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. 2020 Sep 2:11:1250.
doi: 10.3389/fphar.2020.01250. eCollection 2020.

DSTYK Promotes Metastasis and Chemoresistance via EMT in Colorectal Cancer

Jinyu Zhang  1   2 , Zachary Miller  1 , Phillip R Musich  1 , Ashlin E Thomas  1 , Zhi Q Yao  2 , Qian Xie  1 , Philip H Howe  3 , Yong Jiang  1
Affiliations

DSTYK Promotes Metastasis and Chemoresistance via EMT in Colorectal Cancer

Jinyu Zhang et al. Front Pharmacol. .

Abstract

Objective: Tumor metastasis and resistance to chemotherapy are two critical factors that contribute to the high death rate of colorectal cancer (CRC) patients. Metastasis is facilitated by the epithelial-mesenchymal transition (EMT) of tumor cells, which has emerged not only as a fundamental process during metastasis, but is also a key process leading to chemoresistance of cancer cells. However, the underlying mechanisms of EMT in CRC cell remain unknown. Here, we aim to assess the role of dual serine/threonine and tyrosine protein kinase (DSTYK) in CRC metastasis and chemoresistance.

Methods: To study the role of DSTYK in TGF-β-induced EMT, we employed techniques including Crispr/Cas9 knockout (KO) to generate DSTYK KO cell lines, RT-PCR to detect the mRNA expression, immunofluorescence analyses, and western blots to detect protein levels of DSTYK in the following 4 cell lines: control LS411N-TβRII and LS411N-TβRII/DSTYK KO, control LS513 and LS513/DSTYK KO cells, treated with/without TGF-β. The effects of DSTYK on apoptosis were investigated by MTT assays, flow cytometry assays, and TUNEL assays. The expression of DSTYK in CRC patients and its correlation with EMT markers were determined by bioinformatics analysis. For in vivo analysis, both xenograft and orthotopic tumor mouse models were employed to investigate the function of DSTYK in chemoresistance and metastasis of tumors.

Results: In this study, we demonstrate that the novel kinase DSTYK promotes both TGF-β-induced EMT and the subsequent chemoresistance in CRC cells. DSTYK KO significantly attenuates TGF-β-induced EMT and chemoresistance in CRC cells. According to the Gene Expression Omnibus (GEO) database, the expression of DSTYK is not only positively correlated to the expression of TGF-β, but proportional to the death rate of CRC patients as well. Evidently, the expression of DSTYK in the metastatic colorectal cancer samples from patients was significantly higher than that of primary colorectal cancer samples. Further, we demonstrate in mouse models that chemotherapeutic drug treatment suppresses the growth of DSTYK KO tumors more effectively than control tumors.

Conclusion: Our findings identify DSTYK as a novel protein kinase in regulating TGF-β-mediated EMT and chemoresistance in CRC cells, which defines DSTYK as a potential therapeutic target for CRC therapy.

Keywords: chemoresistance; colorectal cancer; dual serine/threonine and tyrosine protein kinase; epithelial-mesenchymal transition; metastasis; transforming growth factor-β.

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Figures

Figure 1
Figure 1
DSTYK is associated with the metastasis of human colorectal cancer. (A) Scatter plot showing the correlation between the mRNA levels of DSTYK and TGF-β, (B) DSTYK and vimentin (VIM), (C) DSTYK and E-cadherin (CDH1), and (D) DSTYK and N-cadherin (CDH2) in the human TCGA colon cancer database. (E) Kaplan-Meier curve assessing the correlation between DSTYK expression and patients’ survival rate obtained from 652 patients in The Gene Expression Omnibus (GEO) database. Blue line represents metastatic colorectal cancer patients with high DSTYK expression (n=154) and the red line represents patients with low DSTYK expression (n=498) in the GSE 17538 dataset. SPSS repeated measures, general linear model. (F) Representative images of human colorectal primary tumors and secondary tumors subjected to immunohistochemical (IHC) analysis with DSTYK antibody. Images were taken after IHC staining. Scale bar, 50 µm. (G) Quantification of the staining signals from IHC analyses of DSTYK in both primary and secondary tumor tissues. Data are presented as means ± SEM. for n= 20.
Figure 2
Figure 2
TGF-β induces EMT in LS411N-TβRII cells and LS513 cells. (A) Morphological change of cells before and after 5 days of TGF-β (1 ng/ml) treatment. Scale bar: 100 µm. (B) Representative immunostaining of E-cadherin in LS411N-TβRII cells and LS513 cells before and after 5 days TGF-β (1 ng/ml) treatment. Scale bar, 50 μm. (C) WB analyses to detect the protein levels of epithelial marker E-cadherin and mesenchymal markers, N-cadherin and vimentin. Hsp90 was used as loading control. All experiments were repeated at least three times and similar results were observed. (D) RT-PCR analyses of DSTYK mRNA levels during TGF-β-induced EMT. β-actin was used as control. (E) Immunoblotting analysis to detect the protein levels of DSTYK during TGF-β-mediated EMT. Hsp90 was used as loading control. All experiments were repeated at least three times and similar results were observed. (F) Real-time PCR analyses to detect the upregulation of DSTYK expression during TGF-β-mediated EMT. Data are shown as mean ± SD, n=3 independent experiments.
Figure 3
Figure 3
DSTYK is required for TGF-β-induced EMT in CRC cells. (A) Immunoblotting analyses were employed to compare the protein levels of DSTYK and the EMT markers E-cadherin, N-cadherin and vimentin between control cells and DSTYK/KO cells during a 5-day TGF-β treatment timecourse. Hsp90 was used as loading control. (B) Representative images of a wound healing assay with LS411N-TβRII cells and LS513 cells. (C) Schematic of the principle of a cell invasion assay. (D) Top: The cells on the basement membrane before invasion at 0h. Middle: The cells migrating to the bottom of membrane after 36h invasion. Bottom: The cells on the bottom of membrane after 36h invasion from 5 days of TGF-β treatment. Representative images of the lower chamber of the trans-well invasion assay in different treatment groups are shown for LS411N-TβRII or LS513 cells. (E) The quantification of cell invasion assays in (D). All the experiments were repeated at least three times. **P < 0.01. Data are shown as mean ± SD, n=3 independent experiments.
Figure 4
Figure 4
TGF-β induces apoptosis in the absence of DSTYK. (A) Morphological differences between control cells and DSTYK/KO cells after 5 days TGF-β treatment. (B) TUNEL assays to detect the apoptotic signals in control cells and DSTYK/KO cells after 5 days TGF-β treatment. The green color indicates DNA fragmentation. Scale bar, 100 μm. (C) Flow cytometry analysis to compare the levels of cell death by staining with cell surface apoptotic marker annexin V and the necrotic marker PI in control and DSTYK/KO cells after 5 days TGF-β treatment. Western blot analysis of apoptosis-related proteins including cleaved-caspase-3, Bcl-2, Bim, and Bcl2-associated X (Bax) in control and DSTYK/KO LS411N-TβRII cells (D) and LS513 cells (E) after 5 days TGF-β treatment. β-actin is used as loading control. All experiments were repeated for at least three times, and similar results were observed.
Figure 5
Figure 5
DSTYK inhibits chemotherapeutic agent-induced apoptosis. (A, B) MTT assays to compare the chemoresistance between control and DSTYK/KO cells after 3 days OXA treatment. Data are presented as means ± SEM, for n > 3 per dosage point. (C, D) Flow cytometry analysis to compare the expression of cell surface apoptotic marker annexin v between control cells and DSTYK/KO cells after a 48 h OXA treatment, 4 µM in (C); 2 µM in (D). Red curves represent control LS411N-TβRII cells or LS513 cells. Blue curves represent DSTYK/KO LS411N-TβRII cells or LS513 cells. ‘-’ means without OXA treatment. ‘+’ means with OXA treatment for 48h. (E, F) Quantification of the flow cytometry analyses in (C, D), respectively. (G, H) Clonogenic assays and the corresponding quantification results. All experiments were repeated at least three times and similar results were observed. *P < 0.05; **P < 0.01.
Figure 6
Figure 6
DSTYK knockout facilitates tumor regression after OXA treatment. For each mouse, control LS411N-TβRII cells were implanted into the right flank and DSTYK/KO LS411N-TβRII cells were implanted into the left flank (n=20 per group). After around 3 weeks, mice carrying two tumors of similar size (tumor diameter is around 50 mm as measured with a caliper) started receiving OXA twice weekly. A tumor volume of 1300 mm3 was defined as the survival endpoint. Tumors were excised (A) and tumors’ weights were evaluated as a box-and-whisker plot (B). Data is presented as mean ± SD. (C) The growth curves of control tumors and DSTYK/KO tumors with and without OXA treatment, n=10 per group. (D) The levels of Ki-67 and cleaved caspase-3 in tumors tissues from LS411N-TβRII control cells and LS411N-TβRII DSTYK/KO cells detected by immunohistochemical analyses. Scale bar, 50 µm. (E) Representative images of liver metastasis (arrows). (F) Kaplan-Meier curve assessing the survival of SCID mice injected with LS411N-TβRII control cells or LS411N-TβRII DSTYK/KO cells in cecum wall (n=10/group, 3x). *P < 0.05; **P < 0.01. (G) The proposed model for the mechanism underlying the expression and function of DSTYK in TGF-β-induced EMT and chemoresistance.
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