Thymidine Kinase 1 Expression Research Articles

A comprehensive analysis and experimental validation of TK1 in uterine corpus endometrial carcinoma

Uterine corpus endometrial carcinoma (UCEC) is becoming a main malignant cancer that threaten to women's health. Thymidine kinase 1 (TK1) is considering to be associated with tumorigenesis and development. Nevertheless, the function of TK1 in UCEC is still unclear. Herein, we analyzed the TK1 expression level in pan-cancer and found that TK1 was upregulated in a variety of cancers including UCEC. Patients of UCEC with high expression of TK1 were related to poor outcome. TK1 was also related to clinical stage, histologic grade and lymph node metastasis. Abnormal expression of TK1 in UCEC was related to promoter methylation while gene mutation was not frequent. TK1 and its associated genes appeared to be prominent in cell cycle and DNA replication, according to GO and KEGG analysis. Analysis of immune infiltration revealed a negative correlation between TK1 and CD8 + T cells, macrophages, and dendritic cells. In vitro experiments, TK1 knockdown resulted in the inhibition of proliferation, migration, invasion and EMT in UCEC cell lines.

TK1 expression influences pathogenicity by cell cycle progression, cellular migration, and cellular survival in HCC 1806 breast cancer cells.

Breast cancer is the most common cancer diagnosis worldwide accounting for 1 out of every 8 cancer diagnoses. The elevated expression of Thymidine Kinase 1 (TK1) is associated with more aggressive tumor grades, including breast cancer. Recent studies indicate that TK1 may be involved in cancer pathogenesis; however, its direct involvement in breast cancer has not been identified. Here, we evaluate potential pathogenic effects of elevated TK1 expression by comparing HCC 1806 to HCC 1806 TK1-knockdown cancer cells (L133). Transcriptomic profiles of HCC 1806 and L133 cells showed cell cycle progression, apoptosis, and invasion as potential pathogenic pathways affected by TK1 expression. Subsequent in-vitro studies confirmed differences between HCC 1806 and L133 cells in cell cycle phase progression, cell survival, and cell migration. Expression comparison of several factors involved in these pathogenic pathways between HCC 1806 and L133 cells identified p21 and AKT3 transcripts were significantly affected by TK1 expression. Creation of a protein-protein interaction map of TK1 and the pathogenic factors we evaluated predict that the majority of factors evaluated either directly or indirectly interact with TK1. Our findings argue that TK1 elevation directly increases HCC 1806 cell pathogenicity and is likely occurring by p21- and AKT3-mediated mechanisms to promote cell cycle arrest, cellular migration, and cellular survival.

Thymidine kinase 1 as a target is regulated by the hsa-let-7b-5p/LINC00665 axis and affects NSCLC prognosis

BackgroundIn the past, multiple studies have offered incremental evidence that indicates that competitive endogenous RNA (ceRNA) regulatory networks are involved in tumor growth and present novel therapeutic targets. Herein, we investigated the impact of thymidine kinase 1 (TK1)-related ceRNA networks on the prognosis of non-small cell lung cancer (NSCLC). MethodsTK1 expression data in NSCLC and normal tissue samples were retrieved from the Cancer Genome Atlas (TCGA) database and were then compared. Thereafter, the findings of the immunohistochemical staining experiments and clinical follow-up data derived from patients with NSCLC were used for conducting prognostic analysis. The starBase database was searched to determine TK1-targeted microRNAs and long non-coding RNAs, and clinical data from TCGA were used for survival analysis to construct a ceRNA network associated with TK1 expression and prognosis. Finally, the roles played by methylation and immunity in the prognosis and treatment of NSCLC were analyzed. ResultsOur findings revealed that the cancer tissues expressed significantly higher TK1 levels than normal tissues, and the follow-up clinical data revealed that the prognosis was generally worse in the high-expression patients than in the low-expression patients. In addition, clinical data collected from the starBase and TCGA databases showed that the LINC00665/has-let-7b-5p/TK1 network could influence the growth and prognosis of NSCLC. It was also noted that the TK1 methylation site was correlated with the prognosis of NSCLC, and immunoprognostic analysis further indicated that patients with higher TK1 expression levels displayed a worse prognosis. ConclusionWhen the regulatory network of LINC00665/has-let-7b-5p/TK1 was assessed, it was observed that elevated TK1 levels may affect the prognosis of NSCLC. Therefore, it could be considered a prognostic biomarker and a probable therapeutic target for predicting NSCLC prognosis.

Transcription factor Dp-1 knockdown downregulates thymidine kinase 1 expression to protect against proliferation and epithelial-mesenchymal transition in cervical cancer.

Thymidine kinase 1 (TK1) level is an independent survival prognostic factor for both premalignant and malignant cervical pathologies. Herein, this study sought to probe the impacts of TK1 on cervical cancer (CC) progression and its underlying mechanism. Transcription factor Dp-1 (TFDP1) and TK1 expression was assessed using qRT-PCR in CC cell lines. After ectopic expression and knockdown experiments, cell counting kit-8 and colony formation assays were adopted to measure cell proliferation, western blot to examine the expression of epithelial-mesenchymal transition (EMT)-related proteins, and Transwell assays to assess cell invasion and migration. The binding of TFDP1 to TK1 was predicted by bioinformatic sites and verified by chromatin immunoprecipitation and dual-luciferase reporter assays. Tumor xenograft experiments in nude mice were performed to validate the influence of TFDP1/TK1 on CC progression in vivo. CC cells had high TK1 and TFDP1 expression. TFDP1 or TK1 knockdown restrained CC cell EMT, invasion, migration, and proliferation. TFDP1 facilitated TK1 expression in CC via transcription. Overexpression of TK1 counteracted the suppressive impacts of TFDP1 knockdown on CC cell malignant behaviors. Moreover, TFDP1 knockdown depressed CC growth in vivo by downregulating TK1. TFDP1 knockdown restricted proliferation and EMT in CC by downregulating TK1 expression.

Regulatory T cells are associated with the tumor immune microenvironment and immunotherapy response in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with a high risk of distant metastasis, an extremely poor prognosis, and a high risk of death. Regulatory T cells (Tregs) contribute to the formation of a tumor immunosuppressive microenvironment, which plays an important role in the progression and treatment resistance of TNBC. A public single-cell sequencing dataset demonstrated increased infiltration of Tregs in TNBC tissues relative to normal breast tissue. Weighted gene co-expression network analysis was used to identify Treg infiltration-related modules for METABRIC TNBC samples. Subsequently, we obtained two Treg infiltration-associated clusters of TNBC by applying consensus clustering and further constructed a prognostic model based on this Treg infiltration-associated gene module. The ability of the selected gene in the prognostic model, thymidine kinase-1 (TK1), to promote the progression of TNBC was evaluated in vitro. We concluded that two Treg infiltration-associated clusters had different prognoses and sensitivities to drugs commonly used in breast cancer treatment, and multi-omics analysis revealed that the two clusters had different copy number variations of key tumor progression genes. The 7-gene risk score based on TNBC Treg infiltration was a reliable prognostic indicator both in the training and validation cohorts. Moreover, patients with TNBC with high Treg infiltration-related scores lacked the activation of immune activation pathways and exhibited resistance to anti-PD1 immunotherapy. Knocking down TK1 led to impaired proliferation, migration, and invasion of TNBC cells in vitro. In addition, specimens from patients with TNBC with high TK1 expression showed significantly higher Treg infiltration in tumors. Results of spatial transcriptome analysis showed that TK1 positive cells mainly localize in tumor area, and Treg cell infiltration in TNBC tissues was associated with high expression of TK1. Pan-cancer analysis also demonstrated that TK1 is associated with poor prognosis and activation of proliferation pathways in multiple cancers. We established a prognostic model related to Treg infiltration and this model can be used to establish a clinically relevant classification of TNBC progression. Additionally, our work revealed the underestimable potential of TK1 as a tumor biomarker and immunotherapeutic target.

Effect of Nucleolin on Lymphoma Proliferation by Regulating Thymidine Kinase 1

To investigate the mechanism of nucleolin (NCL) involved in lymphoma proliferation by regulating thymidine kinase 1 (TK1). Twenty-three patients with diffuse large B-cell lymphoma (DLBCL) were selected and divided into initial treatment group (14 cases) and relapsed/refractory group (9 cases). Serum TK1 and C23 protein in peripheral blood mononuclear cells were detected. Cell models of CA46-NCL-KD (CA46-NCL-knockdown) and CA46-NCL-KNC (CA46-NCL-knockdown negative control) were established by lentivirus vector mediated transfection in Burkitt lymphoma cell line CA46. The half maximal inhibitory concentration (IC50) of CA46-NCL-KD, CA46-NCL-KNC, and CA46 to adriamycin were detected by cell proliferation assay (MTS). The expression of NCL mRNA and protein in CA46-NCL-KD and CA46-NCL-KNC cells were dectected by Q-PCR and Western blot, respectively. The cell cycle of CA46-NCL-KD, CA46-NCL-KNC, and CA46 cells were detected by flow cytometry. The expression of TK1 protein in CA46-NCL-KD and CA46-NCL-KNC cells was detected by an enhanced chemiluminescence (ECL) dot blot assay. The level of serum TK1 in the initial treatment group was 0.43(0-30-1.01) pmol/L, which was lower than 10.56(2.19-14.99) pmol/L in the relapsed/refractory group (P<0-01), and the relative expression level of NCL protein in peripheral blood was also significantly lower. The IC50 of CA46-C23-KD cells to adriamycin was (0.147±0.02) μg/ml, which was significantly lower than (0.301±0.04) μg/ml of CA46-C23-KNC cells and (0.338±0.05) μg/ml of CA46 cells (P<0.05). Compared with CA46-NCL-KNC cells, the expression of NCL mRNA and protein, TK1 protein decreased in CA46-NCL-KD cells, and the proportion of S phase and G2/M phase also decreased, while G0/G1 phase increased in cell cycle. The increased expression of NCL in DLBCL and CA46 cells indicates low sensitivity to drug. NCL may participate in regulation of lymphoma proliferation by affecting TK1 expression, thereby affecting the drug sensitivity.

Visualization research on ENT1/NIS dual-function gene therapy to reverse drug resistance mediated by MUC1 in GEM-resistant pancreatic cancer

PurposeTo use bifunctional target genes to increase the intracellular transport of gemcitabine (GEM) to reverse chemotherapy resistance and to simultaneously use reporter gene imaging to localize therapeutic genes. The therapeutic effect was evaluated by [18F]FLT PET/CT to visualize the effect of gene therapy. MethodsA viral gene vector containing the pancreatic cancer-targeting promoter MUC1 for specific transcription of equilibrative nucleoside transporter 1 (ENT1) and NIS (nuclide transport channel) was employed. [125I]NaI uptake tests and [131I]NaI SPECT imaging were performed to verify the function of NIS and the target function of MUC1. The correlation between [18F]FLT uptake and GEM resistance were assessed, and the influence ENT1 and thymidine kinase 1 (TK1) expression on [18F]FLT micro-PET/CT was measured, which provides a theoretical basis for the use of [18F]FLT micro-PET/CT to evaluate the efficacy of gene therapy. ResultsFirst, functions of gene therapy were confirmed: ENT1 reversed the drug resistance of GEM-resistant pancreatic cancer cells by increasing GEM intracellular transport; MUC1 drove NIS target gene expression in pancreatic cancer; and therapeutic genes could be localized using [131I]NaI SPECT reporter gene imaging. Second, the [18F]FLT uptake ratio was affected by drug resistance and GEM treatment. The mechanism underlying this effect was related to ENT1 and TK1. Increased expression of ENT1 inhibited the expression of TK1 after GEM chemotherapy to reduce the uptake of [18F]FLT. Finally, micro-PET/CT indicated that the SUVmax of [18F]FLT could predict survival time. SUVmax exhibited an increasing trend in resistant pancreatic cancer but a trend of inhibition after upregulation of ENT1, which was more significant after GEM treatment. ConclusionsBifunctional targeted genes can localize therapeutic genes through reporter gene imaging, reverse the drug resistance of GEM-resistant pancreatic cancer and be visually evaluated through [18F]FLT micro-PET/CT.

TK1 is an early screening marker for uterine corpus endometrial carcinoma and promotes its progression

BackgroundThe functional role of thymidine kinase 1(TK1) in uterine corpus endometrial carcinoma (UCEC) is unknown. MethodsThe expression of TK1 between UCEC and adjacent normal tissues was compared using different data sets. Clinical and survival data were used to determine the clinical and prognostic significance of TK1 in UCEC. Serum levels of TK1 were measured by ELISA. The role of TK1 in UCEC cells cultivated in vitro was investigated by loss-of-function tests. GO/KEGG analysis was performed using the TCGA-UCEC dataset to explore possible mechanisms for the role of TK1 in UCEC progression. ResultsWe found that the expression of TK1 increased with the malignancy of UCEC, and TK1 was highly expressed and significantly associated with UCEC prognosis. AUC of ROC curve (0.933) showed that serum TK1 levels may be a potential biomarker to identify UCEC patients. Serum levels of TK1 were higher before surgical treatment and significantly decreased after surgical treatment. Knockdown of TK1 inhibited the growth of UCEC cells. Mechanistically, the function of TK1 may be attributed to its effect on major pathways such as cell cycle. TK1 deletion significantly blocked the S phase in UCEC cells. ConclusionsOverall, TK1 may be a new marker for early screening and prognosis prediction of UCEC, as well as a potential therapeutic target of UCEC.

Abstract 159: Thymidine kinase 1 and cell cycle control in breast cancer

Abstract Recent advances in cancer immunotherapy have transformed biomarkers into diagnostic and prognostic indicators and immunotherapeutic targets in clinical oncology. To ensure higher treatment success, it is critical to identify unique biomarkers to mitigate “off-target” effects. Therefore, the identification of novel biomarkers and appropriate target selection has never been more important to improve cancer immunotherapy. The DNA salvage pathway protein thymidine kinase 1 (TK1) plays an important role in DNA synthesis and repair. Normally, it is highly expressed during S phase and provides recycled nucleotides by phosphorylating thymidine to make thymidine monophosphate. Past studies have suggested that TK1 may influence cell cycle control and, therefore, provides a platform for the understanding of disease progression and possible means of mitigation. The purpose of our study was to verify elevated TK1 levels in breast cancer cells and further investigate TK1’s potential influence over cell cycle control. We hypothesized that TK1 may be important in regulating the cell cycle and other cell checkpoint proteins. Immunohistochemistry analysis was employed to quantify TK1 expression in ductal and lobular primary and matched metastatic breast cancer samples compared to normal tissue. Bioinformatics was then used to analyze RNA-seq data of cell cycle checkpoint factors and TK1 in BRCA patients from the Cancer Genome Atlas. To demonstrate TK1’s potential effect on the cell cycle checkpoint pathway, we utilized HCC1806 breast cancer cells and generated a CRISPR-Cas9 TK1 knockdown (L133) cell line for in vitro testing. These were validated by western blotting and qPCR. Propidium iodide staining and cell cycle analysis were performed via flow cytometry on both cell lines. Immunohistochemistry results indicated that TK1 levels increase as breast cancer progresses and, thus, may correlate with breast cancer aggressiveness. Bioinformatics analysis showed that strong positive correlations exist between cell cycle checkpoint factors and TK1 in BRCA patients, such as BRCA1 and CHEK2. Flow cytometry cell cycle analysis showed that mean differences of L133 cells in S phase were significantly higher than in G1 compared to HCC 1806 cells, suggesting that TK1 levels influence cell cycle arrest. Our preliminary results suggest that TK1 may affect cell cycle checkpoint pathways, which are essential to maintaining homeostasis in normal cells and preventing cancer progression. Further investigation to understand TK1’s potential interactions with important checkpoint factors, such as p53 or Gas6, may help elucidate other therapeutic targets for preventing disease progression. Citation Format: Eliza E. Bitter, Toni Mortimer, Rachel Morris, Kai Barlow, Abby Schekall, Michelle Townsend, Brett E. Pickett, Kim L. O'Neill. Thymidine kinase 1 and cell cycle control in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 159.

Assessing the Potential Prognostic and Immunological Role of TK1 in Prostate Cancer.

Background: It has been reported that thymidine kinase 1 (TK1) was up-regulated in multiple malignancies and participated in the regulation of tumor malignant behavior. However, its specific role in prostate cancer (PCa) remains unclear. Methods: TK1 expression in PCa patients and cell lines was identified via crossover analysis of the public datasets. A series of in vitro experiments and in vivo models was applied to investigate the function of TK1 in PCa. Functional enrichment analyses were further conducted to explore the underlying mechanism. Additionally, TISIDB was applied to explore the correlation between TK1 expression and tumor-infiltrating lymphocytes, immune subtypes, and immune regulatory factors. Results: TK1 expression was significantly up-regulated in PCa patients and cell lines. TK1 ablation inhibited tumor cell proliferation and migration potential, and in vivo experiments showed that TK1 inactivation can significantly restrain tumor growth. Functional enrichment analysis revealed TK1-related hub genes (AURKB, CCNB2, CDC20, CDCA5, CDK1, CENPA, CENPM, KIF2C, NDC80, NUF2, PLK1, SKA1, SPC25, ZWINT), and found that TK1 was closely involved in the regulation of cell cycle. Moreover, elevated mRNA expression of TK1 was related with higher Gleason score, higher clinical stage, higher pathological stage, higher lymph node stage, shorter overall survival, and DFS in PCa patients. Particularly, TK1 represented attenuated expression in C3 PCa and was related with infiltration of CD4+, CD8+ T cells, and dendritic cells as well as immunomodulator expression. Conclusion: Our study indicates that TK1 is a prognostic predictor correlated with poor outcomes of PCa patients, and for the first time represented that TK1 can promote the progression of PCa. Therefore, TK1 may be a potential diagnostic and prognostic biomarker, as well as a therapeutic target for PCa.

A combined strategy of TK1, HE4 and CA125 shows better diagnostic performance than risk of ovarian malignancy algorithm (ROMA) in ovarian carcinoma

BackgroundThe dual marker algorithm Risk of Ovarian Malignancy Algorithm (ROMA) has been widely used in the clinic for the identification of equivocal pelvic masses in ovarian carcinoma. To obtain higher diagnostic efficiency, we created a new diagnostic index, Risk of Ovarian Malignancy Index (ROMI), by combing thymidine kinase 1 (TK1), HE4 and CA125. Methods335 patients with pelvic masses on imaging and 46 healthy controls were enrolled. Serum TK1 was analyzed before further study. ROMI and ROMA were evaluated for diagnostic efficiency. ResultsThe level of TK1 was elevated in malignant ovarian tumors compared to benign masses (p < 0.001) and healthy controls (p < 0.001). TK1 expression was positively correlated with stage, intrapelvic metastasis, lymphatic metastasis and distant metastasis (all p values < 0.001). The area under the receiver operating characteristic curve (AUC) of ROMI was higher than that of ROMA for both pre- and postmenopausal women. ROMI had better sensitivity, specificity, accuracy, and positive and negative predictive values than ROMA in diagnosis of all-stage or stage I + II ovarian carcinoma for both pre- and postmenopausal women. ConclusionsTK1 is a potential biomarker in detection of ovarian carcinoma. ROMI shows better diagnostic performance than ROMA in distinguishing malignant ovarian tumors from benign masses.

Immunohistochemistry for Thymidine Kinase-1 (TK1): A Potential Tool for the Prognostic Stratification of Breast Cancer Patients.

Breast cancer (BC) is the most frequent non-cutaneous malignancy in women. Histological grade, expression of estrogen and progesterone receptors (ER and PgR), overexpression/amplification of the human epidermal growth factor receptor 2 (HER2) oncogene, and proliferative activity measured with ki-67 provide important information on the biological features of BC and guide treatment choices. However, a biomarker that allows a more accurate prognostic stratification is still lacking. Thymidine kinase-1 (TK1), a ubiquitous enzyme involved in the pyrimidine nucleotide recovery pathway, is a cell-proliferation marker with potential prognostic and predictive impacts in BC. Eighty (80) cases of invasive BC with a long-term follow-up were retrospectively selected, and clinicopathological data were collected for each patient. TK1 tissue expression was evaluated immunohistochemically. Data suggested that TK1 expression levels are positively correlated with ER and PgR expression, and negatively correlated with HER2 status and the impact on patients’ distant recurrence-free survival (DRFS): in detail, among patients undergoing adjuvant chemotherapy, lower TK1 levels are correlated with better DRFS. Therefore, these results contribute to furthering the knowledge of TK1, suggesting a possible and important role of this enzyme as a biomarker in the stratification of BC patients.

Abstract 1902: Elevated thymidine kinase 1 (TK1) in cancer serum may suppress immune function

Abstract In this project, we wanted to investigate the role that TK1 may play in macrophage activation and immune suppression. The relationship between immune response and cancer development has been an enigma that researchers have sought to elucidate for decades. Recent and past studies have suggested that the presence and abundance of nucleotides can affect immune response. Thymidine Kinase 1 (TK1) is a salvage pathway enzyme that converts thymidine to thymidine monophosphate, a precursor to DNA synthesis. It is secreted into serum and as such has been suggested to be a tumor biomarker. TK1 has also shown to be present on the surface of many different cancer cell lines including breast, lung, and colorectal cancer. The reason for this upregulation of surface TK1 is unclear, but it has been suspected that the surface expression of TK1 may be beneficial to tumor growth and survival since high TK1 expression and secretion is an early event in tumorigenesis. Although the immunosuppressive properties of purines have been well established, pyrimidines such as thymidine have not been studied as potential immune suppressants. In our investigation, we first confirmed the increased surface expression levels of TK1 in cancerous cell lines. Flow cytometry showed that non-cancerous cells expressed surface TK1 at minimal levels (0.7%), MDA-MB-231 cells expressed TK1 at higher levels (45%), and PC3 cells expressed TK1 at elevated levels as well (64.6%). Bioinformatic analysis of nucleotide transport in relation to TK1 surface expression showed a strong positive correlation between the nucleotide transporter SLC29A2 and surface TK1 expression in colon adenocarcinoma (correlation value 0.455, p-value &amp;lt;0.001), head and neck squamous cell carcinoma (correlation value 0.416, p-value &amp;lt;0.001), rectum adenocarcinoma (correlation value 0.432, &amp;lt;0.001), stomach adenocarcinoma (correlation value 0.405, p-value &amp;lt;0.001), and uterine corpus endometrial carcinoma (correlation value 0.403, p-value &amp;lt;0.001) which further confirms the elevation of TK1 on the surface of cancer cells and suggests a mechanism whereby serum TK1 levels are elevated. Macrophage engulfment assays were then conducted in order to investigate the possibility that exposure to higher concentrations of TK1 leads to immune suppression. We found that macrophages differentiated from THP-1 monocytes that were exposed to varying concentrations of TK1 for 24 hours showed a decrease in macrophage engulfment (29%-37%) compared to controls that were not exposed to TK1(61.2%). These results suggest that TK1 may have some involvement in immune suppression during cancer development, and we intend to further investigate these findings. Citation Format: Kathryn R. Smith, Rachel M. Morris, Edwin J. Velazquez, David M. Bellini, Kim O'Neill. Elevated thymidine kinase 1 (TK1) in cancer serum may suppress immune function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1902.

Abstract LB-149: Serum thymidine kinase 1 levels predict prostate cancer-specific survival

Abstract Background: Thymidine kinase 1 (TK1) is an enzyme involved in DNA synthesis. Thymidine kinases phosphorylate thymidine as part of the DNA synthetic pathway. TK1 expression is a marker of active cellular proliferation; intracellular concentrations are low during the G0/G1 phases of the cell cycle, increasing during the S/G2 phases. Previous studies have suggested that serum TK1 levels could be used as marker for presence of prostate cancer (PCa). Here, we evaluated whether serum TK1 levels could predict risk of PCa prognosis. Materials and Methods: The study population included 40 men with clinically defined T1/T2NxM0 PCa and 43 men with de novo metastatic cancer (M1) at diagnosis confirmed by bone scan imaging. All men were diagnosed and treated at the Tampere University Hospital between 2000-2010. All men provided a serum sample at the time of diagnosis. Information on deaths and causes of death were obtained from national causes of death registry. Serum TK1 concentrations were determined using the AroCell TK210 ELISA kit. Cox regression and Kaplan-Meier Curves with adjustment for tumor risk group (Gleason-grade, TNM stage and PSA level) were used to evaluate the risk of PCa death in relation to TK1 levels (median or below vs. above median). ROC analyses were used to evaluate the predictive accuracy of PSA, TK1 and their combination. Results: M1 cases were older than M0 cases; median age 72 vs 62 years, respectively. M1 cases were mainly managed with androgen deprivation therapy, whereas M0 cases were most often managed with radical prostatectomy. Median serum TK1 levels were significantly higher among M1 cases compared to M0 cases (p for difference 0.001). In the combined study population, serum TK1 levels were a significant predictor of PCa-specific (HR 8.33, 95% CI 2.05-33.88) and overall mortality (HR 5.53, 95% CI 1.93-15.85) after adjustment for established prognostic factors including tumor risk group. Kaplan-Meier survival curves defined by TK1 level started to differ within 24 months from PCa diagnosis. In ROC analyses TK1 alone or in combination with PSA was no more accurate than PSA alone in predicting PCa death. Conclusion: The proliferation biomarker TK1 in serum predicted survival after PCa diagnosis, demonstrating independent predictive value over established clinical risk factors. If confirmed in further and larger studies, this biomarker could be incorporated in PCa risk stratification when selecting optimal treatment and surveillance schedule. Citation Format: Teemu J. Murtola, Paavo Raittinen, Teuvo Tammela. Serum thymidine kinase 1 levels predict prostate cancer-specific survival [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-149.

CCNB2, NUSAP1 and TK1 are associated with the prognosis and progression of hepatocellular carcinoma, as revealed by co-expression analysis.

The mortality rate associated with hepatocellular carcinoma (HCC) is the third highest among all digestive system tumors. However, the causes of HCC development and the underlying mechanisms have remained to be fully elucidated. In the present bioinformatics study, genetic markers were identified and their association with HCC was determined. The mRNA expression datasets GSE87630, GSE74656 and GSE76427 were downloaded from the Gene Expression Omnibus (GEO) database. A total of 96 differentially expressed genes (DEGs) were screened from the 3 GEO datasets, including 25 upregulated and 71 downregulated genes. DEGs were uploaded to the database for Annotation, Visualization and Integrated Discovery to screen for enriched Gene Ontology terms in various categories and the Search Tool for the Retrieval of Interacting Genes/Proteins was used to identify the interactions and functions of the DEGs. A total of 3 genetic markers were identified in a stepwise pathway and functional analysis in a previous study. The association of the genetic markers with prognosis was analysed using the UALCAN online analysis tool. Regression analysis was also performed to identify the relationship between HCC grade and disease recurrence and the expression of genetic markers using The Cancer Genome Atlas HCC dataset. In addition, the expression of the 3 genetic markers in HCC tissues was determined using reverse transcription-quantitative PCR, the Oncomine database and the Human Protein Atlas database. The expression levels of the 3 genetic markers cyclin B2 (CCNB2), nucleolar and spindle-associated protein 1 (NUSAP1) and thymidine kinase 1 (TK1) were significantly correlated with each other and high mRNA expression of CCNB2 was significantly associated with poor overall survival of patients with HCC. Receiver operating characteristic curve analysis indicated that NUSAP1 and TK1 were capable of distinguishing between recurrent and non-recurrent HCC. Furthermore, CCNB2, NUSAP1 and TK1 were highly correlated with the HCC grade. It was also indicated that the mRNA expression of CCNB2, NUSAPA and TK1 was increased in primary HCC tissues when compared with that in adjacent tissues. The present study identified that the CCNB2, NUSAP1 and TK1 genes may serve as prognostic markers for HCC, and may be of value from the perspectives of basic research and clinical treatment of HCC.

Knockdown of Thymidine Kinase 1 Suppresses Cell Proliferation, Invasion, Migration, and Epithelial-Mesenchymal Transition in Thyroid Carcinoma Cells.

Patients with advanced thyroid carcinoma have poor prognosis with low overall survival. Unfortunately, the underlying mechanisms of thyroid carcinoma progression remain unclear. The elevated expression of thymidine kinase 1 (TK1) has been implicated in the progression of thyroid carcinoma, while the role of TK1 in thyroid carcinoma progression has not been explored. The present study aimed to determine the role TK1 in the progression of thyroid cancer and to explore the underlying molecular mechanisms. In this study, it was found that serum TK1 levels were markedly increased in the patients with thyroid nodules. Further online data mining showed that TK1 expression was upregulated in thyroid carcinoma tissues, and higher expression of TK1 was correlated with shorter disease-free survival of patients with thyroid carcinoma. Silencing of TK1 suppressed cell proliferation, invasion, migration, and epithelial–mesenchymal transition, and also induced cell apoptosis in the thyroid carcinoma cell lines. Animal studies showed that TK1 knockdown inhibited in vivo tumor growth of thyroid carcinoma cells. Importantly, miR-34a-5p was found to be downregulated in the thyroid carcinoma cells. Furthermore, miR-34a-5p targeted the 3′ untranslated region of TK1 and suppressed the expression of TK1 in thyroid carcinoma cell lines. In summary, first, these results demonstrated the upregulation of TK1 in thyroid nodules and thyroid carcinoma tissues; second, TK1 promoted thyroid carcinoma cell proliferation, invasion, and migration; lastly, TK1 was negatively regulated by miR-34a-5p. Our study may provide novel insights into the role of TK1 in regulating thyroid carcinoma progression.

Effect of hypoxia on human equilibrative nucleoside transporters hENT1 and hENT2 in breast cancer.

Elevated proliferation rates in cancer can be visualized with positron emission tomography (PET) using 3'-deoxy-3'-l-[18F]fluorothymidine ([18F]FLT). This study investigates whether [18F]FLT transport proteins are regulated through hypoxia. Expression and function of human equilibrative nucleoside transporter (hENT)-1, hENT2, and thymidine kinase 1 (TK1) were studied under normoxic and hypoxic conditions, and assessed with [18F]FLT-PET in estrogen receptor positive (ER+)-MCF7, triple-negative MDA-MB231 breast cancer (BC) cells, and MCF10A cells (human mammary epithelial cells). Functional involvement of hENT2 [18F]FLT transport was demonstrated in all cell lines. In vitro [18F]FLT uptake was higher in MDA-MB231 than in MCF7: 242 ± 9 vs. 147 ± 18% radioactivity/mg protein after 60 min under normoxia. Hypoxia showed no significant change in radiotracer uptake. Protein analysis revealed increased hENT1 (P < 0.0963) in MDA-MB231. Hypoxia did not change expression of either hENT1, hENT2, or TK1. In vitro inhibition experiments suggested involvement of hENT1, hENT2, and human concentrative nucleoside transporters during [18F]FLT uptake into all cell lines. In vivo PET imaging revealed comparable tumor uptake in MCF7 and MDA-MB231 tumors over 60 min, reaching standardized uptake values of 0.96 ± 0.05 vs. 0.89 ± 0.08 (n = 3). Higher hENT1 expression in MDA-MB231 seems to drive nucleoside transport, whereas TK1 expression in MCF7 seems responsible for comparable [18F]FLT retention in ER+ tumors. Our study demonstrates that hypoxia does not significantly affect nucleoside transport as tested with [18F]FLT in BC.-Krys, D., Hamann, I., Wuest, M., Wuest, F. Effect of hypoxia on human equilibrative nucleoside transporters hENT1 and hENT2 in breast cancer.

Overexpression of TK1 and CDK9 in plasma-derived exosomes is associated with clinical resistance to CDK4/6 inhibitors in metastatic breast cancer patients.

Cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) improve progression-free survival (PFS) in patients with hormone receptor-positive (HR+) advanced breast cancer. However, a better knowledge of predictive biomarkers of response and resistance to CDK4/6i is needed. Therefore, the present article addresses the role of the mRNA expression of thymidine kinase 1 (TK1), CDK4, 6 and 9 in plasma-derived exosomes and their relevance in the pharmacologic activity of CDK4/6i. Blood samples of 40 HR+/HER2- advanced breast cancer patients were collected before (T0) the administration of palbociclib plus hormonal therapy and after 3 months (T1). RNA was isolated from exosomes and analysed for the expression of TK1, CDK 4, 6 and 9 by digital droplet PCR (ddPCR). A higher value of TK1 copies/ml at baseline (T0) was significantly associated with the number of previous lines of chemotherapy (p = 0.009). In patients with PD, a significant increase was observed in the number of copies/ml of TK1 (p = 0.01) and CDK9 (p = 0.03) comparing T1 vs. T0 values. No significant correlations between response to treatment and clinical parameters were found at univariate analysis. High baseline CDK4 expression was significantly correlated with longer PFS in patients treated with fulvestrant + palbociclib (low versus high: 6.45months vs. not reached, p = 0.01). The present study demonstrates that, in plasma-derived exosomes, highbaseline CDK4 mRNA levels are associated with response to palbociclib plus hormonal therapy, while the increase in TK1 and CDK9 mRNA copies/ml is associated with clinical resistance.

Abstract 2858: Thymidine kinase 1 variability in primary and metastatic human breast cell lines and its correlation to metastatic potential

Abstract The purpose of this study is to determine the role of Thymidine Kinase 1 (TK1) in the cellular invasion of breast cancer cells in vitro. TK1 is a cytosolic DNA salvage pathway enzyme responsible for the conversion of thymidine to thymidine monophosphate; it is known to increase during G1/S phase of the cell cycle and is significantly elevated in the serum of multiple cancer patients including breast, lung, and colon. As such, TK1 has been implicated as a useful biomarker for cancer prognosis and patient monitoring. In addition to TK1 upregulation in cancer serum, recent findings have shown that TK1 also localizes to the surface of ALL, AML, and colon cancer cells in patients. However, minimal studies have determined how the surface localization of TK1 is an indicator of metastatic potential. In addition, the function and timing of TK1 expression on the cellular surface has not been determined. When comparing the TK1 expression in metastatic cancer cell lines we found that there was a trend of overall elevation in metastatic cell lines (n = 39) when compared to primary cell lines (n = 31). This expression was extremely irregular between cell lines and TK1 showed significant variability between samples. We tested several breast cancer cell lines (primary: HCC 1806, HCC 1937, and JIMT-1; metastatic: T47D, BT 549, and MDA-MB-231) for their TK1 surface localization, more specifically the difference between metastatic cells and primary cells. We found significant TK1 expression in all cell lines tested with the highest expression in HCC 1806 primary breast cancer cells (90.36%; p = .0001) and the lowest expression in MDA-MB-231 metastatic breast cancer cells (47.2%; p = .01). We hypothesized that the expression of TK1 on the surface of cancer cells was dependent on the proliferative state of the cells and would be highest in the S phase of the cell cycle. To evaluate this hypothesis, cell cycle analysis was performed utilizing a PI stain to determine DNA content within the cells. Preliminary analysis of this data indicates that surface localized TK1 may not be dependent on the cell cycle but may be dependent on the proliferative capacity of individual patient’s tumors. Currently under investigation is the direct effect of surface TK1 on invasion potential. To accomplish this, TK1-/-cells are actively being produced and their invasion potential against TK1WT cells will be compared to determine whether TK1 surface localization is influential in the metastatic ability of cancer cells. We plan to test the correlation between TK1 expression for a potential indicator of tumor aggressiveness and invasion potential. Citation Format: Eliza E. Bitter, Michelle H. Townsend, Kelsey A. Bennion, Juan Mejia, Gajendra Shrestha, Kelsey Hirschi, Juan Arroyo, Kim O'Neill. Thymidine kinase 1 variability in primary and metastatic human breast cell lines and its correlation to metastatic potential [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2858.

Cytotoxicity of trifluridine correlates with the thymidine kinase 1 expression level

Trifluridine (FTD), a tri-fluorinated thymidine analogue, is a key component of the oral antitumor drug FTD/TPI (also known as TAS-102), which is used to treat refractory metastatic colorectal cancer. Thymidine kinase 1 (TK1) is thought to be important for the incorporation of FTD into DNA, resulting in DNA dysfunction and cytotoxicity. However, it remains unknown whether TK1 is essential for FTD incorporation into DNA and whether this event is affected by the expression level of TK1 because TK1-specific-deficient human cancer cell lines have not been established. Here, we generated TK1-knock-out human colorectal cancer cells using the CRISPR/Cas9 genome editing system and validated the specificity of TK1 knock-out by measuring expression of AFMID, which is encoded on the same locus as TK1. Using TK1-knock-out cells, we confirmed that TK1 is essential for cellular sensitivity to FTD. Furthermore, we demonstrated a correlation between the TK1 expression level and cytotoxicity of FTD using cells with inducible TK1 expression, which were generated from TK1-knock-out cells. Based on our finding that the TK1 expression level correlates with sensitivity to FTD, we suggest that FTD/TPI might efficiently treat cancers with high TK1 expression.