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. 2021 Dec 1;12(12):1122.
doi: 10.1038/s41419-021-04385-1.

Loss of DSTYK activates Wnt/β-catenin signaling and glycolysis in lung adenocarcinoma

Chenxi Zhong #  1 , Ming Chen #  1 , Yu Chen #  2 , Feng Yao  3 , Wentao Fang  4
Affiliations

Loss of DSTYK activates Wnt/β-catenin signaling and glycolysis in lung adenocarcinoma

Chenxi Zhong et al. Cell Death Dis. .

Abstract

Aberrant activation of Wnt/β-catenin signaling and dysregulation of metabolism have been frequently observed in lung cancer. However, the molecular mechanism by which Wnt/β-catenin signaling is regulated and the link between Wnt/β-catenin signaling and cancer metabolism are not fully understood. In this study, we showed that the loss of dual serine/threonine tyrosine protein kinase (DSTYK) led to the activation of Wnt/β-catenin signaling and upregulation of its target gene, lactate dehydrogenase (LDHA), and thus the elevation of lactate. DSTYK phosphorylated the N-terminal domain of β-catenin and inhibited Wnt/β-catenin signaling, which led to the inhibition of cell growth, colony formation and tumorigenesis in a lung adenocarcinoma mouse model. DSTYK was downregulated in lung cancer tissues, and its expression was positively correlated with the survival of patients with lung adenocarcinoma. Taken together, these results demonstrate that the loss of DSTYK activates Wnt/β-catenin/LDHA signaling to promote the tumorigenesis of lung cancer and that DSTYK may be a therapeutic target.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. DSTYK was downregulated in lung cancer.
A The KmPlot database was mined to confirm the correlation between the expression of DSTYK and the survival of patients with lung cancer. B qPCR was performed to determine the mRNA levels of DSTYK in lung cancer tissues and noncancerous tissues. **P < 0.01. C Immunohistochemistry (IHC) was performed to determine the protein levels of DSTYK in lung cancer tissues and noncancerous tissues. The images with different magnification were shown and the scale bar was indicated. D Statistical analysis of the data in C. The IHC intensity was scored and analyzed. E Western blot and IHC were performed to determine the protein levels of DSTYK in the KP mouse model. Eight-week-old KP mice in the experimental group were treated with Ad-Cre virus (+Ad-Cre) (109 per mouse) to delete the expression of P53 and activate the expression of KrasG12D. Mice in the control group were treated with control virus. Twelve weeks later, the mice were killed, lung tissues were collected, and DSTYK protein levels were examined by western blot (up) and IHC (below). The images of western blot with long exposure and short exposure were shown. F The protein levels of DSTYK in normal lung epithelial cells (Bease-2B) and lung cancer cells.
Fig. 2
Fig. 2. DSTYK inhibited the growth of A549 and SPC-A-1 cells.
A Western blot was performed to examine the overexpression of DSTYK. A549 and SPC-A-1 cells were infected with lentivirus expressing Flag-tagged DSTYK and selected with puromycin. Then, western blotting was performed. B The MTT assay was performed to examine the effects of DSTYK on the growth of A549 and SPC-A-1 cells. C, D A colony formation assay was performed, and the results were quantified. E Sphere formation was assessed to examine the roles of DSTYK in stemness. Details about sphere formation are described in the “Materials and methods”. The spheres were photographed and counted. The scale bar was indicated. *P < 0.05; **P < 0.01.
Fig. 3
Fig. 3. Knockdown of DSTYK promoted the growth of A549 and SPC-A-1 cells.
A Western blot was performed to examine the knockdown efficiency. A549 and SPC-A-1 cells were infected with lentivirus expressing shRNA for DSTYK for 24 hours and selected with puromycin. Then, western blotting was performed. B The MTT assay was performed to examine the effects of DSTYK knockdown on the growth of A549 and SPC-A-1 cells. C, D A colony formation assay was performed, and the results were quantified using control cells and DSTYK-knockdown cells. E–H A tumorigenesis assay was performed to determine the functions of DSTYK in lung cancer. To induce lung cancer, the 8-week-old male KP mice were randomly divided into two groups (5 mice in each group) and anesthetized with 2.5% avertin; full anesthetization and absence of reaction to pain were ensured. A total of 1 × 109 viruses (pSECC/Sg con or pSECC/Sg DSTYK) (OBIO, Shanghai) were administered per mouse using an intranasal/orthotropic infection protocol. The administration of pSECC/Sg con viruses led to the deletion of P53 and activation of KrasG12D. The administration of pSECC/Sg DSTYK viruses led to the deletion of P53 and DSTYK and activation of KrasG12D. Twelve weeks later, the mice were killed, lung tissues were collected and photographed, and the tumors were indicated with “*” (E), the tumors were examined with HE staining and analyzed (F, G), and the expression of DSTYK in the tumors was examined using western blot. The scale bar was indicated. *P < 0.05; **P < 0.01.
Fig. 4
Fig. 4. DSTYK interacts with β-catenin.
A The interaction between exogenously expressed Flag-DSTYK and myc-β-catenin was examined using immunoprecipitation. Cells were harvested 48 h after transfection. B A GST pull-down assay was performed to confirm the interaction between endogenous β-catenin and the fusion protein GST-DSTYK. C The interaction between exogenously expressed Flag-DSTYK and myc-β-catenin was examined using immunoprecipitation. Cells were harvested and lysed with RIPA buffer, and anti-β-catenin antibody was added for immunoprecipitation. D An immunoprecipitation assay was performed to determine the domain of DSTYK that binds myc-β-catenin. N-ter the N-terminus of DSTYK (1–500 aa), C-ter the C-terminus of DSTYK (501–929 aa), FL the full length of DSTYK.
Fig. 5
Fig. 5. DSTYK inhibited Wnt/β-catenin signaling and promoted the phosphorylation of β-catenin.
A The TOPFlash reporter assay was performed to examine the effects of DSTYK on the transcriptional activity of β-catenin. B qPCR was performed to examine the effects of DSTYK on the expression of Axin2 and c-Myc. C Western blot was performed to examine the effects of DSTYK on the accumulation of β-catenin. Control cells and DSTYK-overexpressing cells were treated with Wnt3a for the indicated duration, and the protein levels of β-catenin were examined. D Western blot was performed to examine the effects of DSTYK on the expression of cyclin D1 and c-Myc. E The phosphorylation of β-catenin was examined after overexpression of DSTYK. F. In vitro kinase assay. Flag-DSTYK was immunoprecipitated and incubated with GST-N-β-catenin at 32 °C for 30 min, and the phosphorylation of β-catenin was examined. *P < 0.05; **P < 0.01.
Fig. 6
Fig. 6. β-Catenin regulated the expression of LDHA.
A The heatmap of the RNA-seq was shown. B The protein levels of LDHA in cells with DSTYK overexpression or knockdown. C The sequence of the human LDHA promoter. D The TBE sequence at −1533 to −1528 in the LDHA promoter was indicated and mutated for the luciferase assay. E ChIP was performed to examine the binding of β-catenin to the LDHA promoter. **P < 0.01.
Fig. 7
Fig. 7. DSTYK/Wnt/β-catenin signaling regulated LDHA expression.
A The correlation between DSTYK, β-catenin and LDHA in clinical lung tissues. The protein levels of DSTYK, β-catenin and LDHA in 10 lung cancer tissues were examined. The immunostaining was scored. Representative images of two cases are shown, and the correlations between the expression of DSTYK, β-catenin, and LDHA were analyzed. B Knockdown of β-catenin or LDHA abolished the elevated lactate content induced by DSTYK knockdown. ##P < 0.01; **P < 0.01. C Knockdown of β-catenin or LDHA abolished the enhanced sphere formation induced by DSTYK knockdown. The sphere was counted and the statistical analysis was performed. **P < 0.01; ##P < 0.01. The scale bar was indicated.
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