GALNT2 targeted by miR-139-5p promotes proliferation of clear cell renal cell carcinoma via inhibition of LATS2 activation

Polypeptide N-Acetylgalactosaminyltransferase (GALNTs) are critical enzymes that initiate mucin type-O glycosylation, and are closely associated with the occurrence and development of multiple cancers. However, the significance of GALNT2 in clear cell renal cell carcinoma (ccRCC) progression remains largely undetermined. Based on public multi-omics analysis, GALNT2 was strongly elevated in ccRCC versus adjoining nontumor tissues, and it displayed a relationship with poor overall survival (OS) of ccRCC patients. In addition, GALNT2 over-expression accelerated proliferation of renal cancer cell (RCC) lines. In contrast, GALNT2 knockdown using shRNAs suppressed cell proliferation, and this was rescued by LATS2 knockdown. Similarly, GALNT2 deficiency enhanced p-LATS2/LATS2 expression. LATS2 is activated by phosphorylation (p-LATS2) and, in turn, phosphorylate the downstream substrate protein YAP. Phosphorylated YAP (p-YAP) stimulated its degradation and cytoplasmic retention, as it was unable to translocate to the nucleus. This resulted in reduced cell proliferation. Subsequently, we explored the upstream miRNAs of GALNT2. Using dual luciferase reporter assay, we revealed that miR-139-5p interacted with the 3ʹ UTR of GALNT2. Low miR-139-5p expression was associated with worse ccRCC patient outcome. Based on our experiments, miR-139-5p overexpression inhibited RCC proliferation, and this phenotype was rescued by GALNT2 overexpression. Given these evidences, the miR-139-5p-GALNT2-LATS2 axis is critical for RCC proliferation, and it is an excellent candidate for a new therapeutic target in ccRCC. Supplementary Information The online version contains supplementary material available at 10.1007/s12672-024-00930-4.


Introduction
Renal cell carcinoma (RCC) is a globally widespread malignancy, with the 3rd highest ranking among common urologic cancers.It is only inferior to prostatic and bladder cancers.Clear cell renal cell carcinoma (ccRCC) is the most prevalent histological form of RCC, comprising over 75% of all incidences, and it is the most lethal urological malignancy [1].Localized ccRCC is typically treated with early surgical resection.However, in case of advanced ccRCC, efficacious treatment is lacking [2].The median survival of advanced kidney cancer sufferers is less than 12 months and the 5-year overall survival (OS) rate of the same cohort is no higher than 5% [3].Hence, it is both urgent and critical to examine the mechanisms associated with ccRCC carcinogenesis and progression, as well as explore potential therapeutic targets for improving prognosis of ccRCC patients.
Polypeptide GalNAc Transferase 2 (GALNT2), otherwise known as Polypeptide N-Acetylgalactosaminyltransferase 2 (GalNAc-T2), encodes a member of the glycosyltransferase protein family, which initiates mucin-type O-glycosylation of peptides within the Golgi apparatus [4].Several investigations reported an association between GALNT2 and cancer progression.In colorectal cancer, GALNT2 modified O-glycans on AXL was shown to enhance colorectal cancer infiltration [5].In non-small cell lung cancer, GALNT2 enhances cell proliferation, migration, and infiltration via the O-glycosylation of ITGA5 [6].GALNT2 also facilitates cell proliferation, migration, and infiltration through the O-glycosylation and phosphorylation of EGFR in the glioma [7].However, the precise molecular mechanism underlying the GALNT2-mediated ccRCC progression is not fully elucidated.
The Hippo axis is a highly conserved modulator of cellular growth, proliferation, stem cell function, tissue regeneration, homeostasis, and organ size control [8].LATS1/2 are critical components of the Hippo axis that inversely modulates the downstream effectors, YAP and TAZ.The Hippo axis dysregulation is strongly associated with multiple cancers [9].YAP is a transcriptional co-activator, which induces platinum resistance in ovarian tumors [10].Disturbances in LATS1 and YAP1 expressions are associated with worse ccRCC patient OS [11].Prior investigations revealed that YAP O-GlcNAcylation is essential for high-glucose induced liver tumorigenesis [12].However, there are no reports of a GALNT2 and LATS2 association.
MicroRNAs (miRNAs) are evolutionarily conserved single-stranded noncoding RNAs that are 18-22 nucleotides in length and negatively modulate gene expression via degradation of target mRNAs or repression of mRNA translation [13].Owing to the short-binding motif of miRNAs, one miRNA can effectively silence several genes.MicroRNAs heavily contribute to diverse biological phenomena, namely, cell apoptosis, proliferation, differentiation, and immunity [14,15].Emerging evidences suggest a strong role of miRNAs in tumorigenesis, cancer progression, and aggressiveness [16].Thus, herein, we identified potential miRNAs that serve as upstream modulators of GALNT2.
Herein, we detailed a new modulatory mechanism associated with ccRCC progression.We demonstrated that diminished miR-139-5p levels increased GALNT2, which resulted in worse ccRCC patient OS.Furthermore, we revealed that decreased miR-139-5p and/or elevated GALNT2 enhanced RCC proliferation.Our analysis confirmed that the miR-139-5p-GALNT2-LATS2 axis was potentially critical for the ccRCC oncogenesis and progression.

MTT cell proliferation and cytotoxicity assay
Cell survivability was assessed via the MTT Cell Proliferation and Cytotoxicity Assay Kit (Solarbio, M1020).Approximately 3000 cells/well were seeded in 96-well plates, and incubated at 37 °C in a 5% CO 2 incubator for approximately 24, 48, 72, and 96 h prior to treatment with specified compounds.Following treatment, 20 μl MTT solution was introduced to the cells, followed by a 4 h incubation at 37 °C in a 5% CO 2 incubator.The reaction was terminated by removing the media, then 150 μl dimethylsulfoxide (DMSO) was introduced and optical density was determined using Spectramax (Molecular Devices) at 490 nm.All samples were assayed thrice.

Colony formation assays
Cells were maintained on 6-well plates (200 cells per well), with fresh medium every 3rd day for 14 days.Next, they underwent a 15 min fixation in 4% paraformaldehyde (PFA), and staining with crystal violet.They were then imaged and quantified.

Luciferase assay
The dual-luciferase vectors were acquired from Jiangsu Dongxuan Gene Technology Co. Ltd., and harbored the 3ʹ-UTR of GALNT2 with seed sequences.The corresponding mutant vectors were generated by ligating the 3-bp mutations with the aforementioned seed sequences.Next, the GALNT2/mutant vectors and miR-139-5p mimics were co-incorporated into Caki-1 and OS-RC-2 cell lines by Gene Pharma (Gene Pharma; Shanghai GenePharma Co. Ltd., CHINA).Luciferase activity quantification was performed 48 h later with the use of Dual-Luciferase Reporter Assay System (Molecule Device, Molecular Devices, LLC., U.S.A.).

Statistical analysis
All experiments were performed in three biological replicates.Data were analyzed using SPSS 16.0, and are provided as mean ± SD.Inter-group assessments utilized the unpaired Student's t-test.GALNT2 content and clinicopathological features were assessed using the χ 2 test.Survival curves were plotted and assessed following the Kaplan-Meier technique and the log-rank test, respectively.Association between GALNT2 and miR-139-5p contents was assessed via the Spearman's correlation coefficient.ns p > 0.05.was not significant.*p < 0.05, **p < 0.01, ***p < 0.001 were set as the significance threshold.

GALNT2 overexpression augmented RCC cell proliferation
To further elucidate the physiological mechanisms behind the GALNT2-mediated acceleration of ccRCC progression, we examined the GALNT2 transcript levels in RCC cell lines and renal proximal tubular epithelial cell using RT-qPCR (Fig. 2A).We overexpressed GALNT2 in different RCC lines in vitro (Fig. 2B).Then, using MTT, colony formation, and Brdu assays,

GALNT2 modulated LATS2 activation in ccRCC
To explore the mechanism behind the GALNT2-mediated regulation of cell proliferation, we examined the downstream signaling pathways.We acquired the GALNT2 protein expression data, in correlation with multiple signaling network, from the UALCAN database.The Hippo pathway showed the most significant p-value, and was therefore chosen for further analysis.The GALNT2 proteomic expression profile in UALCAN indicated that dysregulation of the Hippo pathway was related to GALNT2 levels in ccRCC (Fig. 3A).Next, we knocked down GALNT2 in RCC lines (Fig. 3B), and assessed Hippo pathway-relevant key protein expressions.We revealed that the LATS2 and p-YAP proteins were strongly enhanced in the GALNT2-knockdown group (Fig. 3C).In addition, phosphorylated LATS2 (p-LATS2) is the active form, and the p-LATS2 protein was markedly enhanced in the GALNT2-knockdown group (Fig. 3D).Next, using MTT, colony formation, and Brdu assays, we revealed that the GALNT2 knockdown severely suppressed cell proliferation.However, LATS2 knockdown rescued this phenotype (Fig. 3E-G).Collectively, these evidences suggested that GALNT2 reduced LATS2 activation to enhance RCC proliferation.

Discussion
Glycosylation was a widespread modification in various cellular and biological processes.GALNT2 belonged to the GalNAc-type O-glycosylation gene family, and modulated multiple forms of cancers.Glycosyltransferases were excellent prognostic bioindicators of pan-cancer patient OS [20].The contents of varying GalNAc-transferases were potentially relevant to the progression and poor outcome of epithelial ovarian cancer patients [21].GALNT2 modified O-glycosylation and EGFR activity enhanced oral squamous cell carcinoma migration and invasion [22].GALNT2 also activated the Notch/Hes1-PTEN-PI3K/Akt axis to induce cell proliferation, migration, and invasion in lung adenocarcinoma [23].Despite these reports, the mechanism underlying GALNT2 action remained unclear in ccRCC progression.Herein, we demonstrated that GALNT2 was ubiquitous among ccRCC tissues, and strongly associated with poor ccRCC patient outcome.We next examined the significance of GALNT2 in ccRCC progression.Elevated GALNT2 levels accelerated RCC proliferation in a series of in vitro experiments.Then elevated GALNT2 levels stimulated the Hippo pathway to enhance RCC proliferation.GALNT2 knockdown marked enhanced LATS2, p-LATS2 and p-YAP levels.Moreover, GALNT2 knockdown strongly suppressed cell proliferation, and this was rescued by LATS2 knockdown.Our analysis identified LATS2 as a key molecule that was targeted by GALNT2 in ccRCC progression.Unfortunately, owing to limitations in experimental condition, we failed to identify the direct interaction sites between GALNT2 and LATS2.However, we have several possible hypotheses.LATS2 may be glycosylated by GALNT2, which, in turn, may inhibit degradation or induce activation of LATS2.However, one study revealed that O-GlcNAcylation of LATS2 by OGT (O-Linked N-Acetylglucosamine (GlcNAc) Transferase) strongly inhibits its activity [24].GALNT2 (GalNAc-Transferase) and O-GlcNAcylation (GlcNAc Transferase) may ecounter competitive inhibition.Alternately, the Hippo pathway-related upstream proteins may be glycosylated and activated by GALNT2, which may result in LATS2 activation.LATS1/2 and YAP/TAZ are key molecules in ccRCC progression.Prior investigation revealed that LATS1/2 and active YAP dysregulation are sufficient to initiate renal tumor formation in mice [25].Overall, LATS2 is a potentially promising target of GALNT2 in ccRCC progression.MiR-139-5p exhibited scarce expression in ccRCC tissues, and indicated a poor ccRCC prognosis.Based on our dual luciferase reporter assay analysis, miR-139-5p interacted with the 3ʹ UTR of GALNT2.Hence, it was identified as an upstream modulatory factor of GALNT2.MiR-139-5p is a tumor suppressor, and its expression is strongly diminished in the vast majority of human tumors [26].MiR-139-5p targeted GABRA1 to suppress glioma cell proliferation [27].Exosomal miR-139-5p suppressed bladder cancer malignancy by targeting PRC1 [28].MiR-139-5p was also a tumor suppressor, which targets TOP2A and PXN in RCC [29,30].Herein, we presented GALNT2 as a new target of miR-139-5p, thereby interpreting a potential mechanism of cell proliferation in ccRCC.Based on this, we reasoned that the miR-139-5p-GALNT2-LATS2 axis accelerated cell proliferation in ccRCC (Fig. 5).Based on our findings, the 3ʹ-UTR of several Hippo pathway-related proteins, namely, STK3, STK4, LATS1, and YAP1, potentially bind miR-139-5p (Additional file 2).In our future investigation, we plan to further explore the modulatory role between miR-139-5p and the Hippo pathway.
In this paper, OS and DSS indicators were used to evaluate the impact of GALNT2 on ccRCC prognosis.However, disease specific survival (DSS) was not collected by the TCGA study.The source of DSS came from a publication [31] that estimated DSS data, and the authors of that study cautioned on the utilization of this data point, depending on the specific tumor type.Therefore, the DSS was estimated and that this was a limitation of this study.
Tumor cells were controlled by multiple regulatory mechanisms during initiation and progression.In this study, we found that GALNT2 acted as a key regulator for the initiation and progression of ccRCC.In further mechanistic studies, we found that the disorder of the miR-139-5p-GALNT2-LATS2 regulatory axis were widespread in ccRCC and play important roles in the cell proliferation of ccRCC.Our study provided insight into new cancer therapeutic strategies for ccRCC targeting the miR-139-5p-GALNT2-LATS2 axis.

Conclusions
GALNT2 was highly expressed in tumor versus NT samples.Hence, GALNT2 may be a robust stand-alone indicator of worse ccRCC patient OS.Using functional and molecular experiments, we revealed that GALNT2 accelerated RCC proliferation in vitro, as well as tumor growth in vivo.We also identified LATS2 as a key downstream signaling

Fig. 1
Fig. 1 GALNT2 was elevated in ccRCC tissues, and was correlated with worse patient outcome.A-C GALNT2 mRNA levels in tumor and adjoining normal tissues (NTs) in TCGA, GSE105261, and GSE66276.D GALNT2 protein expressions in tumor and NTs in UALCAN.E The single cell GALNT2 content in kidney in HPA.F The single cell GALNT2 content in varying ccRCC stages in Single cell Portal.Red circle indicated single cell of late stage ccRCC; blue circle indicated single cell of early stage ccRCC.G GALNT2 protein expression in tumor and adjoining normal tissues in HPA, as evidenced by immunohistochemistry. H The Sankey diagram display of the proportions of TNM stages in TCGA.I-K The GALNT2 expression in TNM stages in TCGA.L Elevated GALNT2 expression correlates with poor overall survival (OS) and disease-free survival (DFS) in GEPIA, as evidenced by univariate analysis

Fig. 2 4 Fig. 3
Fig. 2 GALNT2 overexpression induced RCC cell proliferation in vitro.A GALNT2 expression analysis in RCC cell lines via RT-qPCR.B GALNT2 expression analysis in RCC cell lines via Western blot.C MTT cell proliferation assay.D Colony formation assay.E Brdu cell proliferation assay.F Flow cytometric analysis of cell apoptosis