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

Cell culture and transfection

Normal bronchial epithelial cell line Bease-2B and lung cancer cell lines were obtained from the Cell Bank of the Chinese Academy of Science (Shanghai, China) and maintained in DMEM (Gibco) containing 10% FBS (Gibco) and antibiotics in a 5% CO₂ incubator. Cells were transfected with Lipofectamine 8000 (Invitrogen) according to manufacturer instructions. Stable cell lines were selected using puromycin (Sangon Biotechnology, A610593) for one week. Cell lines were authenticated using STR profile analysis and used within 3–20 passages of thawing the original stocks.

qPCR

Total RNA was extracted from cells or tissues using TRIzol and reverse transcribed to cDNA using a kit (Takara). RT-PCR analysis was performed using an Applied Biosystems Prism 7900 HT sequence detection system with Universal SYBR qPCR Master Mix (Vazyme, China). The expression levels of each mRNA relative to the β-actin expression level were calculated based on the 2^-ΔΔCt method. The primer sequences were as follows: F, 5’-ttgcatactgatcctcgg-3’; R, 5’-tgtgcactagttcatact-3’.

Immunohistochemistry (IHC)

30 clinical samples were collected from Shanghai Chest Hospital after the informed consent was obtained was obtained from all subject. This study was approved by the ethical committee of the Shanghai Chest Hospital. The clinical information was listed in Table S1. Tissues were embedded in paraffin and cut into 5 µm sections. Dewaxing and rehydration were performed by placing slides in a decreasing gradient series of xylol and ethanol. Antigen recovery was performed by incubating the sections in a pH 6.0 citrate sodium solution at 100 °C for 20 min. After blocking endogenous peroxidase activity using a kit (ZSGB-BIO, PV-8000), the tissues were incubated with primary antibody (anti-DSTYK, 1;100, Proteintech, 20102-1-AP; anti-β-catenin, 1;100, CST, 8480; or anti-LDHA, 1;100, Proteintech, 19987-1-AP) at 4 °C overnight. Then, secondary antibody was added, and the tissues were incubated at room temperature for 1 h. The signals were developed using DAB (ZSGB-Bio, Beijing, China), and the sections were stained with hematoxylin. Tissues were examined under a microscope, and scoring was performed as described.

KP mouse model

p53^f/f; LSL-Kras^G12D (KP) model C57BL/6 mice were housed under standard conditions with 12 h dark and 12 h light cycles.

To study the roles of DSTYK in the tumorigenesis in vivo, the sg con or sg DSTYK were inserted into the cassette of pSECC lentivirus vector which expressed cas 9 enzyme and Cre recombinase. 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 (OBIO, Shanghai) were administered per mouse (8 weeks old) using an intranasal/orthotropic infection protocol as described [22]. The administration of pSECC/Sg con viruses led to the deletion of P53 and activation of Kras^G12D. The administration of pSECC/Sg DSTYK viruses led to the deletion of P53 and DSTYK and activation of Kras^G12D. Twelve weeks later, the lungs were harvested, and western blot assays were performed.

For the examination of DSTYK protein levels in the KP tumors, the mice were treated with/without Ad-Cre virus (adenovirus). This study was approved by the ethical committee of the Shanghai Chest Hospital, and complied with the ethical regulations of ethical committee of the Shanghai Chest Hospital.

Western blot

Cells were harvested with RIPA buffer. The protein concentration was determined using BCA. Then, SDS-PAGE was performed, and the proteins were transferred to a PVDF membrane. After blocking with 5% BSA at room temperature for 1 h, the membrane was incubated with the primary antibody for 4 h and sequentially with the secondary antibody for 1 h. Then, the signals were examined using an ECL kit. The information about the antibodies were: Anti-DSTYK (Proteintech, 20102-1-AP), anti-GAPDH (Santa Cruz, sc-47724), ant-β-catenin (CST, 8480), anti-phosphorylated β-catenin (CST, 9561), anti-GST (Santa Cruz, sc-138), anti-Flag (Proteintech, 80010-1-RR), anti-Cyclin D1 (Santa Cruz, sc-8396), anti-c-Jun (Santa Cruz, sc-74543), anti-c-Myc (Santa Cruz, sc-40), anti-LDHA (Proteintech, 19987-1-AP), anti-Tubulin (Santa Cruz, sc-166729).

MTT assay

An MTT (Merck, 11465007001) kit was used. Cells were plated in 96-well plates at a density of 1000 cells per well, and each well was loaded with 200 µl of medium. On days 1, 3, 5, and 7, 20 µl of MTT solution were added to each well, and the plates were incubated for 4 h. Then, supernatants were removed, precipitates were dissolved in 200 µl of DMSO, and absorbance was measured.

Colony formation assay

Cells were suspended in 2× DMEM at a density of 10⁶ cells/L. A bottom agar layer was prepared with an equal volume of 1.2% agar and 2× DMEM containing antibiotics and 20% FBS, and 3 ml of the mixture was added to a 6 cm dish. Thirty minutes later, a bottom agar layer was prepared with an equal volume of 1.2% agar and 2× DMEM with an additional 0.2 ml of the cell suspension. After softly mixing, the mixture was added to the dish with the bottom agar layer. Fourteen days later, colonies were counted.

Sphere formation assay

Cells from each group were collected and digested into single cells. Subsequently, for each group, 2 × 10⁴ viable cells per well were counted and seeded in low-adherent 6-well plates (Corning, USA) in serum-free F12/MEM containing B27, EGF (20 ng/ml) and FGF (40 ng/ml). After incubation at 37 °C for 14 days, pictures were taken under a microscope, and tumor spheres in five separate fields were counted. EGF (R&D, 236-EG), FGF (R&D, 233-FB), and B27 (R&D, AR008) were obtained from R&D.

Immunoprecipitation

Cells were scraped and proteins were extracted from the cells with 1 ml RIPA buffer. After centrifugation (4 °C, 12,000 rpm) for 20 min, supernatant was divided into two tubes and immunoprecipitated with the indicated antibody and IgG as control at 4 °C. On the next day, protein A beads (Sangon, Shanghai) were added to the supernatant and incubated for another 4 h. Then, the beads were collected through centrifugation and washed three times. After as much wash buffer as possible was removed, the beads were mixed with 30 µl of loading buffer and boiled for 5 min at 100 °C. The precipitates were examined using western blot.

GST pull-down assay

Cells were scraped, and proteins were extracted from the cells with 1 ml RIPA buffer. After centrifugation (4 °C, 12,000 rpm) for 20 min, the supernatants were divided into two tubes and immunoprecipitated with 5 µg of GST or GST-DSTYK fusion protein at 4 °C. The next day, Sepharose 4B beads (GE Healthcare) were added to the supernatants and incubated for another 4 h. Then, the beads were collected through centrifugation and washed three times. After as much wash buffer as possible was removed, the beads were mixed with 30 µl of loading buffer and boiled for 5 min at 100 °C. The precipitates were then examined using western blot.

Reporter assay

Cells at 70% confluence were plated in a 24-well plate. For each well, the cells were transfected with 0.01 µg of TOPFlash, 0.01 µg of TK Renilla, and 0.25 µg of DSTYK expression vector or empty vector. Forty-eight hours later, the cells were lysed with RIPA buffer, and reporter activity was examined using a dual reporter assay kit (Promega).

In vitro kinase assay

Fusion protein GST-β-catenin (N) (the N-terminal domain of β-catenin) was purified and incubated with Flag-DSTYK protein immunoprecipitated from a stable A549 cell line in the presence of kinase assay buffer and ATP (CST, 9802). The reaction was performed at 32 °C for 30 min and stopped by the addition of loading buffer. The phosphorylation of β-catenin was examined using western blot.

ChIP

ChIP was performed using a kit (Cell Signaling Technology, 9004) according to manufacturer instructions. The primers for qPCR were as follows: F, 5’-TCAAAACCAAGAAACTCAG-3’; F, 5’-GGGAAAGACCAAATGATTA-3’. qPCR was performed using a 2× SYBR qPCR mixture according to manufacturer instructions.

Lactate measurement

A lactate assay was performed using a kit (Abcam, ab65331). Briefly, cell culture medium was collected. The culture medium and standards were added to the wells of a plate, after which reaction mixture was added and incubated for 30 min at room temperature. The samples were analyzed with a microplate reader according to manufacturer instructions.

RNA sequencing

For RNA sequencing, A549 control cells and cells with the knockdown of DSTYK were harvested. The libraries were performed by Novogene (Tianjin, China), and an Illumina HiSeqX10 (Illumina, San Diego, CA, USA) was used for high-throughput sequencing. Quality control and removal of adapters of raw paired-end reads were performed using fastp (version 0.20.1). The gene expression counts were analyzed using HTseq-count software. The differently expressed genes were identified using DEseq2 (version 3.12) in R environment (version 3.6.3). Data were uploaded, and the GEO number is GSE178487.

Statistical analysis

All experiments in this study were performed in triplicate and error bars represent the standard deviation (±S.D) of triplicate samples. Statistical analysis was conducted using GraphPad Prism (version 7.0). Comparisons between groups were performed using two-tailed independent sample Student’s t-tests analysis. Data were expressed as mean ± S.D, P < 0.05 was considered a significant difference (*P < 0.05; **P < 0.01; ***P < 0.001).

Low DSTYK expression was observed in lung cancer

To clarify the expression pattern of DSTYK in lung cancer, we initially searched the Kmplot database for correlations between DSTYK expression and lung cancer patient survival. Bioinformatics analysis showed that DSTYK expression in lung cancer was positively correlated with patient survival time (Fig. 1A). Next, we detected the mRNA and protein levels of DSTYK in 30 lung cancer tissues and 30 paracancerous tissues by qPCR. The mRNA level of DSTYK was low in lung cancer (Fig. 1B), and immunohistochemistry indicated that the protein level of DSTYK was low in lung cancer (Fig. 1C, D). The KP (LSL-Kras^G12D;P53 ^loxp/loxp) mouse model is a classic model for investigating lung cancer. The western blot (Fig. 1E, up) and IHC (Fig. 1E, below) results showed that DSTYK expression levels was decreased in the tumorigenesis of KP mice (the mice were treated with Ad-Cre virus to induce the tumor). Finally, DSTYK expression in lung cancer cell lines was detected by Western blot. As shown in Fig. 1F, DSTYK was highly expressed in the normal pulmonary bronchial epithelial cell line Bease-2B (Fig. 1F). These results indicate that DSTYK expression is downregulated in lung cancer.

DSTYK inhibited the growth, colony formation, and sphere formation of lung cancer cells

Downregulated DSTYK expression in lung cancer implies that DSTYK plays an important role in lung cancer progression. We overexpressed DSTYK in A549 and SPC-A-1 cells to further explore the function of DSTYK in the progression of lung cancer (Fig. 2A). Figure 2B shows that DSTYK overexpression inhibited the growth of lung cancer cells in liquid medium. Anchorage-independent growth is a basic characteristic of tumor cells. Therefore, we evaluated the effect of DSTYK expression on the anchorage-independent growth of lung adenocarcinoma cells via soft agar colony formation and sphere formation assays. The results showed that DSTYK overexpression inhibited colony formation on soft agar (Fig. 2C, D) and sphere-forming abilities of lung cancer cells (Fig. 2E). However, when DSTYK expression was knocked down in A549 and SPC-A-1 cells (Fig. 3A), various functional tests indicated that DSTYK knockdown promoted growth (Fig. 3B) and colony formation (Fig. 3C, D) of A549 and SPC-A-1 cells. Moreover, knockout of the expression of DSTYK in the KP mouse model promoted tumorigenesis (Fig. 3E-H). These results confirm that DSTYK inhibits the progression of lung tumors.

Interaction between DSTYK and β–catenin in lung cancer cells

We detected the interactions among DSTYK and key components of the Wnt/β-catenin and TGFβ signaling pathways to explore the molecular mechanism by which DSTYK regulates lung cancer and found an interaction between exogenous DSTYK expression and β-catenin (Fig. 4A). A GST pull-down test showed the same result (Fig. 4B). Moreover, endogenously expressed DSTYK in lung adenocarcinoma cells interacted with β-catenin (Fig. 4C), and an interaction between the N-terminal domain of DSTYK and β-catenin was observed by immunoprecipitation (Fig. 4D).

DSTYK phosphorylated β-catenin and inhibited the Wnt/β-catenin signaling pathway

We first investigated the effect of DSTYK expression on the TOPFlash reporter to elucidate the influence of DSTYK on the Wnt/β-catenin signaling pathway. The results showed that DSTYK overexpression inhibited the Wnt3a-induced activation of the TOPFlash reporter (Fig. 5A), the mRNA levels of Axin2 and c-Myc (two targets of β-catenin) (Fig. 5B) and the accumulation of β-catenin (Fig. 5C). Similarly, DSTYK overexpression inhibited the expression of target genes downstream of β-catenin (Fig. 5D). Moreover, DSTYK overexpression elevated the phosphorylation level of β-catenin (Fig. 5E). Because DSTYK has serine/threonine kinase activity and interacts with β-catenin, we conducted an in vitro kinase reaction to detect the phosphorylation effect of DSTYK on β-catenin. The results indicate that DSTYK phosphorylated β-catenin in vitro (Fig. 5F).

LDHA is a target gene of the Wnt/β-catenin signaling pathway

Next, the RNA-seq was performed using A549 control cells and A549 cells with DSTYK knockdown, which suggested that knockdown of DSTYK upregulated the mRNA levels of LDHA (Fig. 6A, The GEO number is GSE178487). Therefore, we next verified the results of RNA-seq. It was found that the LDHA protein level was inhibited by DSTYK overexpression and upregulated by knockdown of DSTYK expression (Fig. 6B). Moreover, we found a binding site for β-catenin/TCF (TBE, TCF-binding element) in the LDHA promoter region and analyzed the LDHA promoter (Fig. 6C). After TBE was removed or mutated, β-catenin had no significant activation effect on the LDHA promoter (Fig. 6D). In addition, chromatin immunoprecipitation showed that β-catenin formed a complex with the LDHA promoter (Fig. 6E). These results suggest that LDHA is a target gene of the Wnt/β-catenin signaling pathway.

DSTYK exerts biological functions through Wnt/β-Catenin/LDHA signaling

We analyzed whether the biological function of DSTYK depends on the Wnt/β-catenin/LDHA signaling pathway. In clinical lung cancer specimens, DSTYK protein level was inversely correlated with the expression of β-catenin and LDHA (Fig. 7A). Moreover, the lactate content was upregulated in lung cancer cells in which DSTYK expression was disturbed; this upregulation could be restored by knockdown of β-catenin or LDHA expression (Fig. 7B). In terms of biological function, disturbing DSTYK expression promoted sphere formation, which could be restored in the manner stated above (Fig. 7C). These results indicate that the biological effects of DSTYK occur via Wnt/β-catenin/LDHA signaling.