Suppressing the MLK3 promotes glutamine metabolism: mechanism and implications in progression of colon cancer

This study was designed to explore the potential role of mixed-lineage protein kinase 3 ( MLK3 ) in colorectal cancer (CRC) progression and its relationship with glutamine metabolism. The immunohistochemical staining results of MLK3 were primarily collected through 100 CRC patients. Wound healing and transwell assays were used to detect migration ability of CRC cells by transfecting cells with siMlk3 . Gene set variation analysis (GSVA) and Spearman’s rank correlation coefficient were used as bioinformatics tools to explore the signaling pathways related to MLK3 . Western blotting was performed to analyze the downstream of glutamine metabolism. The results suggested an increased expression of MLK3 in CRC tissues, which was related to adverse clinicopathological characteristics in those CRC patients. Knockdown of MLK3 inhibited the proliferative and migratory potential of CRCs. Bioinformatics analysis confirmed the relationship between MLK3 expression and cancer malignancy related signaling pathways. CRC cell lines transfected with siMlk3 suppressed glutamine metabolism by downregulating the glutamine transporter alanine-serine-cysteine transporter 2 (ASCT2). These results suggested the vital role of MLK3 in CRC progression, which may be related to the suppression of glutamine transporter, namely alanine, serine, cysteine transporter 2 (ASCT2).


Introduction
Colorectal cancer (CRC) is characterized by high migration capacity and poor prognosis, and is one of the most common cancer types globally [1].The 5-year relative survival rate for CRC is relatively poor, i.e., approximately 64% from 2009 to 2015 [2].Therefore, more effective biomarkers are needed for CRC prognosis.
Mixed lineage kinase 3 (MLK3), one of the mitogenactivated protein kinases (MAPK) members, has recently attracted attention as a chief adaptor molecule due to its role in inflammation and neurodegenerative diseases [3,4].It activates the MAPKs family, including c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK), extracellular-signaling regulated kinase (ERK), and p38 MAPK signaling pathways.Also, it can negatively regulate Ras homolog gene family member A (RhoA), and the guanosine triphosphate phosphohydrolase (GTPase), during disease progression [5].Increased MLK3 expression has been detected in various human cancers, including breast cancer, pancreatic cancer, as well as ovarian cancer tissues with undesirable outcomes [6][7][8].MLK3 expression was significantly upregulated in breast cancer cell lines as compared to adjacent normal tissues [9].Schroyer et al. [10] suggested a promoting role of MLK3 in CRC progression under oxidative stress.Notably, MLK3 is upregulated in various types of cancers and is closely associated with poor prognosis.Based on an immune-competent mouse model, Kumar et al. [11] demonstrated that MLK3 could be a potential therapeutic target in breast cancer by increasing T cell cytotoxicity.However, the role of MLK3 in CRC progression has not been fully elucidated.
It is widely acknowledged that glutamine metabolism is required for progression of cancer cells based on various biological processes [12], including biosynthesis, antioxidative defense, epigenetic and posttranslational modifications as well as regulation of cell signaling pathways [13].The intestinal mucosal cells are dependent on glutamine metabolism and rapidly undergo necrosis after glutamine depletion [14].Interfering the glutamine metabolism is a promising anti-cancer approach [15].Alanine-serinecysteine amino acid transporter (ASCT2), encoded by solute carrier family 1, member 5 (SLC1A5) gene, is important for transferring glutamine into the cellular microenvironment [16].Van et al. [17] suggested that knockdown of ASCT2 alone led to the death of breast cancer cells in vitro besides reducing the growth of xenografted cells in vivo.In kirsten rat sarcoma viral oncogene (KRAS)-mutant CRC, inhibiting the glutamine uptake by blocking ASCT2 suppresses the migration of malignant cells, suggesting ASCT2 as a potential therapeutic target [18].Therefore, it is important to explore the relationship between MLK3 and glutamine transporter ASCT2, as targeting MLK3 could suppress CRC progression partly by reducing glutamine intake.
Current study reports that MLK3 is highly expressed in CRC and its knockdown could decrease the malignancy of CRC cells by inhibiting the glutamine transporter ASCT2.

Bioinformatics analysis
Spearman's rank correlation coefficient analysis was used to detect MLK3 related signaling pathways.RNA-sequencing information was acquired from the cancer genome atlas (TCGA) dataset (https://portal.gdc.cancer.gov/).The enrichment analysis of MLK3 was performed based on the Kyoto encyclopedia of genes and genomes (KEGG) database (https: //www.kegg.jp)and Gene Ontology (GO) database (http: //geneontology.org/).

Collection of clinical samples
Subsequent collecting of fresh or after paraffin-embedding blocks of colorectal tissue was performed.A total of 100 patients enrolled in this study for colorectal cancer (CRC) surgical treatment from April 2008 to December 2008, and with a follow up of 6.6 to 7.2 years until July 2015 (for detailed information is provided in Supplementary Table 1).

Immunohistochemistry
Formalin-fixed and paraffin-embedded tissues were prepared for dewaxing, hydration and antigen extraction.After 3,3 ′ -Diaminobenzidine (DAB) staining, tissues were counterstaining with hematoxylin, and sealed with neutral silicone resin.The slides were observed under microscope.

Quantitative real-time PCR
Total RNA was extracted and quantified by the Qiagen RNeasy kit (74106, Qiagen Bioinformatics, Dusseldorf, Germany).Then, cDNA was obtained by the RT Reagent kit (RR037A, Takara Bio Lnc., Shiga, Japan).The primers used are shown in Supplementary Table 3.The relative amount of MLK3 mRNA was normalized to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Wound healing assay and transwell assay
When cells grew to 100% confluency, they were scratched by a 20 µL pipette tip, and photographed at 0 and 24 h, respectively.For the transwell migration assay (6.5 mm diameter, 8.0 mm pore size; Corning Inc.), the upper chamber was cultured with serum-free cultured cells, while lower chamber was loaded with 20% FBS medium for 48 h.Cells were enriched with methanol (30 min) and crystal violet for staining.The migrated cells were finally photographed and counted.

Statistical analysis
Statistical analysis was analyzed by GraphPad Prism 9 software (v9.0.0.121,GraphPad Software, San Diego, CA, USA) and R package.Kaplan-Meier survival curve and log-rank test was used to represent the overall survival rate with CRC patients.Data were analyzed by one-way analysis of variance (ANOVA) and presented as means ± standard deviations (SEM).It was considered statistically significant when p < 0.05.

MLK3 is upregulated in human CRC tissues
Primarily an immunohistochemical study was performed with MLK3 in collected samples.As compared to the normal subjects, MLK3 expression is significantly upregulated in colorectal cancer tissues (Fig. 1A), and positive staining of MLK3 was mostly observed in the nucleus of colorectal cancer.Furthermore, MLK3 protein level in CRCs was analyzed with various pathological markers.As shown in Fig. 1B, higher grade cancer samples expressed with higher expression of MLK3.The Kaplan-Meier survival result (n = 100) suggested that an increased MLK3 expression indicated an undesirable outcome as well (p = 0.0015, Fig. 1C).

MLK3 knockdown suppresses malignancy of CRC
Since elevated MLK3 expression is associated with poorer clinical outcomes, we examined functional effect of MLK3 in CRC progression by transfecting human RKO and SW480 cells with siMlk3.Transfection with siMlk3 suppressed the migration ability of CRC (p < 0.05) (Fig. 2A,B).To further explore the promoting role of MLK3 in CRCs stemness or epithelialmesenchymal transition (EMT) program, their related markers, including β-catenin, Oct4, Notch-1 and Nanog were measured.As shown in Fig. 2C, the expression of these markers decreased substantially after MLK3 knockdown.

MLK3 gene-mediated signaling pathways
To comprehend the role of MLK3 in CRC activity, a GSVA-based bioinformatic analysis was performed.The results revealed significant correlations between reactive oxygen species (ROS), EMT program, p53 pathway, tumor proliferation signature, MYC targets, degradation of extracellular matrix (ECM), and ferroptosis in group having elevated level of MLK3 (p < 0.05).Similarly, KEGG analysis suggested that MLK3 was highly enriched in the cyclic adenosine monophosphate (cAMP) signaling pathway, cell cycle and chemokine signaling pathway (Fig. 3).

The effect of MLK3 on ASCT2 expression
As shown in Fig. 4, knockdown of MLK3 in cancer cell lines suppressed expression of ASCT2 proteins.It indicates that MLK3 can target ASCT2 to exhibit a pro-tumor role in CRC progression.

Discussion
It is well recognized that mitogen-activated protein kinase (MAPKs) family plays a vital role in progression of cancer, e.g., cancer invasion and migration.Current study focused on the biological significance of MLK3, which is one of the mitogen-activated protein kinase (MAP3K) subfamily [19].Previous studies confirmed that MLK3 can regulate a broad range of cancer cell characteristics, including cellular invasion, EMT program and migration ability [5].However, few studies have addressed the effect of MLK3 with glutamine metabolism on biological behavior of CRC.
The immunohistochemical results in current study suggested a significant upregulation of MLK3 level in CRC tissues, indicating a direct relationship between increased expression of MLK3 and adverse prognosis.The potential correlation between age, sex, and the expression of MLK3 in colorectal cancer was not confirmed.In vitro experiments confirmed that MLK3 could stimulate stemness of cancer cells, EMT and migration capacity.To explore the role of MLK3 in CRC, Spearman's rank correlation coefficient was applied, which revealed its positive correlation with ROS, EMT-related markers, the p53 pathway, the tumor proliferation signature, and ferroptosis activity.Through illumina whole genome arrays, Velho et al. [20] revealed that MLK3 played a critical role in the progression of CRC related signaling pathways including MAPK, Wnt, TGF-β, p53 and Notch.Additionally, MLK3 exhibits ERK1/2-dependent phosphorylation under oxidative  stress, accelerating the invasion of CRC by activating the B-Raf and ERK1/2 in a positive loop [10].
Previous studies have uncovered the function of MLK3 in fatty acid metabolism, suggesting a positive regulatory role in saturated fatty acid metabolism by JNK activation in vivo.Meanwhile, MLK3-deficient mice showed improved insulin resistance and decreased hepatic steatosis [21].Current study concentrated on MLK3 and glutamine metabolism, which is an indispensable regulator in cellular metabolism.It can biosynthesize metabolites such as α-ketoglutarate (α-KG) and generate nicotinamide adenine dinucleotide (NADH) and flavine adenine dinucleotide, reduced (FADH 2 ) for adenosine triphosphate (ATP) needed for cell growth [22].In cancer cells, increased glutamic acid uptake and glutamine concentration have been widely observed, and glutamine deprivation leads to decreased survival rate [23], and apoptosis in glutamineaddicted cancers [24].Glutamine is imported from the microenvironment by the SLC family, and ASCT2 is considered as the primary one [25].A previous study suggested that ASCT2 can be a prognostic marker in KRAS-mutant CRC clinical specimens, and its overexpression is positively related with cancer invasion and migration [18].Besides, ASCT2 knockdown is involved in the apoptosis of cancer cells manifested by decreased BCL-2 levels or increased BAX levels [26].Current study showed that MLK3 knockdown led to the suppression of ASCT2 expression, indicating a pro-tumor role of MLK3 in glutamine metabolism (Fig. 5).Current research has some limitations.Firstly, the clinical subjects and cancer cell lines used in this study are less, which makes the results less convincing.Secondly, the detailed relationship between MLK3 and glutamine metabolism or ASCT2 needs further demonstration.Hence, further studies focused on more detailed mechanisms are necessary in the future to comprehend the mechanism of action in detail.

F I G U R E 5. Schematic diagram of the role of MLK3
in CRC progression.MLK3 can promote growth, invasion and migration ability of colorectal cancer cells (CRC).The potential mechanism may be due to its suppression with glutamine metabolism by directly targeting ASCT2.MLK3, mixed-lineage protein kinase 3; ASCT2, alanine-serinecysteine transporter 2.

Conclusions
The current study suggests an oncogenic role of MLK3, as increased expression of this gene signifies an independently undesirable prognosis in CRCs.Suppression of glutamine transporter ASCT2 can be the potential mechanism.

AVA ILABILITY OF DATA AND MATERIALS
Data will be available on request.

A UTHOR CONTRIBUTIONS
SQW and ZQS-designed the research study and methodology, wrote the manuscript.ZQS, KJW and RPS-contributed to formal analysis, contributed to data curation, revised the manuscript, supervised the study.All authors contributed to editorial changes in the manuscript, read and approved the final manuscript.

E THICS APPROVAL AND CONSENT TO PA R TICIPATE
This study was performed in line with the principles of the Declaration of Helsinki.Approval was granted by the Ethics Committee of the First People's Hospital of LinPing District (No. 20080102002).Informed consent was obtained from all individual participants included in the study.

ACK NOWLEDGMENT
Not applicable.

F UNDING
This work was supported by grants from the Excellent Subject Team Project of Jiamusi University (No. JDXKTD-2019002).

F I G U R E 1 .
Immunohistochemical staining of MLK3 in CRC specimens.(A) Representative images of MLK3 expression in CRC samples and normal tissues.(B) Representative images of MLK3 expression with various CRC status (scale bar, 500 µm and 100 µm).(C) MLK3 expression and overall survival rate.MLK3, mixed-lineage protein kinase 3.

F
I G U R E 3. MLK3 associated signaling pathways in CRC progression.(A) Increased expression of MLK3 was correlated with ROS, EMT markers, p53 pathway, cancer proliferation signature, MYC targets, collagen formation, degradation of EMC and ferroptosis.(B) KEGG analysis suggested that MLK3 was highly enriched in cAMP, cell cycle and chemokine signaling pathway.