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. 2022 Mar 3:12:802807.
doi: 10.3389/fonc.2022.802807. eCollection 2022.

Thymidine Kinase 1 Drives Skin Cutaneous Melanoma Malignant Progression and Metabolic Reprogramming

Sipeng Zuo  1   2 , Huixue Wang  1   2 , Lin Li  1   2 , Hui Pan  1   2 , Linna Lu  1   2
Affiliations

Thymidine Kinase 1 Drives Skin Cutaneous Melanoma Malignant Progression and Metabolic Reprogramming

Sipeng Zuo et al. Front Oncol. .

Abstract

Background: Thymidine kinase 1 (TK1) is a cell cycle-dependent kinase that catalyzes the addition of a gamma-phosphate group to thymidine. The protumorigenic role of TK1 has been reported in various malignancies. However, the role of TK1 in skin cutaneous melanoma (SKCM) remains unclear. This study aimed to explore the molecular function of TK1 in SKCM progression.

Methods: Bioinformatics data were acquired from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). Subcutaneous xenografts were established to observe the effect of TK1 knockdown on the proliferation of SKCM cells in vivo. RNA sequencing (RNA-seq; deposited in Sequence Read Archive, SRX10950283-SRX10950285 for A375 control cells and SRX10950286-SRX10950288 for TK1-silenced A375 cells) and immunoprecipitation-mass spectrometry (IP-MS) were used to analyze TK1-related genes and pathways. Seahorse XF Cell Mito tests and glycolysis stress assays were conducted for metabolic testing.

Results: TK1 was upregulated in malignant SKCM compared to that in normal tissues and cell lines. Elevated expression of TK1 was associated with poor prognosis. In vitro and in vivo assays demonstrated that TK1 promoted the proliferation and migration of SKCM cells. Moreover, TK1 was strongly associated with multiple intracellular metabolic pathways, facilitating cell mitochondrial respiration and glycolysis in SKCM malignant progression.

Conclusions: TK1 drives SKCM malignant progression and supports metabolic reprogramming, indicating that TK1 serves as a therapeutic target for SKCM.

Keywords: bioinformatics; cell metabolism; skin cutaneous melanoma; thymidine kinase 1; tumorigenesis.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Upregulation of thymidine kinase 1 (TK1) in skin cutaneous melanoma (SKCM) in databases. (A) Pan-Cancer analysis of TK1 in The Cancer Genome Atlas (TCGA) database. (B) Structural variants of TK1 in diversified tumors in 10,953 patients/10,967 samples. (C) The elevated TK1 level in 25 kinds of malignancies. (D, E) Relative TK1 expression in normal and tumor tissues. The tumor tissues were 461, and the normal tissues were 558. (F) Relative TK1 expression in different pTNM stages from IIIA to IV. (G, H) Unfavorable prognosis due to higher TK1 levels in Gene Expression Omnibus (GEO) and TCGA cohorts. (I) Gene Ontology (GO) analysis to reveal TK1 function in cancer. *p < 0.05, ***p < 0.001.
Figure 2
Figure 2
A protumor role of thymidine kinase 1 (TK1) was confirmed in skin cutaneous melanoma cell lines. (A) Immunohistochemistry (IHC) staining on dozens of nevi, primary skin cutaneous melanomas (SKCMs), and metastatic SKCMs with respective fold change of TK1. (B) Relative mRNA expression of TK1 in different melanoma cell lines by quantitative real-time PCR (qRT-PCR). The experiments were performed in triplicate. (C) Relative protein expression of TK1 in different melanoma cell lines by Western blot. The experiments were performed in triplicate. (D, E) Cell Counting Kit-8 (CCK-8) assay and colony formation assay were conducted to confirm the influence of TK1 on cell proliferation. The number at the first day was set to 1 for every group. The experiments were performed in triplicate. (F, G) Transwell assay and statistics were analyzed to explore the influence of TK1 on SKCM cell migration. The experiments were performed in triplicate. **p < 0.01, ***p < 0.001.
Figure 3
Figure 3
Thymidine kinase 1 (TK1) silencing reduced skin cutaneous melanoma (SKCM) oncogenic progression in vivo. (A) Tumors excised from nude mice after 3 weeks. (B, C) Tumor volumes and weights. (D) Ki-67 and TK1 index of tumors in the control group and TK1 knockdown group. (E) Downstream effectors of the TK1 pathway from tumor tissues were downregulated. *p < 0.05, ***p < 0.001.
Figure 4
Figure 4
Thymidine kinase 1 (TK1) was associated with skin cutaneous melanoma (SKCM) cell metabolism disturbance. (A) Altered genes with TK1 knockdown in SKCM. |log2FC| >1, p < 0.05. (B) Integrative Genomics Viewer (IGV) shows the expression of TK1 in knockdown/control group. The data range was set to 1,274. (C) Fifteen pathways of top enrichment of changed genes with TK1 knockdown. (D) Western blot confirmed that TK1 was purified successfully in lysate and had no impurity. (E) Fifteen top enrichment pathways of all genes that corresponded to proteins interacting with TK1; most were relevant to intracellular metabolic processes.
Figure 5
Figure 5
The oxygen consumption rate (OCR) was suppressed in thymidine kinase 1 (TK1) knockdown group. (A) The mitochondrial respiration curve of control group and TK1 downregulation group. (B–G) The basal respiration, maximal respiration, spare respiratory capacity, ATP production, proton leak, and non-mitochondrial oxygen consumption were inhibited in TK1 knockdown group. ***p < 0.001, ****p < 0.0001.
Figure 6
Figure 6
The extracellular acidification rate (ECAR) was suppressed in thymidine kinase 1 (TK1) knockdown group. (A) The glycolytic function curve of control group and TK1 downregulation group. (B, C) The glycolysis and glycolytic capacity were inhibited in TK1 knockdown group. ***p < 0.001, ****p < 0.0001.
Figure 7
Figure 7
Oxidative phosphorylation (OxPhos) inhibitor attenuated proliferation and migration of skin cutaneous melanoma (SKCM) cells. (A) The growth ability of SKCM was attenuated after treatment with OxPhos inhibitor by Cell Counting Kit-8 (CCK-8) assay. (B) Colony formation assay demonstrated that SKCM formed smaller and fewer colonies after treating with 0.25 and 0.5 μM rotenone. (C, D) Cellular migration capacity was significantly attenuated by Transwell and wound healing assay after treatment with OxPhos inhibitor. *p < 0.05, **p < 0.01
Figure 8
Figure 8
A schematic diagram of functions of thymidine kinase 1 (TK1) in skin cutaneous melanoma (SKCM).
See this image and copyright information in PMC

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