer cells.

a Human phospho-kinase antibody array and western blot analysis to determine kinases differentially regulated in KATOIII cells cultured as spheroids vs. monolayers. b Western blot analysis demonstrating levels of CDK5RAP3, p-ERK1/2, ERK2, p-JNK1/2, JUK2, p-p38 and p38 in monolayers and spheroid cells of AGS and KATOIII. c Photos of single cell assay in AGS and KATOIII cells showing diameter of spheroids at selected time points following treatment of ERK inhibitor U0126 or carrier (DMSO) and grown in spheroid formation conditions. d Western blot analysis of AGS and KATOIII gastric cancer spheroid cells treated with U0126 or DMSO in spheroid formation condition for levels of phospho-ERK1/2, ERK2 and CDK5RAP3. e Western blot demonstrating levels of CDK5RAP3, p-ERK1/2, ERK2, p-JUK1/2, JNK2, p-p38 and p38 in spheroids of AGS and KATOIII transduced with CDK5RAP3 or Vector. β-actin was used as a loading control.

Fig. 6. ERK-signalling inhibition and CDK5RAP3 negatively regulate EMT and metastasis in gastric CSCs.

a Western blot analysis of AGS and KATOIII spheroid cells treated with U0126 or DMSO in spheroid formation condition for levels of phospho-ERK1/2, ERK2, CDK5RAP3, Snail, Slug and Zeb1. b Photos and graphs of migration and invasion assays for AGS and KATOIII spheroid cells treated with U0126 or DMSO. c Immunofluorescence of AGS and KATOIII spheroid cells for DAPI (blue), CD44 (green) and snail (red) following transduction with sh.Kras or sh. Scr. Scale bar 50 μm. d Graphs of migration and invasion assays for AGS and KATOIII spheroid cells transduced with sh.Kras or sh.Scr. e Western blot analysis and immunofluorescence staining of AGS and KATOIII spheroid cells transduced with CDK5RAP3 or Vector for levels of CDK5RAP3, Snail, Slug and Zeb1. Scale bar 50 μm. β-actin was used as a loading control. *p < 0.05 compared to control.