KIF20A promotes cellular malignant behavior and enhances resistance to chemotherapy in colorectal cancer through regulation of the JAK/STAT3 signaling pathway

Abstract

Background/aims: Kinesin family member 20A (KIF20A) is upregulated in multiple cancers and plays important roles in promoting malignant behavior, whereas its exact role in CRC remains unknown.

Results: Both genomic and protein expression levels showed that KIF20A was upregulated in CRC. Further functional analyses revealed that KIF20A had a crucial role in improving cell proliferation and resistance to chemotherapy in CRC. Finally, we provided distinct mechanistic evidence that KIF20A achieved all of its pathological functions in CRC by activating the JAK/STAT3 pathway.

Conclusion: Our results suggested that KIF20A regulated a set of malignant characteristics in CRC by activating the JAK/STAT3 pathway. Our findings indicate a new direction for the development of more effective therapeutic treatments for CRC.

Methods: Three Gene Expression Omnibus datasets and The Cancer Genome Atlas datasets were used to investigate the expression level of KIF20A in CRC. Further experiments included immunohistochemical staining, western blot analysis, qRT-PCR, gene silencing, and a cell-injected xenograft mouse model to investigate the interaction between KIF20A and the JAK/STAT3 signaling pathway in both patient-derived specimens and CRC cell lines.

Keywords: JAK/STAT3; KIF20A; chemotherapy resistance; colorectal cancer; malignant characteristics.

Figure 1

KIF20A is dysregulated in CRC according to online database information. (A) The overlap of the dysregulated genes from GSE10972, GSE18105 and GSE41657 using GRO2R online analysis. Screen standard: P value < 0.05, fold change > 1.5. (B) Protein-protein interaction network analysis of 338 overlap genes. Red color represents upregulated genes. Green color represents downregulated genes. Size of the circle reflects the expression fold change of genes. (C) Enriched pathway analysis, biological process and molecular function analysis of 338 overlap genes. (D) Analysis of the expression pattern of KIF20A in normal colorectal tissue, colon adenoma and CRC based on the data from GSE20916. (E) Analysis of the expression pattern of KIF20A in normal colorectal tissue and different types of intestinal cancers based on the data from TCGA.

Figure 2

KIF20A is a prognostic predictor in CRC. (A) Survival analysis of CRC patients with high/low expression of KIF20A based on the data from GSE17536. (B) Survival analysis of CRC patients with high/low expression of KIF20A based on the data from GSE17538. (C) Survival analysis of CRC patients with high/low expression of KIF20A based on the data from TCGA.

Figure 3

KIF20A is significantly upregulated in CRC. (A) Immunohistochemical analysis of KIF20A in paraffin tissues from CRC tissues and normal colorectal tissues of two patients (case 1 and case 2). (B) Western blot analysis of KIF20A in five pairs of CRC and paracancerous tissues. (C) mRNA level of KIF20A in twelve pairs of CRC and paracancerous tissues were detected by RT-PCR. Data are presented as mean ± SEM. *P < 0.05. (D–E) Western blot and RT-PCR analysis of KIF20A in a normal colorectal cell line (NCM) and seven CRC cell lines. Data are presented as mean ± SEM.

Figure 4

KIF20A improves malignant behaviors in CRC. (A and B) Western blot and RT-qPCR analysis of KIF20A in constructed cell lines. Mock represents blank groups. Sh-NC represents lentivirus-mediated control groups. Sh-KIF20A represents lentivirus-mediated KIF20A silencing groups. (C and D) Western blot and RT-qPCR analysis of KIF20A in constructed cell lines. Mock represents blank groups. Lv-NC represents lentivirus-mediated control groups. Lv-KIF20A represents lentivirus-mediated KIF20A overexpression groups. (E) The relative growth rates in KIF20A knockdown CRCs were measured using CCK-8 analysis and compared between 3 groups at indicated times in two different cell lines. Data are presented as mean ± SEM. *P < 0.05; **P < 0.01. (F) The relative growth rates in KIF20A overexpression CRCs were measured using CCK-8 analysis and compared between three groups at indicated times in two different cell lines. Data are presented as mean ± SEM. *P < 0.05; **P < 0.01. (G–J) Colony formation of 4 different cell lines transfected with different treatments. Data are presented as mean ± SEM. *P < 0.05; **P < 0.01.

Figure 5

KIF20A promotes colorectal cancer cells growth in vivo. The volume and weight of xenografted tumor from different groups were compared. (A) Representative images of tumor form in each group. (B–D) Growth curve of tumors quantification of volume and weight in each group Data are presented as mean ± SEM. *P < 0.05; **P<0.01.

Figure 6

KIF20A induces chemo-resistance in CRC. (A, C) Western blot analysis of apoptosis-related factors (cleaved-caspase 3, bax, bcl-2) in different transfected groups in HCT-116 cell line treated with 4ug/ml 5-FU (left) or 8 ug/ml oxaliplatin (right) and LOVO cell line treated with 1.5 ug/ml 5-FU (left) or 1 ug/ml oxaliplatin (right). (B, D) Cell viability was measured using CCK-8 analysis and compared between different groups with different treatments at indicated times. (E–H) The apoptotic rates of different transfected groups with different treatments were measured by flow cytometry. Data are presented as mean ± SEM. *P < 0.05; **P < 0.01.

Figure 7

KIF20A mediates malignant behaviors in CRC via the JAK/STAT3 signaling pathway. (A) Phosphorylation of JAK and STAT3 in different transfected groups was determined by western blot analysis. (B) Western blot analysis of phosphorylation of JAK and STAT3 in different transfected groups with or without the administration of JAK inhibitor, INCB. (C) Western blot analysis of phosphorylation of JAK and STAT3 in different transfected groups with or without the silence of STAT3. (D, E) Relative growth rate of different transfected groups with or without the administration of INCB or silence of STAT3. The comparison was made at indicated times. (F–I) Colon formation assay of different transfected groups with or without the administration of INCB or silence of STAT3. (J–K) The apoptotic rates of different transfected groups with or without silence of STAT3 were measured by flow cytometry. (L) Immunohistochemical staining of KIF20A and p-STAT3 in an independent set of CRC tumor samples with good or poor responses to 5-FU and oxaliplatin therapy. Scale bar=50 μm. Data are presented as mean ± SEM. *P < 0.05; **P < 0.01.