
==== Front
Redox Biol
Redox Biol
Redox Biology
2213-2317
Elsevier

S2213-2317(24)00267-2
10.1016/j.redox.2024.103289
103289
Research Paper
Transcriptional Isoforms of NAD+ kinase regulate oxidative stress resistance and melanoma metastasis
Cascio Graciela a
Aguirre Kelsey N. a
Church Kellsey P. a
Hughes Riley O. ad
Nease Leona A. a
Delclaux Ines a
Davis Hannah J. a
Piskounova Elena elp2025@med.cornell.edu
abc⁎
a Sandra and Edward Meyer Cancer Center, 413 East 69th Street, Belfer Research Building, Weill Cornell Medicine, 10021, New York, NY, USA
b Department of Dermatology, Weill Cornell Medicine, New York, NY, USA
c Department of Biochemistry, Weill Cornell Medicine, New York, NY, USA
d Department of Pharmacology, Weill Cornell Medicine, New York, NY, USA
⁎ Corresponding author. Sandra and Edward Meyer Cancer Center, 413 East 69th Street, Belfer Research Building, Weill Cornell Medicine, 10021, New York, NY, USA. elp2025@med.cornell.edu
28 7 2024
10 2024
28 7 2024
76 10328912 6 2024
24 7 2024
26 7 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Metastasizing cancer cells encounter a multitude of stresses throughout the metastatic cascade. Oxidative stress is known to be a major barrier for metastatic colonization, such that metastasizing cancer cells must rewire their metabolic pathways to increase their antioxidant capacity. NADPH is essential for regeneration of cellular antioxidants and several NADPH-regenerating pathways have been shown to play a role in metastasis. We have found that metastatic melanoma cells have increased levels of both NADPH and NADP+ suggesting increased de novo biosynthesis of NADP+. De novo biosynthesis of NADP+ occurs through a single enzymatic reaction catalyzed by NAD+ kinase (NADK). Here we show that different NADK isoforms are differentially expressed in metastatic melanoma cells, with Isoform 3 being specifically upregulated in metastasis. We find that Isoform 3 is more potent in expanding the NADP(H) pools, increasing oxidative stress resistance and promoting metastatic colonization compared to Isoform 1. We have found that Isoform 3 is transcriptionally upregulated by oxidative stress through the action of NRF2. Together, our work presents a previously uncharacterized role of NADK isoforms in oxidative stress resistance and metastasis and suggests that NADK Isoform 3 is a potential therapeutic target in metastatic disease.

Keywords

NADK
Antioxidants
Oxidative stress
Metastasis
NADP+
==== Body
pmc1 Introduction

Oxidative stress limits metastasis [[1], [2], [3], [4]]. Cancer cells, both in circulation and during metastatic colonization, experience high levels of oxidative stress and exhibit decreased levels of the cellular antioxidant, reduced glutathione (GSH) [5]. While most cancer cells do not survive the metastatic cascade, successful metastasizers are able to rewire their metabolism to upregulate antioxidant pathways and overcome oxidative stress by regenerating GSH from its oxidized form, GSSG. To reduce GSSG to GSH cells must utilize NADPH, in the process converting it to NADP+ (Supp Fig. 1A). It has been shown previously that metastasizing cells rely on different NADPH regeneration pathways including one carbon metabolism and pentose phosphate pathway, to increase their antioxidant capacity. However, the process by which metastasizing cells reinforce their NADP(H) pool is unknown.

NADP+ is generated de novo by NAD+ kinase (NADK) by phosphorylating NAD+ [6,7]. Due to a high cellular demand for reduction reactions, cells maintain a high NADPH/NADP+ ratio to ensure that these reactions are favorable [8]. Since NADP+ and NADPH are membrane impermeable cells have developed organelle-specific NADPH synthesis. In mammals, NADK is the cytoplasmic form of the enzyme, while NADK2 localizes to the mitochondria [9,10]. NADK contains a bifunctional NADP+ phosphatase/NAD kinase domain, while NADK2 contains a diacylglycerol kinase catalytic domain [11]. Phylogenetic analysis indicates that NADK and NADK2 have developed independently during evolution [11]. Presence of other NADKs in organelles such as peroxisomes and endoplasmic reticulum remains unknown. Recent studies demonstrated the importance of NADK in the antioxidant defense, while NADK2 has been mainly implicated in proline biosynthesis [12,13].

Cytoplasmic NADK exists in three isoforms. Isoform 1 and 2 are the two longer isoforms that contain an autoinhibitory N-terminal domain [14]. It has been previously shown that both AKT and PKC regulate the activity of Isoform 1 through phosphorylation of three terminal serine residues: Ser44, Ser46 and Ser48 [14,15]. This phosphorylation relieves the autoinhibition of NADK and stimulates NADP+ production. In contrast, Isoform 3 is a shorter isoform, generated through an alternative transcriptional start site with a unique N-terminal domain that does not have autoinhibitory activity or AKT/PKC phosphorylation sites. Consequently Isoform 3 has much higher enzymatic activity than Isoform 1 at steady state [14]. However, the differential role of NADK isoforms in NADP+ generation is unknown.

Here we examine the role of NADK isoforms in oxidative stress resistance and melanoma metastasis to address how NADK activity contributes to de novo NADP(H) generation as an antioxidant defense mechanism in metastasizing melanoma cells. We specifically focused on Isoforms 1 and 3 as Isoform 2 has the same regulatory domain as Isoform 1. We generated loss of function melanoma cell lines of these NADK Isoforms which allowed us to address how these differential activities impacted melanoma cell survival under oxidative stress and during metastasis. We demonstrated that metastasizing melanoma cells increase NADP(H) biosynthesis and increase levels of Isoform 3 compared to the primary tumor. Loss of Isoform 3 caused a greater increase in both cytoplasmic and mitochondrial oxidative stress levels than loss of Isoform 1, and sensitized cells to prooxidant treatment. Finally, loss of either Isoform 1 or 3 had no effect on the growth of subcutaneous tumors but lead to a significant decrease in metastasis. Interestingly, overexpression of Isoform 1 or 3 had no significant effect on primary tumor growth either, but overexpression of truncated Isoform 1 or Isoform 3 caused a much higher increase in metastatic burden in the organs of the mice compared to full-length Isoform 1. We found that transcriptional upregulation of Isoform 3 is driven by the transcription factor NRF2. Our findings suggest that metastasizing melanoma cells increase expression of NADK Isoform 3 to combat oxidative stress and increase their survival during the metastatic cascade. Our data indicate that increased levels of Isoform 3 promote melanoma cell extravasation and colonization of distant sites. Our findings identify NADK isoform 3 as a potential therapeutic target for metastatic disease.

2 Methods

2.1 Plasmid construction

miR-E shRNA knockdown constructs were generated by the splashRNA program and ordered from Invitrogen as oligos. Hairpins were ligated (T4 Ligase, NEB) into SiREP lentiviral plasmid behind an SFFV promoter and iRFP fluorescent protein using XhoI and EcoRI sites. For overexpression constructs, the template plasmids for NADK isoforms were kindly provided by Gerta Hoxhaj and the coding sequences of NADK isoforms 1 and 3 for the rescue experiments were ordered as gene fragments (IDT) with an N-terminus Kozack sequence and 3xHA. Constructs were ligated (In-Fusion HD, Takara or T4 Ligase, NEB) into pLenti vector behind an EFS promoter using BamHI and NsiI sites. A cleavable P2A site links the protein expression to a fluorescent protein tag. CRISPR knockout sgRNA sequences were generated using Geneious cloning software and ordered as single stranded oligos from IDT. Vector backbones were kindly provided by Lukas Dow, and all plasmids were sequence verified.

2.2 Lentiviral transduction and generation of stable cell lines

For virus production, 0.9 μg of plasmid was combined with 1 μg of packaging plasmids (0.4 μg pMD2G and 0.6 μg psPAX2) and transfected into HEK 293T cells using Polyjet (SignaGen) according to manufacturer's instructions. Fresh media was added the following day and viral supernatants were collected 72hr after transfection and filtered through a 0.45 μM filter. Approximately 1 million cells were infected with viral supernatant and 10 μg/mL polybrene (Sigma-Aldrich). Cells then underwent antibiotic selection with puromycin or hygromycin (Inviogen) depending on the antibiotic resistance of each plasmid. For CRISPR knockout, cells stably-expressing Cas9 were infected with the gRNA construct. To generate single clones, cells were diluted and plated into a 10 cm dish. Glass rings were used to isolate single cells and grow colonies. All single clones were verified by Western blot analysis and Synthego genomic DNA sequencing.

2.3 Real- time PCR quantification of mRNA

mRNA was extracted using a Direct-zol RNA Purification Kit (Zymo Research). Reverse transcription was performed using iScript cDNA synthesis kit (Rio-Rad) as described by manufacturer. cDNA was then diluted and used for qPCR analysis with SYBR Green PCR master mix (Thermo Fisher). IDT online primer design tool was used to generate qPCR primers and UCSC In-Silico PCR database was then used to verify human-specificity of primers. qPCR analysis was performed on an Applied Biosystems QuantStudio 6 Real-Time PCR System. All targets were normalized to Actin in a standard ΔΔCt analysis.

2.4 Analysis of cell survival under prooxidant treatment

10,000 cells were plated in 200ul of normal media/well in a white TC-treated 96 well plate (Corning). After 24 h, the media was removed and the cells were treated in 100ul of normal media with prooxidant H2O2 (Sigma-Aldrich) or pyocyanin (Cayman Chemical Company). After 24 h of treatment, CellTiter-Glo 2.0 (Promega) was used according to manufacturers instructions to quantify cell viability.

2.5 Lentiviral transduction of human melanoma cells

A lentiviral construct with luciferase and dsRed (luc P2A dsRed) was used to label patient-derived melanoma cells as described in Piskounova et al. Nature (2015). Virus was produced as described above. For lentiviral transduction, 500,000 freshly dissociated melanoma cells were infected with viral supernatant and supplemented with 10 μg/mL polybrene (Sigma). The following day, the media was replaced and 48hrs post-infection cells were either injected subcutaneously into mice as bulk tumors or FACS sorted for positive infection.

2.6 Flow cytometry of melanoma cells

All melanoma cells in this study stably express dsRed and luciferase so that melanoma cells could be distinguished by flow cytometry or bioluminescent imaging. When preparing cells for sorting by flow cytometry, cells were stained with antibodies against mouse CD45 (30-F11-VioletFluor, Tonbo), mouse CD31 (390-VioletFluor, eBiosciences), Ter119 (Ter-119-VioletFluor, Tonbo) and human HLA-A, -B, –C (BD Biosciences) to select live human melanoma cells and exclude mouse endothelial and hematopoietic cells. Antibody labelling was performed for 20 min on ice, followed by washing and centrifugation. Before sorting, cells were resuspended in staining medium (L15 medium containing bovine serum albumin (1 mg/mL), 1 % penicillin/streptomycin, and 10 mM HEPES, pH 7.4) containing 4′6-diamidino-2-phenylindole (DAPI; 5 μg/mL; Sigma) to eliminate dead cells from sorting. Live human melanoma cells were isolated by flow cytometry by sorting cells that were positive for dsRed and HLA and negative for mouse CD45, CD31, Ter-119 and DAPI.

2.7 Animal studies

The tissue bank protocol used for this study was developed and approved jointly by the clinical director of the University of Michigan (UM) melanoma program, UM Cancer Center director of tissue procurement, UM chief of anatomic pathology, and UM director of the section of dermatopathology. The protocol was developed to avoid any compromise in patient care, pathologic diagnosis, tumor staging, or treatment. Patient confidentiality was maintained by password- and firewall-protected access to all pertinent databases. Melanoma specimens were obtained with informed consent from all patients according to protocols approved by the Institutional Review Board of UM Medical School (IRBMED approvals HUM00050754 and HUM00050085). All patients included in this study had clinically apparent melanoma disease (biopsy-proven stage II, III, or IV, or obvious clinical stage IV) from which a small (typically 2–5 mm) tissue sample not required for standard-of-care pathology assessment was obtained. Most of the melanomas in this study were regional stage III lymph node or skin/soft tissue disease with palpable, clinically enlarged node(s) or soft tissues, undergoing definitive surgical resection, with biopsy-proven (most often needle core) diagnosis confirmed before surgery.

Tumors were mechanically dissociated with a McIlwain tissue chopper (Mickle Laboratory Engineering) before sequential enzymatic digestion in collagenase IV (200 U/ml) (Worthington) for 20 min followed by 0.05 % trypsin-EGTA for 2 min, both at 37 °C. Deoxyribonuclease (50–100 U/ml) was added to reduce clumping of cells during digestion. Cells were filtered (40-μm cell strainer) to obtain a single-cell suspension. Dead cells and debris were depleted by density centrifugation [OptiPrep (1.1 g/ml); Sigma] when necessary. Cells were always passaged in vivo (in immunocompromised mice as detailed below), not in vitro.

After sorting, cells were counted and resuspended in staining medium [L15 medium containing bovine serum albumin (1 mg/ml), 1 % penicillin/streptomycin, and 10 mM Hepes (pH 7.4)] with 25 % high-protein Matrigel (product 354248; BD Biosciences). Subcutaneous injections of human melanoma cells were performed in each flank and the interscapular region of NOD.CB17-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice (Jackson Laboratory) in a final volume of 50 μl.

For a standard metastasis assay, 100 cells, for patient-derived melanoma cells, or 50 for A375, were injected subcutaneously into the right flank of the mice. Tumor formation was evaluated by regular palpitation of the injection site and tumors were measured weekly until any tumor in the mouse cohort reached 2.5 cm in its largest diameter. Mice were monitored weekly for signs of distress according to a standard body condition score or within 24hr of their tumors reaching 2.5 cm in diameter – whichever came first. These experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee at Weill Cornell Medicine (protocol 2017-0033).

2.8 Bioluminescence imaging

Mice injected subcutaneously with melanoma cells expressing luciferase were monitored until tumors reached 2.5 cm in diameter. For bioluminescent imaging, mice were injected intraperitoneally with 100 μL of DPBS containing D-luciferin monopotassium salt (40 μg/mL, Goldbio) 5 min before imaging, followed by general anesthesia with isoflurane 2 min before imaging. IVIS Imaging System 200 Series (Caliper Life Sciences) with Living Image Software was used with the exposure time set to 10 s. After imaging the whole body, mice were euthanized and individual organs were dissected and imaged. The bioluminescent signal was quantified with ‘region of interest’ measurement tools in Living Image (PerkinElmer) software.

After imaging, tumors and metastatic nodules were collected to perform protein and/or molecular analysis.

2.9 Flow cytometric analysis of oxidative stress

Cells were plated in low attachment plates (3D system) or in TC-treated plates and treated with a prooxidant pyocyanin (Cayman). We stained the dissociated cells for 30 min at 37 °C with 5 μM CellROX Green, or CellROX DeepRed in HBSS-free (Ca2+ and Mg2+ free) to assess mitochondrial and cytoplasmic oxidative stress.

2.10 NADPH/NADP+ and NAD+/NADH measurement

For the mice results, subcutaneous tumors and metastatic nodules were surgically excised as quickly as possible after euthanizing the mice then melanoma cells were mechanically dissociated. For the melanoma cell lines, cell were plated the day before in 24 wells TC-treated plates. NADPH, NADP+, NADH and NAD+ were measured using NADPH/NADP+ Glo-Assay (Promega) and NADH/NAD+ Glo-Assay (Promega) following the manufactures instructions. Luminescence was measured using a Omega plate reader (BMG Labtech FLUOstar). Values were normalized to protein concentration, measured using The Pierce Rapid Gold BCA Protein Assay Kit (Thermo Fisher Scientific).

2.11 Western blot analysis

Tissue or cell lines lysates were prepared using Triton lysis Buffer (Triton X-100 1 %, 20 mM Tris pH8, 137 mM NaCl, 1 mM EDTA, 10 % glycerol, 1.5 mM MgCl2) supplemented with phenylmethylsulphonyl fluoride (Sigma-Aldrich), and 2 cocktails of protease and phosphatase inhibitor (Halt™, Fisher Scientific; Phosstop™- phosphatase inhibitor tablets, Roche). The Pierce Rapid Gold BCA Protein Assay Kit (Thermo Fisher Scientific) was used to quantify protein concentrations. Equal amounts of protein (15–30 μg) were separated on 4–20 % Tris Glycine SDS gels (BioRad) and transferred to polyvinylidene difluoride membranes (BioRad). Membranes were blocked for 30 min at room temperature with 5 % milk in TBS supplemented with 0.1 % Tween20 (TBST) then incubated with primary antibodies overnight at 4 °C. After incubating with horseradish peroxidase conjugated secondary antibodies (Cell Signaling Technology), membranes were developed using SuperSignal West Pico or Femto chemiluminescence reagents (Thermo Fisher Scientific). The following primary antibodies were used for Western blot analyses: NADK (Cell Signaling Technologies; 55948S), NADK (Sigma-Aldrich; HPA048909), NADK2 (Abcam; ab181028), HA-Tag (Cell Signaling Technologies; 14031), NRF2 (Cell Signaling Technologies; 14409), GAPDH HRP Conjugate (Cell Signaling Technologies; 8884), HIF1 α (Cell Signaling Technologies; 36169) and β-Actin HRP Conjugate, (Cell Signaling Technologies; 12262).

2.12 Cell culture

Melanoma cell line A375 was culture using DMEM (Corning; 10-017-CV) supplemented with 10 % FBS and Penicillin: Streptomycin solution 100x (Corning; 45000-652).

2.13 Nuclear and cytoplasmic extraction

Subcutaneous tumors and metastatic nodules were surgically excised as quickly as possible after euthanizing the mice then melanoma cells were mechanically dissociated. Nuclear and cytoplasmic extraction was performed using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents kit and the final samples were analyzed by Western Blot.

2.14 Statistical analysis

No statistical methods were used to predetermine sample size. The data in most figure panels represents several independent experiments performed on different days. Variation is always indicated using standard deviation. For analysis of statistical significance, we first tested whether there was homogeneity of variation across conditions (as required by ANOVA) using Levene's test, or when only two conditions were compared, using the F-test. In cases where the variation was significantly different among conditions, we used a non-parametric Kruskal-Wallis test or a non-parametric Mann-Whitney test to assess significance of difference among populations and conditions. Usually variation did not significantly differ among conditions. Under those circumstances, two-tailed Student's t-tests were used to test significance of differences between two conditions. When more than one conditions were compared, a one-way ANOVA followed by Dunnett's multiple comparisons test was performed. A two-way ANOVA followed by Dunnett's multiple comparisons test were used in cases where more than two groups were compared with repeated measures. In all xenograft assays, we injected 5–8 week old NSG mice, 5 per condition. Both male and female mice were used. When mice died before the end of the experiment due to opportunistic infections the data from those mice was excluded.

3 Results

Metastasis is limited by oxidative stress across different cancer types [1,5]. It has been previously shown that metastasizing melanoma cells rely on NADPH regenerating pathways such as one carbon metabolism and pentose phosphate pathway as part of their antioxidant defense system [5,16]. We have used a previously characterized patient-derived xenograft model of melanoma metastasis to quantify levels of NADPH and NADP+ in subcutaneous tumors and metastatic nodules in two different patient derived melanoma xenografts (PDX) with different driver mutations, M405 (NRAS Q61H) and M481 (BRAF V600E) [17]. We find that both NADPH and NADP+ are elevated in metastatic nodules in different organs compared to the primary tumor in both PDX models (Fig. 1 A and B left and middle). The ratio of NADPH/NADP+ is an indicator of oxidative stress levels, where a low NADPH/NADP+ ratio indicates high levels of oxidative stress in the cells. We have found that the ratio of NADPH/NADP+, was decreased in metastatic nodules compared to the subcutaneous tumor in both PDX models, indicating higher levels of oxidative stress (Fig. 1 A and B right). Overall, these data suggest that while metastases are under increased levels of oxidative stress, they increase regeneration of NADPH from NADP+ as well as de novo biosynthesis of NADP+ to sustain increased antioxidant capacity. We have also found increased levels of both NAD+ and NADH in metastatic nodules compared to subcutaneous tumors across two different PDX models of melanoma metastasis, however the NAD+/NADH ratio remained unchanged (Supp Figs. 1B and C).Fig. 1 Metastatic nodules exhibit increased synthesis of NADP and NADPH as well as upregulation of NAD Kinase

(A) Levels of NADP+ (left), NADPH (middle) and ratio of NADPH/NADP+ (right) in subcutaneous melanoma tumors compared to metastatic nodules from different organs in PDX tumor M481.

(B) Levels of NADP+ (left), NADPH (middle) and ratio of NADPH/NADP+ (right) in subcutaneous melanoma tumors compared to metastatic nodules from different organs in PDX tumor M405.

(C) Expression levels of NAD+ Kinase and NAD+ Kinase 2 in subcutaneous melanoma tumors compared to metastatic nodules in using PDX melanoma tumors M481 and M405.

(D) Levels of NADP+ (left), NADPH (middle) and ratio of NADPH/NADP+ (right) in NADK knock down A375 cells.

(E) Fold increase of CellROX Deep Red dye fluorescence measured by flow cytometry in basal conditions (left) and after treatment with the prooxidant pyocyanin (right) in NADK knock down A375 cells.

(F) NADK knock down in A375 cells survival after 24hr treated with 10uM pyocyanin.

Fig. 1

Since the main mechanism of de novo NADP+ biosynthesis is through NAD+ Kinase (NADK) activity, we tested expression levels of both cytoplasmic NADK and mitochondrial NADK2 in metastatic nodules and subcutaneous tumors in two different PDX models of melanoma metastasis (Fig. 1C). Metastatic nodules showed a significant increase in cytoplasmic NADK levels but no significant increase in mitochondrial NADK2 levels, indicating increased NADP+ biosynthesis in the cytoplasm during metastasis. We genetically depleted NADK in A375 melanoma cells using shRNAs targeting all three NADK isoforms (Supp Fig. 1D Western blot). Levels of both NADP+ and NADPH were decreased (Fig. 1D left and middle); the ratio of NADPH/NADP+ also decreased indicating that these cells are under higher levels of oxidative stress (Fig. 1D right). In addition, we found higher levels of Reactive Oxygen Species (ROS) by flow cytometry in NADK-depleted cells, further confirming higher levels of oxidative stress at steady state (Fig. 1E left). Loss of NADK lead to higher levels of ROS induced by prooxidant pyocyanin (Fig. 1E right) and decreased cell survival, indicating increased sensitivity to oxidative stress (Fig. 1F). No significant differences were found in the levels of NAD+, NADH and NAD+/NADH ratio (Supp Fig. 1D graphs).

To functionally test the role of total NADK in vivo we knocked down NADK in two PDX melanoma tumors and injected them subcutaneously. There was no difference in the growth of the subcutaneous tumors and a trend towards a decrease in the metastatic burden (Supp Figs. 1E and F). We speculate that incomplete depletion of NADK by shRNAs leaves sufficient level of the enzyme to control levels of oxidative stress in order to metastasize (Supp Fig. 1D Western blot).

NADK has three different isoforms [14]; Isoform 1 contains a long autoinhibitory N-terminal domain, while Isoform 3 has a different transcriptional start site and has a unique N-terminal domain (Supp Fig. 2A). Due to the difference in the N-terminus domains, Isoform 3 has been previously shown to have a much higher activity than Isoform 114. We tested the mRNA levels of the different isoforms in metastatic nodules compared to the primary tumor in 2 different PDX models of melanoma metastasis. We found that Isoform 3 was greatly upregulated in metastatic nodules compared to Isoform 1 suggesting that the observed increase in NADP + biosynthesis in metastasis is due to increased activity of Isoform 3 (Fig. 2A).Fig. 2 Loss of NADK Isoform 3 increases oxidative stress levels in melanoma cells

(A) mRNA levels of Isoform 1 and 3 in PDX tumor M481 (left) and M4051 (right) in subcutaneous tumor compared to metastatic nodules.

(B) NADK Isoform 3 mRNA levels after treatment with 10uM pyocyanin for 2hr.

(C) Levels of NADP+ (left), NADPH (middle) and ratio of NADPH/NADP+ (right) in A375 melanoma cells with Isoform 1 or Isoform 3 deletion.

(D) Fold increase in CellROX Green (mitochondrial) and CellROX DeepRed (cytoplasmic) fluorescence as measured by flow cytometry in A375 melanoma cells with either Isoform 1 or Isoform 3 deletions.

(E) Fold increase in CellROX Green dye fluorescence measured by flow cytometry with either Isoform 1 or Isoform 3 deletion treated with 50uM pyocyanin for 2 h

(F) Survival of A375 melanoma cells with either Isoform 1 or Isoform 3 deletion under oxidative stress from H2O2 treatment for 24 h.

Fig. 2

To establish the role of Isoform 3 in oxidative stress regulation, we induced oxidative stress using a prooxidant pyocyanin (Supp Fig. 2B) and we saw a transcriptional increase in Isoform 3 but no effect on Isoform 1 (Fig. 2B). This suggests that Isoform 3 is specifically upregulated in response to oxidative stress. To establish how the loss of individual NADK isoforms affects melanoma cells we used CRISPR/Cas9 technology to generate isoform-specific knockout A375 melanoma cell lines. Isoform 1 knockout cells were validated by western blotting (Supp Fig. 2C l Western blot), however due to lack of Isoform 3-specific antibodies, genomic DNA sequencing was used to validate Isoform 3 knockouts (Supp Fig. 2C right). Interestingly, we saw an upregulation of Isoform 1 in the Isoform 3 deleted cells suggesting that melanoma cells may compensate for the loss of Isoform 3 with Isoform 1 upregulation (Supp Fig. 2C Western blot). Measurement of NADP+ showed an increase in Isoform 3 knockout cells, confirming that there is a compensation with Isoform 1 expression due the lack of Isoform 3 (Fig. 2C left). The levels of NADPH showed no change in both knockouts but the NADPH/NADP+ ratio showed a greater decrease in cells lacking Isoform 3 (Fig. 2C middle and right), suggesting higher levels of oxidative stress upon loss of Isoform 3. Consistent with decreased NADP+ biosynthesis, we found increased levels of both NAD+ and NADH in cells lacking either Isoform 1 or Isoform 3 (Supp Fig. 2D left and middle). However, we did not find any changes in the NAD/NADH ratio (Supp Fig. 2D right). We assessed levels of oxidative stress by flow cytometry using a ROS reactive dye and we found that knockout of Isoform 3 specifically lead to a higher increase in both mitochondrial and cytosolic ROS levels than Isoform 1 knockout (Fig. 2D), suggesting that the endogenous compensation by Isoform 1 is not enough to rescue the ROS levels. Oxidative stress induced by prooxidant pyocyanin was also higher in cells lacking Isoform 3 than Isoform 1 as measured by flow cytometry (Fig. 2E). We measured the ability of knockout cells to survive under oxidative stress by treating them with the prooxidant H2O2 and found that both Isoform 1 and 3 knockouts showed decreased cell survival (Fig. 2F and Supp Fig. 2E). Taken together, these data show that loss of Isoform 3 greatly elevates cellular oxidative stress through decreased NADPH/NADP+ ratio and sensitizes melanoma cells to oxidative stress compared to Isoform 1 loss. It also indicates that melanoma cells depend on both isoforms for oxidative stress resistance with Isoform 3 having a greater effect.

Since we observed differential expression of NADK isoforms in metastasis, we wanted to functionally test the role of NADK isoforms in enabling survival of metastasizing melanoma cells during organ colonization in vivo. We transplanted luciferase-labelled A375 melanoma cells lacking either NADK Isoform 1 or Isoform 3 subcutaneously into immunocompromised mice. We evaluated metastatic burden in the organs of the mice using bioluminescence signal once the subcutaneous tumor reached the end point of 6 weeks. We found that subcutaneous tumor growth was not affected by either Isoform 1 or Isoform 3 loss (Fig. 3 A). However, metastasis was greatly reduced by loss of either Isoform 1 or Isoform 3 suggesting that both isoforms are involved in metastatic colonization (Fig. 3B).Fig. 3 NADK Isoform 3 is necessary and sufficient for metastasis in vivo

(A) Subcutaneous tumor growth of A375 melanoma cells with either Isoform 1 or Isoform 3 deletion in NSG mice.

(B) Metastatic burden in the organs of NSG mice xenografted with A375 melanoma cells with either Isoform 1 or Isoform 3 deletion.

(C) Subcutaneous tumor growth of A375 melanoma cells overexpressing different NADK isoforms in NSG mice.

(D) Metastatic burden in the organs of the metastatic nodules in NSG mice xenografted with A375 melanoma cells overexpressing different NADK isoforms.

(E) Organ distribution of the metastatic nodules in NSG mice xenografted with A375 melanoma cells overexpressing different NADK isoforms.

Fig. 3

To test the role of the NADK isoforms in our PDX model M405 (NRAS Q61H) we generated an isoform specific knockdown, that we verified by flow cytometry using an isoform specific reporter in A375 melanoma cells (Supp Figs. 3A and B). We injected PDX melanoma cells with either Isoform 1 or Isoform 3 knockdown subcutaneously and measured both primary tumor growth and metastatic burden. At the end point of the experiment, we found no difference in the growth of the primary tumor between control and Isoform 1 and a slight decrease in Isoform 3 (Supp Fig. 3C left), but there was a significant decrease in metastatic burden upon loss of either isoform (Supp Fig. 3C right).

In order to determine whether NADK isoforms can differentially drive metastasis in vivo, we overexpressed NADK isoforms 1 and 3 in Luciferase labelled A375 melanoma cells. In addition, we overexpressed a truncated version of Isoform 1, (ΔIsoform 1) (Supp Fig. 2A and Suppl Fig. 3D). Truncated Isoform 1 lacks the regulatory N-terminus domain, and it has been shown to have a similar activity level as Isoform 3 in vitro14. We tested the levels of NADP+, NADPH and the ratio NADPH/NADP+ in these cells, and as expected Isoform 3 and ΔIsoform 1 had the highest levels of NADP, NADPH and NADP/NADPH ratio (Supp Fig. 3E). Consistent with this we observed increased survival of A375 melanoma cells overexpressing Isoform 3 when treated with Pyocyanin (Supp Fig. 3F).

We transplanted A375 cells subcutaneously and we found that none of the isoforms had a significant impact on the growth of the subcutaneous tumor (Fig. 3C). Overexpression of wild type Isoform 1 had no effect on the amount of metastatic burden in the organs of the mice, however, overexpression of either truncated Isoform 1 or Isoform 3 significantly increased the metastatic burden in the organs of the mice (Fig. 3D). This suggests that overall increased de novo biosynthesis of NADP+ drives metastasis. When we analyzed the metastatic organotropism of melanoma cells overexpressing different isoforms, we found that both truncated Isoform 1 and Isoform 3 showed significantly less metastasis to the lung and more metastasis to other organs such as the liver, pancreas and the gut, compared to Isoform 1-overexpressing cells and control cells (Fig. 3E). This suggests that increased de novo NADP+ biosynthesis by truncated Isoform 1 or Isoform 3 drives broad metastatic colonization to different organs. This data also suggests that cells with low NADP+ biosynthesis may get lodged in the lung and are unable to metastasize more widely to other sites.

Since we observed a transcriptional upregulation of Isoform 3 in both metastatic nodules and upon pyocyanin treatment, we examined the Isoform 3 promoter for known transcriptional factor binding elements. The NFR2 binding sequence also known as antioxidant response element (ARE) was found in the Isoform 3 promoter (Fig. 4A). We deleted the ARE using a CRISPR/Cas9 system in A375 melanoma cells and showed that treatment with pyocyanin had no effect and expression of Isoform 3 was greatly reduced (Fig. 4A). Since NRF2 is the major transcriptional regulator of the antioxidant response, we validated that its canonical transcriptional targets, NQO-1 and HO-1 are upregulated in the metastatic nodule compared to the primary tumor in a PDX model of melanoma metastasis suggesting increased NRF2 activity (Fig. 4B). We also validated that both cytoplasmic and nuclear NRF2 levels were increased in metastatic nodules using a PDX model of melanoma metastasis (Fig. 4B Western blot). Next, we analyzed existing ChIP data from the ENCODE database and found that NRF2 was specifically found to bind the putative promoter of Isoform 3 but not Isoform 1 in Hela-S3 cells (Fig. 4C) and A549 cells (Data not shown). This suggested that NRF2 might be driving the transcriptional upregulation of Isoform 3 in response to oxidative stress and in metastasizing cells. To test this hypothesis functionally, we used the NRF2 activator Ki696 to test its effect on Isoform 3 induction in A375 melanoma cells. We found that NRF2 activation leads to a rapid increase in Isoform 3 level, but not in Isoform 1 levels (Fig. 4D and Supp 4A). We knocked NRF2 down in A375 melanoma cells using an shRNA. We showed that treatment of A375 melanoma cells with Ki696 activator was able to induce canonical targets such as NOO1 and HO-1 but this was not observed in NRF2 KD cells (Supp Fig. 4B). More importantly, levels of Isoform 3 were not increased by Ki696 activator and were significantly reduced in NRF2 KD cells compared to Isoform 1 (Fig. 4E). We also found an increase of Isoform 1, suggesting again that there is a level of compensation between the two isoforms. In addition, we have generated reporters in which we fused either Isoform 1 or Isoform 3 promoters to GFP and showed that while Isoform 1 promoter was able to respond to pyocyanin treatment and increase GFP signal in both Control and NRF2 KD A375 melanoma cells, Isoform 3 promoter was only able to increase GFP signal in Control cells but not in NRF2 KD cells (Fig. 4F). This suggests that Isoform 3 promoter specifically responds to oxidative stress through activity of NRF2 while transcriptional increase of Isoform 1 is independent of NRF2. In conclusion, this data suggests that Isoform 3 is upregulated in response to oxidative stress, specifically through the action of NRF2, and that in the absence of Isoform 3 the cell compensates by inducing the expression of Isoform 1, confirming the importance of Isoform 3 in the antioxidant response.Fig. 4 NRF2 specifically regulates NADK Isoform 3

(A) Schematic of the sequence of the ARE in the Isoform 3 promoter. mRNA levels of Isoform 3 in A375 melanoma cells with ARE deletion.

(B) mRNA levels of NRF2 targets NQO-1 and HO-1 in PDX tumor M481 (left) and M405 (right) in subcutaneous tumor compared to metastatic nodules. Expression levels of NRF2 PDX tumor M481 after nuclear fractionation.

(C) ChIP seq data looking at Pol2 and activating histone marks.

(D) NADK Isoform 1 and 3 mRNA expression in A375 melanoma cell line treated with Ki676 (5uM) for 30 min

(E) NADK Isoform 1 and 3 mRNA expression in A375 melanoma cell line NRF2 knock down treated with Ki676 (5uM) for 30 min

(F) GFP mRNA levels in A375 melanoma cell line NRF2 knock down expressing NADK Isoform 1 or Isoform 3 reporter and treated with pyocyanin (10uM) for 24 h.

Fig. 4

4 Discussion

Metastasizing cancer cells have increased plasticity and are able to rewire their metabolism to increase survival during metastatic colonization. Increased antioxidant capacity enables metastasis of cancer cells by increasing regeneration of GSH from GSSG through use of NADPH and its consequent conversion to NADP+. While there are numerous metabolic pathways that can recycle NADPH from NADP+ to drive the cellular antioxidant cycle, such as the pentose phosphate pathway, malic enzyme and one carbon metabolism, there are limited ways that the NADP+ pool can be replenished de novo. NAD kinase (NADK) is the main route of generating NADP+ from NAD+. Since NADPH is used for reduction reactions, cells maintain a high NADPH/NADP+ ratio and therefore convert most of NADP+ into NADPH. Increased activity of NADK would contribute to increased antioxidant capacity of cancer cells, through de novo synthesis of NADP+ and therefore generation of NADPH for GSH regeneration.

It has been previously shown that NADK has a role in cancer and metastasis. In breast cancer NADK levels are increased due to a histone H3.3 variant-mediated epigenetic regulation of the NADK promoter helping the cancer cells to metastasize [18]. An increased occurrence of mutant NADK (NADKI90F) with increased activity has been found to increase cancer cell growth in pancreatic adenocarcinoma [19]. Consistent with these findings, we have found higher levels of NADK in metastatic nodules compared with the primary tumor in a patient-derived xenograft model of melanoma. Increased expression of NADK would increase the supply of NADP+ that is reduced to NADPH allowing cells to survive under high levels of oxidative stress. We have shown loss of NADK makes melanoma cells more sensitive to prooxidant treatment and less metastatic.

NADK has been shown to exist in several isoforms. Isoforms 1 has a long-term autoinhibitory N-terminus domain, whose inhibition is alleviated upon post-translational phosphorylation. One of the major regulators of NADK activity has been shown to be AKT signaling as well as PKC signaling [14,15]. Both kinases have been shown to phosphorylate specific sites in the autoinhibitory N-terminus, which means that increased signaling through the AKT and PKC pathways leads to increased NADK activity and increased NADP+ production. In contrast, Isoform 3 of NADK has a unique short N-terminal domain, generated by an alternative transcriptional start site, lacking autoinhibitory properties and remaining outside of upstream kinases regulation. This also means that Isoform 3 has a much higher activity than the other Isoforms at baseline. AKT signaling through AKT1 has been shown to decrease during epithelial-mesenchymal transition [20,21]. In fact, genetic depletion of AKT1 has been shown to promote an invasive phenotype in breast cancer [20]. This suggests that there may not be sufficient AKT signaling to alleviate the autoinhibition of NADK Isoform 1 during metastasis, however metastasizing cells are still highly dependent on the NADP(H) pool for antioxidant defenses. Our findings suggest that Isoform 3 due to its high activity, which is independent of AKT signaling, is a potentially novel mechanism to increase the NADP(H) pool and increase oxidative stress resistance.

Our results in vitro reveal that Isoform 3 expression increases in response to oxidative stress and it is highly upregulated in metastatic nodules. Lack of both isoforms in melanoma cells increases levels of oxidative stress having a higher impact in Isoform 3 knock out cells. Our data suggest that both Isoform 1 and 3 are required for metastatic colonization, while loss of either isoform does not have an impact on the primary tumor growth. This may indicate that the demand for NADPH is much higher in metastasizing melanoma cells compared to proliferating cells in the primary tumor. This also suggests that even a minor decrease in NADP+ levels has a strong effect on the ability of melanoma cells to colonize distant sites.

Interestingly, both truncated Isoform 1 and Isoform 3 overexpression led to an increase in the metastatic burden in the organs of the mice, while Isoform 1 overexpression had no effect. Since it has been previously shown that truncated Isoform 1 and Isoform 3 have similar activities in vitro, and they both increase metastatic colonization, this data suggests that increased de novo NADP + generation is a major metastatic driver. Intriguingly, when we analyzed metastatic organotropism we saw increased metastasis to the liver, gut and pancreas and decreased metastasis to the lung with cells overexpressing truncated Isoform 1 and Isoform 3. On the other hand, control cells and full-length Isoform 1 overexpressing cells showed increased metastasis to the lung and limited metastasis to other organs. This suggests that during metastasis, first many cells get lodged in the lung and tend to metastasize there, however increased NADP+ biosynthesis enables cells to metastasize more widely. It is possible that after dissemination, cells with increased NADP+ biosynthesis are able to survive better across many different organs, and therefore cause more widespread metastasis. More broadly, this indicates that there are different mechanisms of oxidative stress resistance that are required for melanoma cells at different steps of the metastatic cascade and in different organotropic environments.

Metastasizing cancer cells experience many different stresses during the metastatic cascade. It is likely that these stress cues induce specific stress resistance mechanisms. Our work specifically identified NRF2, a master regulator of the antioxidant response as the transcription factor responsible for driving Isoform 3 expression. Our data suggests that Isoform 3 is rapidly upregulated by NRF2. This suggests that Isoform 3 may play a role in the initial response to acute oxidative stress due to its elevated activity, while Isoform 1 may play a role in the maintenance of oxidative stress resistance, suggesting there are different mechanisms between acute and chronic oxidative stress resistance during metastasis. Upregulation of Isoform 3 maybe part of a broader network of oxidative stress adaptations that occur during metastatic colonization.

In conclusion, our work has discovered an antioxidant mechanism through differential transcriptional induction of various NADK isoforms. We find Isoform 3 is specifically upregulated in metastatic nodules compared to the primary tumor and provides antioxidant protection to melanoma cells increasing extravasation and organ colonization by metastasizing melanoma cells, driven by coordinated action of NRF2. Taken together, our data highlights NADK Isoform 3 as a potential novel mechanism that drives metastatic colonization that could be exploited for therapeutic inhibition of metastatic disease.

Funding

This work has been supported by grants from the 10.13039/100000054 National Cancer Institute (R01CA270885 ) as well as the MRA Dermatology Fellow Award.

CRediT authorship contribution statement

Graciela Cascio: Writing – review & editing, Writing – original draft, Supervision, Investigation, Formal analysis, Data curation, Conceptualization. Kelsey N. Aguirre: Investigation, Formal analysis. Kellsey P. Church: Investigation. Riley O. Hughes: Investigation. Leona A. Nease: Investigation. Ines Delclaux: Investigation. Hannah J. Davis: Investigation. Elena Piskounova: Writing – review & editing, Writing – original draft, Supervision, Resources, Project administration, Funding acquisition, Formal analysis, Data curation, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A Supplementary data

The following is the Supplementary data to this article:

Supplementary Figure 1: (A) Levels of NAD+ (left), NADH (middle) and ratio of NAD+/NADH (right) in subcutaneous melanoma tumors compared to metastatic nodules from different organs in PDX tumor M481.(B) Levels of NAD+ (left), NADH (middle) and ratio of NAD+/NADH (right) in subcutaneous melanoma tumors compared to metastatic nodules from different organs in PDX tumor M405.(C) Expression levels of NADK in NADK knock down A375 cells.(D) Levels of NAD+ (left), NADH (middle) and ratio of NAD+/NADH in NADK knock down A375 cells.(E) Subcutaneous tumor growth of PDX tumor M481with NADK expression knock down in NSG mice (left). Metastatic burden in the organs of the metastatic nodules in these NSG mice (right).(F) Subcutaneous tumor growth of PDX tumor M405 with NADK expression knock down in NSG mice (left). Metastatic burden in the organs of the metastatic nodules in these NSG mice (right).

Supplementary Figure 2: (A) Schematic of NAD+ Kinase Isoforms 1, truncated Isoform 1 and Isoform 3.(B) Mitochondrial oxidative stress levels in A375 melanoma cells by flow cytometry treated with pyocyanin (10uM for 2 h).(C) Western blot for NADK Isoform 1 levels (left) and sequencing results (right) in A375 melanoma cells with Isoform 1 or Isoform 3 deletions.(D) Levels of NAD+ (left), NADH (middle) and ratio of NAD+/NADH in A375 melanoma cells with Isoform 1 or Isoform 3 deletions.(E) Survival measured by trypan blue of A375 melanoma cells with either Isoform 1 or Isoform 3 deletion under oxidative stress from H2O2 treatment (120uM) for 48 h.

Supplementary Figure 3:(A) Schematic of NADK Isoform 1 reporter (top). Levels of NADK Isoform 1 by flow cytometry in NADK Isoform 1 and Isoform 3 knock down in A375 cells.(B) Schematic of NADK Isoform 3 reporter (top). Levels of NADK Isoform 1 by flow cytometry in NADK Isoform 1 and Isoform 3 knock down in A375 cells.(C) Left. Subcutaneous tumor growth of A375 NADK Isoform 1 and Isoform 3 knock down in NSG mice. Right. Metastatic burden in the organs of the metastatic nodules of A375 NADK Isoform 1 and Isoform 3 knock down in NSG mice.(D) Western blot with NADK Isoform overexpression cell lines.(E) Levels of NADP+ (left), NADPH (middle) and ratio of NADPH/NADP+ in A375 melanoma cells with Isoform 1, truncated Isoform 1 and Isoform 3 deletions.(F) Survival of A375 melanoma cells overexpressing Isoform 1, and Isoform 3 under oxidative stress from pyocyanin treatment for 24 h.

Supplementary Figure 4:(A) NADK Isoform 1 and 3 mRNA expression in A375 melanoma cell line treated with Ki676 (5uM) for 30 min and 1hr.(B) mRNA levels of NRF2 targets NQO-1 and HO-1 in A375 melanoma cell line (left) and in A375 melanoma cell line NRF2 knock down (right) after 4hr treatment with Ki696 5uM.Multimedia component 1

Multimedia component 1

Data availability

Data will be made available on request.

Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.redox.2024.103289.
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