Role of Nudt2 in Anchorage-Independent Growth and Cell Migration of Human Melanoma

Simple Summary Melanoma is one of the deadliest types of skin cancer, accounting for the majority of skin cancer-related deaths. The function of Nudt2 in melanoma is unknown. The goal of this study was to examine the role of Nudt2 in melanoma cells and in vivo model. In melanoma, Nudt2 appears to be a tumor-promoting gene that could be utilized as a cancer therapy target. Abstract Nudt2 encodes a diadenosine tetraphosphate (Ap4A) hydrolase that catalyzes the hydrolysis of Ap4A and is involved in the lysyl tRNA synthetase-Ap4A-Nudt2 (LysRS-Ap4A-Nudt2) signaling pathway. We have previously demonstrated that this pathway is active in non-small cell lung cancer. Nudt2 was shown to be involved in cell proliferation in breast cancer, making it an important target in cancer therapy. Currently, the function of Nudt2 in malignant melanoma has not been demonstrated. Therefore, we investigated the role played by Nudt2 in the growth of human melanoma. Our study showed that Nudt2 knockdown suppressed anchorage-independent growth of human melanoma cells in vitro. The in vivo effect of Nudt2 was determined by investigating the role played by Nudt2 knockdown on the ability of the cells to form tumors in a mice xenograft model. Nudt2 knockdown significantly suppressed tumor growth in this model. Moreover, overexpression of Nudt2 resulted in an increase in anchorage-independent growth of these cells, whereas Nudt2 knockdown decreased their migration. In addition, Nudt2 knockdown reduced vimentin expression. Vimentin is one of the mesenchymal markers that are involved in the epithelial mesenchymal transition (EMT) process. Thus, Nudt2 plays an important role in promoting anchorage-independent growth and cell migration in melanoma.


Introduction
Nudix (nucleoside diphosphate linked moiety X)-type motif 2 (Nudt2) is a member of the MutT family of nucleotide pyrophosphatases, a subset of the larger Nudix hydrolase family which is widespread among bacteria, eukaryotes, and viruses [1]. Nudt2 encodes an Ap 4 A hydrolase that is involved in the hydrolysis of Ap 4 A (diadenosine 5 , 5 -p1, p4tetraphosphate) to AMP and ATP, thus regulating the intracellular level of Ap 4 A. Ap 4 A is found in all living cells, both prokaryotic and eukaryotic, and studies conducted in our lab have previously identified it as a bona fide second messenger [2][3][4][5]. It is a small molecule composed of two adenosine moieties joined in a 5 -5 linkage by a chain of four phosphates and was discovered as an in vitro synthesis product of lysyl-tRNA synthetase (LysRS) [6]. In activated eukaryotic cells, Ap 4 A is synthesized by serine 207 phosphorylated LysRS as part of the S 207 LysRS-Ap 4 A signaling pathway that was discovered and characterized by 2 of 10 our team [3,[7][8][9]. Our studies showed that in activated cells both microphthalmia transcription factor (MITF) and upstream stimulating factor 2 (USF2) are positively regulated by Ap 4 A, allowing the transcription of their respective target genes [4,5] (Scheme 1). Ap 4 A has been implicated in the function of many cellular process including apoptosis [10], DNA damage response [11], and many other signaling pathways [12,13]. Nudt2 promotes proliferation of breast carcinoma cells in vitro, and is a potent prognostic factor in human breast carcinomas, under a different mechanism of estrogen [14]. RNAseq analysis in KBM-7 chronic myelogenous leukemia cells with or without Nudt2 knockdown indicated Nudt2 involvement in cell proliferation, invasion and metastasis [15]. Knockdown of Nudt2 suppressed proliferation of several breast carcinoma cell lines by regulating mTROC1 activity via physical interaction with RagGTPases [16], suggesting that Nudt2 inhibition could have strong anti-tumor effects. Furthermore, we have shown that LysRS is phosphorylated on serine 207 in EGFR-mutated non-small cell lung cancer, and that this phosphorylation is correlated with increased disease-free survival [17]. We have also demonstrated that the LysRS-Ap 4 A pathway is active in various melanoma cell lines [2]. During the process of epithelial mesenchymal transition (EMT), epithelial markers, such as E-cadherin, will decrease and there will be an increase in mesenchymal markers, such as vimentin, N cadherin, and matrix metalloproteases (MMPs), in addition to the involvement of many transcription factors that regulate this process including snail, slug, and twist [18]. characterized by our team [3,[7][8][9]. Our studies showed that in activated cells both microphthalmia transcription factor (MITF) and upstream stimulating factor 2 (USF2) are positively regulated by Ap4A, allowing the transcription of their respective target genes [4,5] (Scheme 1). Ap4A has been implicated in the function of many cellular process including apoptosis [10], DNA damage response [11], and many other signaling pathways [12,13]. Nudt2 promotes proliferation of breast carcinoma cells in vitro, and is a potent prognostic factor in human breast carcinomas, under a different mechanism of estrogen [14]. RNAseq analysis in KBM-7 chronic myelogenous leukemia cells with or without Nudt2 knockdown indicated Nudt2 involvement in cell proliferation, invasion and metastasis [15]. Knockdown of Nudt2 suppressed proliferation of several breast carcinoma cell lines by regulating mTROC1 activity via physical interaction with RagGTPases [16], suggesting that Nudt2 inhibition could have strong anti-tumor effects. Furthermore, we have shown that LysRS is phosphorylated on serine 207 in EGFR-mutated non-small cell lung cancer, and that this phosphorylation is correlated with increased disease-free survival [17]. We have also demonstrated that the LysRS-Ap4A pathway is active in various melanoma cell lines [2]. During the process of epithelial mesenchymal transition (EMT), epithelial markers, such as E-cadherin, will decrease and there will be an increase in mesenchymal markers, such as vimentin, N cadherin, and matrix metalloproteases (MMPs), in addition to the involvement of many transcription factors that regulate this process including snail, slug, and twist [18].
Scheme 1. LysRS-Nudt2-Ap4A-Hint-1-MITF pathway in activated mast cells. Following mast cell activation, LysRS is phosphorylated on the serine 207 residue through the MAPK pathway. This phosphorylation dissociates LysRS from the MultiSynthetase complex (MSC), which then translocate into the nucleus. The phosphorylated form of LysRS at serine 207 can produce an Ap4A inside Scheme 1. LysRS-Nudt2-Ap4A-Hint-1-MITF pathway in activated mast cells. Following mast cell activation, LysRS is phosphorylated on the serine 207 residue through the MAPK pathway. This phosphorylation dissociates LysRS from the MultiSynthetase complex (MSC), which then translocate into the nucleus. The phosphorylated form of LysRS at serine 207 can produce an Ap4A inside the nucleus. Ap4A will bind Hint-1 and MITF complex cause a dissociation of Hint-1 from MITF. Upon dissociation, MITF will be able to transcribe its target genes. Upon mast cell activation, Ap4A hydrolase (Nudt2) will associate with importin beta and it will translocate into the nucleus. The presence of Ap4A hydrolase in the nucleus leads to hydrolysis of Ap4A into AMP and ATP decreases its levels. When Ap4A level is decreased, Hint-1 will associate again with MITF, leading to the resting state in the cells.
These observations indicate that the Nudt2 may have profound importance in melanoma proliferation. Thus, the main goal of the present study is to explore the role played by Nudt2 in melanoma cell function by using in vitro assays and the xenograft model. In addition, we followed the effect of Nudt2 on EMT by checking vimentin expression.

Generation of Stable Nudt2 Knockdown Melanoma Cell Lines
We have recently identified that the LysRS-Ap 4 A-Nudt2 pathway is involved both in non-small cell lung cancer and melanoma. Here, we investigated whether Nudt2 plays a role in melanoma cell growth by first lowering Nudt2 levels in these cells. Control and Nudt2-knocked down human melanoma cell line (CHL−1) were produced using either non-targeting or Nudt2-specific shRNA lentiviral particles. The stable knockdown of Nudt2 was verified in this cell line via the Western blot analysis and mRNA expression level was verified using real-time PCR (Figure 1 and Figure S1). the nucleus. Ap4A will bind Hint-1 and MITF complex cause a dissociation of Hint-1 from M Upon dissociation, MITF will be able to transcribe its target genes. Upon mast cell activation, A hydrolase (Nudt2) will associate with importin beta and it will translocate into the nucleus presence of Ap4A hydrolase in the nucleus leads to hydrolysis of Ap4A into AMP and AT creases its levels. When Ap4A level is decreased, Hint-1 will associate again with MITF, leadi the resting state in the cells.
These observations indicate that the Nudt2 may have profound importance in m noma proliferation. Thus, the main goal of the present study is to explore the role pl by Nudt2 in melanoma cell function by using in vitro assays and the xenograft mod addition, we followed the effect of Nudt2 on EMT by checking vimentin expression.

Generation of Stable Nudt2 Knockdown Melanoma Cell Lines
We have recently identified that the LysRS-Ap4A-Nudt2 pathway is involved bo non-small cell lung cancer and melanoma. Here, we investigated whether Nudt2 pl role in melanoma cell growth by first lowering Nudt2 levels in these cells. Control Nudt2-knocked down human melanoma cell line (CHL−1) were produced using e non-targeting or Nudt2-specific shRNA lentiviral particles. The stable knockdow Nudt2 was verified in this cell line via the Western blot analysis and mRNA expres level was verified using real-time PCR (Figures 1 and S1).

Nudt2 Is Required for Anchorage-Independent Growth of Melanoma Cells
Tumorigenesis and carcinogenesis require the acquisition of many tumor feat including increased cell growth rate and anchorage-independent growth potential The soft agar colony formation assay is an in vitro assay that is used to assess tumor esis capacity. In this assay, transformed cells gain the ability to expand in the absen anchorage environment. The colony formation assay was used to assess the effe Nudt2 knockdown on anchorage-independent development. Nudt2 knockdown resu in a reduction in colony number and size CHL−1 cell line ( Figure 2A). The numb colonies ( Figure 2B) decreased by 88% in CHL−1 cell (p = 0.0312). These findings sho that Nudt2 plays an important role in promoting anchorage-independent melanom mor cell growth and tumorigenesis.

Nudt2 Is Required for Anchorage-Independent Growth of Melanoma Cells
Tumorigenesis and carcinogenesis require the acquisition of many tumor features, including increased cell growth rate and anchorage-independent growth potential [19]. The soft agar colony formation assay is an in vitro assay that is used to assess tumorigenesis capacity. In this assay, transformed cells gain the ability to expand in the absence of anchorage environment. The colony formation assay was used to assess the effect of Nudt2 knockdown on anchorage-independent development. Nudt2 knockdown resulted in a reduction in colony number and size CHL−1 cell line ( Figure 2A). The number of colonies ( Figure 2B) decreased by 88% in CHL−1 cell (p = 0.0312). These findings showed that Nudt2 plays an important role in promoting anchorage-independent melanoma tumor cell growth and tumorigenesis.

Nudt2 Knockdown Suppress Xenograft Tumor Growth In Vivo
In order to explore whether Nudt2 knockdown affects tumor growth in vivo, we a xenograft tumor model. For this purpose, we prepared luciferase-expressing cells Nudt2 knockdown and control cells by infecting the cells with lentiviral particles con luciferase-expressing vector; luciferase will help us to monitor tumor growth in in imaging as mentioned in detail in the Materials and Methods section. NOD/SCID were subcutaneously injected with either control CHL − 1 melanoma cells or N knocked down IVIS; the analysis revealed a significant difference in tumor growth tween the control and Nudt2 knockdown groups (p = 0.032; Figure 3D,F). The tumo mice injected with CHL−1 Nudt2 knockdown cells grew at a slower rate and had a sm volume than the tumors in mice injected with control melanoma cells ( Figure 3G,H) mor volume in the Nudt2 knockdown group was lower than that of the control gr with a reduction of 96%. The difference in tumor volume between the control and N knockdown groups was significant, with a p value of 0.006. These results suggest Nudt2 suppressed tumor growth in this in vivo xenograft model.

Nudt2 Knockdown Suppress Xenograft Tumor Growth In Vivo
In order to explore whether Nudt2 knockdown affects tumor growth in vivo, we used a xenograft tumor model. For this purpose, we prepared luciferase-expressing cells from Nudt2 knockdown and control cells by infecting the cells with lentiviral particles contains luciferase-expressing vector; luciferase will help us to monitor tumor growth in in vivo imaging as mentioned in detail in the Materials and Methods section. NOD/SCID mice were subcutaneously injected with either control CHL−1 melanoma cells or Nudt2 knocked down IVIS; the analysis revealed a significant difference in tumor growth between the control and Nudt2 knockdown groups (p = 0.032; Figure 3D,F). The tumors in mice injected with CHL−1 Nudt2 knockdown cells grew at a slower rate and had a smaller volume than the tumors in mice injected with control melanoma cells ( Figure 3G,H). Tumor volume in the Nudt2 knockdown group was lower than that of the control group, with a reduction of 96%. The difference in tumor volume between the control and Nudt2 knockdown groups was significant, with a p value of 0.006. These results suggest that Nudt2 suppressed tumor growth in this in vivo xenograft model.

Nudt2 Overexpression Increased Anchorage-Independent Growth in a Melanoma Cell Line
Recent research supports Nudt2's role in the regulation of cellular proliferation in breast cancer [14,16]. The purpose of this study was to find out whether Nudt2 has an effect on anchorage-independent growth in melanoma cell lines. To investigate this, lentiviral infection of human melanoma CHL−1 was used to produce stable cell lines overexpressing wild-type (WT) Nudt2 by using PLX304-V5-Nudt2 expressing vector. As a control, infection with an empty vector (PLX304-EV) was used. Western blot was used to compare the expression levels of WT vs. EV. V5 antibody was used to check the overexpression of Nudt2, which is tagged to V5 in the expression vector: it showed one band in overexpressed cells compared with none in control. When Nudt2 antibody was used, two bands were detected in overexpressed cells, corresponding to V5−Nudt2 fusion and to endogenous Nudt2. In control, only endogenous Nudt2 was detected ( Figure 4A). The colony formation assay was used to determine the effect of WT Nudt2 overexpression (PLX304−V5−Nudt2) on anchorage-independent growth ( Figure 4B and Figure S2). Overexpression of WT Nudt2 increased the colony number and colony size in CHL−1 cell compared to cells transfected with EV (PLX304−EV). These findings suggest that Nudt2 is important to support anchorage-independent growth in human melanoma cells.

G. H.
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Nudt2 Overexpression Increased Anchorage-Independent Growth in a Melanoma Cell Line
Recent research supports Nudt2's role in the regulation of cellular proliferation in breast cancer [14,16]. The purpose of this study was to find out whether Nudt2 has an effect on anchorage-independent growth in melanoma cell lines. To investigate this, lentiviral infection of human melanoma CHL−1 was used to produce stable cell lines overexpressing wild-type (WT) Nudt2 by using PLX304-V5-Nudt2 expressing vector. As a control, infection with an empty vector (PLX304-EV) was used. Western blot was used to compare the expression levels of WT vs. EV. V5 antibody was used to check the overexpression of Nudt2, which is tagged to V5 in the expression vector: it showed one band in overexpressed cells compared with none in control. When Nudt2 antibody was used, two bands were detected in overexpressed cells, corresponding to V5−Nudt2 fusion and to endogenous Nudt2. In control, only endogenous Nudt2 was detected ( Figure 4A). The colony formation assay was used to determine the effect of WT Nudt2 overexpression (PLX304− V5− Nudt2) on anchorage-independent growth (Figures 4B and S2). Overexpression of WT Nudt2 increased the colony number and colony size in CHL−1 cell compared to cells transfected with EV (PLX304−EV). These findings suggest that Nudt2 is important to support anchorage-independent growth in human melanoma cells. . Colony numbers were counted using the Image J software(ij153-win-java8); n = 7. The data are represented as mean ± SEM. For statistical analysis, Wilcoxon signed-rank test was used. * p < 0.01.

Nudt2 Knockdown Decreased Cell Migration
Cells that have developed the ability to grow in an anchorage-independent environment will migrate and invade [20]. The aim of this study was to see if Nudt2 knockdown affected cell migration. A wound scratch model was used to test the migration of mela- . Colony numbers were counted using the Image J software(ij153-win-java8); n = 7. The data are represented as mean ± SEM. For statistical analysis, Wilcoxon signed-rank test was used. * p < 0.01.

Nudt2 Knockdown Decreased Cell Migration
Cells that have developed the ability to grow in an anchorage-independent environment will migrate and invade [20]. The aim of this study was to see if Nudt2 knockdown affected cell migration. A wound scratch model was used to test the migration of melanoma Nudt2 knockdown cell lines ( Figure 5A). Every 2 h for 48 h, cell migration was calculated as a percentage of relative wound density (RWD) ( Figure 5A) (p = 0.03). Protein expression of vimentin, a mesenchymal marker involved in EMT during metastasis, was detected in Nudt2 knockdown and control cells of CHL−1. It was observed that vimentin expression was reduced in Nudt2 knockdown cells compared to control ( Figure 5B and Figure S3). Moreover, expression level of N cadherin, MMP9 and snail was checked. The results showed that there is no change in protein expression level of these proteins in Nudt2 knockdown cells compared with control ( Figure S4).

Discussion
High expression levels of Nudt2 has been observed in breast cancer and it has shown to affect cell proliferation [14,16]. In this study, Nudt2 was shown to have a si icant role in promoting breast cancer proliferation by different mechanisms from estr [16]. Nudt2 has been demonstrated to have a role in breast cancer proliferation by r lating mTORC1 localization and activity which are regulated by Nudt2 and RagGT interactions [16]. This suggests that it is a tumor-promoting gene, and its high expres in breast cancer cells could make it a prognostic marker for that cancer. A previous re showed that Nudt2 knockout in chronic myelogenous leukemia cells regulate many g involved in tumorigenesis and metastasis indicating that Nudt2 may have a major ro tumorigenic potential of cancer cells [15]. All these studies showing the importan Nudt2 in regulation of breast cancer proliferation under different mechanisms led u study its roles in melanoma. In this study, we found that Nudt2 knockdown inhib

Discussion
High expression levels of Nudt2 has been observed in breast cancer and it has been shown to affect cell proliferation [14,16]. In this study, Nudt2 was shown to have a significant role in promoting breast cancer proliferation by different mechanisms from estrogen [16]. Nudt2 has been demonstrated to have a role in breast cancer proliferation by regulating mTORC1 localization and activity which are regulated by Nudt2 and RagGTPase interactions [16]. This suggests that it is a tumor-promoting gene, and its high expression in breast cancer cells could make it a prognostic marker for that cancer. A previous report showed that Nudt2 knockout in chronic myelogenous leukemia cells regulate many genes involved in tumorigenesis and metastasis indicating that Nudt2 may have a major role in tumorigenic potential of cancer cells [15]. All these studies showing the importance of Nudt2 in regulation of breast cancer proliferation under different mechanisms led us to study its roles in melanoma. In this study, we found that Nudt2 knockdown inhibited anchorage-independent melanoma growth but without any effect on cell proliferation (Figure 2), and its knockdown reduced tumor growth in vivo ( Figure 3). Moreover, we showed that Nudt2 overexpression increased anchorage-independent growth in human melanoma cells (Figure 4). We wanted to understand the molecular mechanism behind this effect. As previously shown, Nudt2 regulates cell proliferation in breast cancer cell lines via its effect on mTORC1 activity [16]. In this study, we investigated the effect of Nudt2 knockdown on mTROC1 downstream targets, such as p-P70 s6 kinase, but we did not see any change in these proteins' expression level, which indicates that Nudt2 knockdown has no effect on mTROC1 activity in melanoma and the effect of Nudt2 on anchorage-independent growth in melanoma is not mediated by mTROC1 activity. Anchorage-independent growth is a hallmark of cancer and a contributor to cancer metastasis [20]. Anchorage-independent growth is connected to EMT in many cancers [21]. EMT involves many epithelial and mesenchymal markers, such as E cadherin, N cadherin, vimentin, slug, snail, and MMP9. During EMT, epithelial markers, such as E cadherin, will decrease while mesenchymal protein expression will increase [18]. In this study, we checked whether Nudt2 effect on anchorage-independent growth is accompanied by cell migration and changes in EMT markers. Our results showed that Nudt2 knockdown decreased cell migration in CHL−1and Nudt2 knockdown reduced the expression of vimentin without any change in the expression level of N-cadherin, snail, and MMP9 ( Figure S4). These results indicate that Nudt2 effect on anchorage-independent growth is not fully regulated by EMT. These results confirmed that Nudt2 is a potent regulator of tumorigenicity in melanoma cell lines and in a xenograft model, indicating that Nudt2 is very important to support anchorage-independent growth in melanoma and that Nudt2 could serve as a target for cancer therapy. Further investigation is needed to understand the molecular mechanism behind the effect of Nudt2 on anchorage-independent growth in melanoma. Moreover, it is very important to determine whether Ap 4 A is involved in this process, and if it has role, what is the mechanism behind it.

Stably Transfected Melanoma Cell Lines
Human melanoma cells (1 × 10 6 ) were cultured in 6-well plates to reach 60% confluence on the day of infection. They were infected with either Nudt2 shRNA lentiviral particles (#sc-60188-V; Santa Cruz Biotechnology, Almog, Israel) or control shRNA lentiviral particles (# sc-108080; Santa Cruz Biotechnology) using polybrene transfection reagent (#TR-1003-G; Merck, Herzliya, Israel) at a final concentration of 8 µg/mL. The cells were then incubated at 37 • C, in a 5% CO 2 incubator for 24 h, after which the medium was replaced with complete medium. Another 24 h later, the medium was replaced by a puromycin selection medium, which was optimized for each cell line according to their killing curves. Selected colonies of the cells were expanded and maintained.

Expression Vector, Lentiviral Production, Cell Infection and Preparation of Luciferase Expressing Cells
Lentiviral production was performed using X-tremeGENE HP Transfection Reagent. A mixture of vsvg/viral envelope plasmid, dvpr/viral packaging vector, PLX304 empty Int. J. Mol. Sci. 2023, 24, 10513 8 of 10 vector (Addgene) or Nudt2 WT vector or PLX304-Luciferase-V5 vector were co-transfected into the 293T cells. Supernatants containing viral particles were collected and used for infection. The PLX304-Blast-V5 Nudt2 (V5-Nudt2) lentiviral expression vector (CCSB-Broad Lentiviral Expression Library, Dharmacon) was used as the expression vector. The empty PLX304 vector (pLX304 was a gift from David Root (Addgene plasmid # 25890; http://n2t.net/addgene:25890 (accessed on 6 June 2018); RRID: Addgene_25890) was used as control. CHL−1 cells was infected with either PLX304 empty vector or Nudt2 WT using polybrene transfection reagent at a final concentration of 8 µg/mL. 1 × 10 6 cells were cultured in 6-well plates to reach 60% confluence on the day of infection. The cells were then incubated at 37 • C, in a 5% CO2 incubator for 24 h, after which the medium was replaced with complete medium. Another 24 h later, the medium was replaced by a blasticidine selection medium, which was optimized for the cell line according to the killing curve. The selected colonies of the cells were expanded and maintained. To prepare luciferaseexpressing cells, 1 × 10 6 cell of CHL−1 human melanoma stable Nudt2 knockdown and control were cultured in 6-well plates to reach 60% confluence on the day of infection. They were infected with lentiviral particles prepared using PLX304 Luciferase-V5 plasmid blast (a gift from Kevin Janes; Addgene plasmid # 98580; http://n2t.net/addgene:98580 (accessed on 19 January 2021); RRID: Addgene_98580) using polybrene transfection reagent at a final concentration of 8 µg/mL. The cells were then incubated at 37 • C, in a 5% CO 2 incubator for 24 h, after which the medium was replaced with complete medium. Another 24 h later, the medium was replaced by a blasticidine selection medium. The selected colonies of the cells were expanded and maintained. Please see Table 1.