tRNA-Uridine Aminocarboxypropyltransferase DTW Domain Containing 2 Suppresses Colon Adenocarcinoma Progression

Background DTW Domain Containing 2 (DTWD2) is a newly identified transfer RNA-uridine aminocarboxypropyltransferase. Dysregulated expression of DTWD1 has been reported in several malignancies, nevertheless, the role of DTWD2 in cancers remains completely unknown. Here, we aimed to initially investigate the expression and role of DTWD2 in colon adenocarcinoma. Methods We first evaluated the transcription and mRNA levels of DTWD2 using data from The Cancer Genome Atlas. Besides, we tested its mRNA and protein expression in our enrolled retrospective cohort. Univariate and multivariate analyses were conducted to assess its prognostic value. Cellular experiments and xenografts were also performed to validate the role of DTWD2 in colon cancer progression. Results DTWD2 was downregulated in colon adenocarcinoma and associated with poor prognosis. Lymph node metastasis, distant metastasis, and advanced tumor stage are all characterized by lower DTWD2 levels. Furthermore, Cox regression analysis demonstrated that DTWD2 is a novel independent prognostic factor for colon cancer patients. Finally, cellular and xenograft data demonstrated that silencing DTWD2 significantly enhanced colon cancer growth. Conclusion Low expression of DTWD2 may be a potential molecular marker for poor prognosis in colon cancer.


Introduction
Transfer RNA (tRNA) links a specific codon in mRNA with its corresponding amino acid during protein synthesis.Similar to proteins, tRNAs can be post-transcriptionally modified by various enzymes [1].For example, the 3-(3amino-3-carboxypropyl)uridine (acp3U) is a conserved modification in bacteria and eukaryotes.In humans, acp 3 U is identified at positions 20 and 20a in the D-loop of certain tRNAs [2].As for the upstream regulators, DTW Domain Containing 1 (DTWD1) is responsible for acp 3 U 20 modification, whereas DTWD2 was responsible for acp 3 U 20a [3].Therefore, DTWD proteins were also named as tRNA-Uridine aminocarboxypropyltransferases.
It is well-acknowledged that the defection of tRNA modification is responsible for numerous diseases, such as neurological disorders and malignancies [4].Therefore, dysregulated DTWDs are also involved in human diseases.For example, DTWD1 is speculated as a candidate gene in the development of the bipolar disorder and depressive disorder [5,6].Ma et al. reported that DTWD1 was a tumor suppressor in gastric cancer, which functioned by suppressing cyclin B1 expression and modulated by histone deacetylase 3 [7].Consistently, DTWD1 mRNA was significantly downregulated in breast cancer compared with non-cancerous breast tissues, suggesting its diagnostic significance [8].Another study also reported the correlation between DTWD1 expression and melanoma prognosis [9].Moreover, transcriptome data revealed DTWD1 as a critical gene for subgrouping clear cell renal cell carcinoma into different risk groups [10].
However, few studies reported the clinical relevance of DTWD2 since it has been a newly identified enzyme in the past decade.Copy number variation of DTWD2 was reported in primary open-angle glaucoma [11].The single nucleotide polymorphisms of DTWD2 may also be involved in scrub typhus according to bioinformatics analysis [12].Until now, there is no reported evidence suggesting the involvement of DTWD2 in any malignancy.
Colorectal cancer is recognized as one of the most prevalent malignancies globally [13].Despite advances in curative surgical resection and adjuvant therapies, the prognosis for regional advanced colon adenocarcinoma, specifically TNM stage III colon adenocarcinoma (COAD), remains suboptimal.Hence, the identification of specific biomarkers is not only crucial for prognostic prediction but also imperative for innovative therapeutic strategies.In this study, we have presented, for the first time, the involvement of DTWD2 in COAD.We have conducted a comprehensive analysis of the clinical relevance of DTWD2 in both The Cancer Genome Atlas (TCGA) cohort and a retrospective COAD cohort from our hospital.Additionally, we have explored the detailed function of DTWD2 in COAD progression through meticulous cellular experiments and mice models.The discovery of novel prognostic biomarkers carries significant significance in the context of chemotherapy and the risk of therapeutic failure in COAD [14].Chemotherapy, being a standard treatment for COAD, can face challenges, such as the development of resistance, leading to therapeutic failure, and disease progression [15].Hence, the identification of reliable prognostic biomarkers, such as DTWD2, has the potential to play a pivotal role in predicting treatment outcomes and guiding personalized therapeutic strategies for COAD patients [16].This highlights the urgent need for improved prognostic tools in the management of COAD.

Methods
2.1.Ethical Approval.Written informed consent was obtained from each participant.This study was approved by the Ethics Committee of Taizhou People's Hospital (No. KY 2022-169-01).

Patient Enrollment and Follow
Up.We retrospectively enrolled adult COAD patients who underwent surgical intervention in our hospital.The inclusion criteria included adult patients with COAD who underwent surgical intervention at Taizhou People's Hospital, and resected specimens confirmed as TNM stage III based on pathological tests.The exclusion criteria were patients who did not underwent radical surgery, lacked intact follow-up information, combined with other malignancy disease history, accepted preoperative chemotherapy or radiotherapy, and disagreed with the written informed consent.After exclusion, 176 cases were enrolled in the final cohort from our medical center (Taizhou, China).Besides, the mRNA expression level of DTWD2 was retrieved from TCGA datasets [17], and compared by Pearson's Chi-square test to evaluate its clinical relevance.

Protein Expression Test by Immunohistochemistry
Staining.Immunohistochemistry (IHC) staining was performed to evaluate the protein expression level of DTWD2 in COAD tissues.Formalin-fixed, paraffin-embedded specimens were cut into 4 μm slides, deparaffinized, and hydrated.Then, slides were subjected to epitope retrieval, followed by peroxidase incubation to block endogenous reactions.Specific anti-DTWD2 primary antibody (#PA5-62751, Thermo Fisher Scientific; 1 : 100 dilution) was used to incubate with specimen slides overnight at 4 °C.Slides were then incubated with horseradish peroxidase (HRP)-linked secondary antibody and subjected to diaminobenzidine staining, followed by counterstaining using Hematoxylin.IHC results were independently assessed by two pathologists to distinguish the high-DTWD2 expression or low-DTWD2 expression of each specimen.

Protein Expression
Test by Western Blotting.Total protein in cultured cells was extracted with radioimmunoprecipitation assay (RIPA) buffer supplemented with Phenylmethylsulfonyl fluoride (PMSF) and separated by Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and followed by transfer to PolyVinylidene DiFluoride (PVDF) membranes.The membranes were blocked with 5% FBS for 30 minutes at room temperature and incubated with the primary antibodies (anti-DTWD2, #PA5-65902, Thermo Fisher Scientific; anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), #2118, Cell Signaling Technology; both 1 : 1000 dilution) at 4 °C overnight.Afterward, the membranes were washed and then incubated with HRP-labeled anti-rabbit secondary antibody (#7074, Cell Signaling Technology. 1 : 10,000) for an additional 1 hour at room temperature.Membranes were finally developed using chemiluminescent HRP substrate [19].

Proliferation Test by Colony Formation. COAD cells
were cultured with a complete medium and seeded in 6-well plates (800 cells/well).Cells were cultured in routine culturing conditions for 14 days to allow colony growth.Afterward, cells were stained with 0.1% crystal violet for 30 minutes and washed [21].Clones with diameters larger than 1 mm were counted and compared.International Journal of Genomics COAD cells (n = 7) or control cells (n = 7) into the nude mice [22].The tumor size was monitored every three days.After three weeks, mice were sacrificed by intraperitoneal injections of 150 mg/kg sodium pentobarbital, and subcutaneous mice xenografts were resected.
2.9.Statistics.Cancer-specific survival (CSS) was defined as the time period from disease diagnosis to the date of COAD-related death or the date of last follow-up.Prognostic evaluation was performed using univariate log-rank analysis and multivariate Cox hazard regression analysis, respectively.The SPSS 20.0 and the GraphPad Prism 5.0 software were utilized for data analyses.P < 0 05 was defined as statistical significance.* indicates P < 0 05, ** indicates P < 0 01, and *** indicates P < 0 001.Consistently, patients with more advanced invasion depth, namely the T stage, showed lower DTWD2 expression levels (P < 0 001).

Survival Analysis.
Prognoses of the enrolled COAD cohort were assessed by CSS using univariate analysis (Table 3) and multivariate analysis (Table 4), respectively.As indicated by Kaplan-Meier univariate analysis, patients' survival was negatively correlated with their age at the time of diagnosis (P = 0 023).The CSS time of younger patients was 79 5 ± 2 9 months with a 5-year CSS rate of 80.5%, whereas was only 64 9 ± 3 7 months with a 5-year CSS rate of 68.5% (Figure 2(a); Table 3).In contrast with patients' age, our cohort did not find any significant survival difference between females and males (Figure 2(b); P = 0 175).
Although previous studies suggested an indispensable effect of tumor sidedness on COAD prognosis, our cohort did    ).In detail, patients with the N1 stage showed a 5-year CSS time of 72 4 ± 2 4 months and a 5-year CSS rate of 81.0%, whereas patients with the N2 stage showed a 5-year CSS time of 64 1 ± 5 3 months and a 5-year CSS rate of 57.5%.Our data also confirmed that chemotherapy treatment was beneficial for patients' CSS (Figure 2(h), P = 0 017).In the subgroup that accepted chemotherapy, patients' 5-year CSS time was 78 6 ± 2 8 months with a 5year CSS rate of 79.6%; whereas in the subgroup without chemotherapy or unsure about the treatment, patients' 5year CSS time was only 62 7 ± 4 0 months with 5-year CSS rate of 67.0%.Importantly, here we identified the significant effect of DTWD2 protein expression in COAD prognosis for the first time (Figure 2(i), P = 0 032).Patients with high-DTWD2 protein level showed a median 5-year CSS time of 78.5 ± 2.9 months and a 5-year CSS rate of 79.6%, whereas those with low-DTWD2 level showed a   4, poor differentiation grade and lower DTWD2 protein level were identified as two independent unfavorable risk factors for CSS of COAD.The hazard ratio of grade III-IV versus grade I-II was 2.25 (95% CI 1.11-4.57,P = 0 024).The hazard ratio of high DTWD2 versus low DTWD2 expression was 0.50 (95% CI 0.25-0.97,P = 0 040), indicating that DTWD2 may be a tumor-suppressive factor for COAD.

Clinical Relevance and Prognostic
Role of DTWD2-mRNA.Considering that our data was collected from a sin-gle medical center and only contained COAD cases with TNM stage III, we further investigated the role of DTWD2 in the TCGA cohort, which contains 41 normal colon specimens and 478 COAD specimens.In silico analysis firstly suggested that DTWD2-mRNA level was lower in COAD tissues than in normal colon tissues (Figure 3(a), < 0 001).Second, DTWD2-mRNA was negatively correlated with N stage, M stage, lymphovascular invasion, and disease mortality (Figures 3(b), 3(c), 3(d), and 3(e), all P < 0 05).Thirdly, Kaplan-Meier survival analysis indicated that low DTWD2-mRNA level was significantly correlated with worse overall survival of COAD (Figure 3(f), P = 0 038), which was consistent with our findings regarding its protein expression level.
3.5.DTWD2 Inhibits COAD Cell Proliferation.Based on the clinical data, we hypothesized that DTWD2 may exert tumor-suppressing effects in COAD.Therefore, we next conducted cellular experiments by ectopic expression of DTWD2.Plasmids containing the coding gene of DTWD2 were transfected into SW480 and SW620 cells, using scrambled vector plasmids as controls.After confirming the overexpressing efficiency of plasmids by western blotting (WB) (Figures 4(a) and 4(b)), cells were further subjected to phenotype analyses.For example, cell proliferation capacity was evaluated by MTT experiments, which demonstrated that overexpressing DTWD2 resulted in decreased cell proliferation (Figures 4(c) and 4(d)).Consistently, colony formation assay indicated that overexpressing DTWD2 could significantly attenuate the colony formation capacity of both SW480 and SW620 cells (Figures 4(e) and 4(f)).
3.6.DTWD2 Suppresses COAD Growth in Mice Model.Furthermore, we established a mice xenograft model in nude mice to validate the in vivo effects of DTWD2 during COAD progression.COAD cells with ectopic high expression of DTWD2 or control cells were subcutaneously injected into nude mice to monitor xenograft growth, which showed that DTWD2-overexpression inhibits COAD growth in mice model (Figures 5(a) and 5(b)).Three weeks later, all xenografts were resected and exhibited consistent size differences with growth curves above (Figures 5(c) and 5(d)).In addition, we tested the weights of all resected tumors and came to the same conclusion (Figures 5(e) and 5(f)).Finally, we lysed the resected tumors and tested protein expression level of DTWD2 to validate the overexpression efficiencies.As expected, xenografts generated by DTWD2-overexpressing cells showed significantly higher DTWD2 immunoreactivities than the control ones (Figures 5(g) and 5(h)).

Discussion
There are hundreds of tRNA modifications in mammals, which are similar to several well-known protein modifications, such as phosphorylation and ubiquitination [23].DTWD protein family refers to a specific protein family that can catalyze the formation of acp 3 U of tRNAs, an important post-translational modification of tRNA.Abnormal regulation of tRNA modifications leads to impaired cell growth [24].Previous studies suggested tumor-related roles of  8 International Journal of Genomics DTWDs in different malignancies.For example, DTWD1 has been discovered as a tumor suppressor, whose inactivating mutations had been identified in colorectal cancer [25].
Here, we initially discovered the significant role of DTWD2 in human cancer.According to the TCGA dataset and another COAD cohort from our medical center, we confirmed that DTWD2 was significantly downregulated in COAD specimens compared to non-tumorous colon tissues.Meanwhile, low mRNA levels or protein level of DTWD2 can help predict a worse prognosis of COAD cases.
Besides the novel finding regarding the prognostic predictive role of DTWD2 in COAD, our cohort revealed several interesting points in TNM stage III COADs.Firstly, patients' age affected the CSS of COAD.This is unexpected  9 International Journal of Genomics since most published studies reported the correlation between patients' age and OS instead of CSS.In our opinion, this finding is also reasonable because older age leads to decreased cancer immunity, subsequently resulting in a higher risk of disease relapse or heterochronous metastasis.
Moreover, our study provided the initial evidence that overexpressing DTWD2 could significantly attenuate COAD cell proliferation and colony formation capacities.The in vitro experimental data was further validated by in vivo mice experiments, highlighting that targeting DTWD2 may   International Journal of Genomics be a novel direction for COAD treatment, especially for those with dysregulated DTWD2 expression.
Our study has several limitations.First, the retrospective cohort from our hospital only contained TNM stage III COAD cases, thus our conclusion may only apply to COADs with locally advanced stages.However, we retrieved the DTWD2-mRNA information in the TCGA cohort which contains COAD cases in TNM stage I-IV.Of note, the major conclusion that lower DTWD2 predicted a worse prognosis was consistent in our stage III-COAD cohort and COAD-TCGA cohort.Therefore, the tumor-related role of DTWD2 in more malignancy types deserves further investigation.Second, despite our data confirming the tumor-suppressing effects of DTWD2 in COAD cells and mice models, its detailed signaling mechanisms remain unclear.Considering that transfected cancer cells exhibited a lower proliferation rate and a reduced colony formation capacity compared to the control group.We further examined several signaling pathways known to regulate cell proliferation, such as the MAPK/ERK and PI3K/Akt pathways, as well as cell cycle-related proteins, including cyclins and CDKs [26].Unfortunately, we did not observe any significant alteration of the abovementioned pathways (data not shown).To further dig into the potential downstream signaling mechanisms, high throughput screening experiments, such as RNA-sequencing and mass-spectrometry, will be necessary.In our hypothesis, as a tRNA-uridine aminocarboxypropyltransferase, DTWD2 may participate in tRNA post-transcriptional modification and play multiple functions in various aspects.WB may be incapable of detecting slight changes due to its low resolution.Furthermore, multipleomic strategies will be essential to map and elucidate the detailed signaling network of DTWD2 in tumor cells.

2. 8 .
Mice Model.BALB/c nude mice were obtained from the Shanghai Animal Center (Shanghai, China).The xenograft model was generated by subcutaneously injecting transfected 2

Figure 1 :
Figure 1: Immunoreaction of DTWD2 in tissue specimens.(a) Representative high protein expression of DTWD2 in adjacent nontumorous colon tissue.(b) Representative high protein expression of DTWD2 in COAD specimen.(c) Representative medium expression of DTWD2 in COAD.(d) Representative low expression of DTWD2 in COAD.(e) Representative negative expression of DTWD2 in COAD.Magnification: 400×.

Figure 4 :
Figure 4: DTWD2 exerts tumor-suppressing effects in COAD cells.(a, b) Overexpression of DTWD2 was achieved by transfecting its coding gene into SW480 and SW620 cell lines, using scrambled vector as control.The transfection efficiency was examined by WB. (c, d) To evaluate proliferation alterations of cells transfected with DTWD2 or vector, MTT method was conducted to plot their proliferation curves.(e, f ) Colony formation experiments were conducted to compare colony formation capacities of COAD cells transfected with DTWD2 or vector.

Figure 5 :
Figure 5: DTWD2 attenuates COAD growth in mice model.(a, b) Cells transfected with DTWD2 plasmids or vector plasmids were subcutaneously injected into nude mice to establish xenograft mice model.Then the growth curves were plotted every three days.(c, d) The images showed size difference of resected xenografts.(e, f) The weights of resected xenografts were tested and compared by unpaired Student's t-test.(g, h) WB was conducted to double-confirm DTWD2's expression difference in resected xenografts.

10
DTWD2 inhibits COAD progression by suppressing tumor growth both in vitro and in vivo.Low expression of DTWD2 in tumor tissues predicts worse clinical outcomes of COAD.

Table 2 :
Correlations between characteristics of TNM Stage III COAD patients and DTWD2 protein expression.

Table 3 :
CSS analyses of enrolled TNM Stage III COAD patients.

Table 4 :
CSS analyses of enrolled TNM Stage III COAD patients.