Thyroid Hormone Receptor Interactor 13 Research Articles

TRIP13 is Critical for the Survival of B-cell Lymphomas and Myeloid Leukemias In Vitro

Hematologic malignancies represent a diverse group of blood tumors accounting for approximately 10% of all cancer cases in the US. Based on the site of manifestation, presumed cell of origin, genetic abnormalities, and clinical features, we recognize more than 100 clinically important subtypes of hematologic malignancies broadly categorized into three main groups: leukemia, lymphoma, and multiple myeloma. While the prognosis for patients with specific subtypes of hematologic malignancies, such as Hodgkin lymphomas or chronic lymphocytic leukemia has significantly improved over the last several years, other subtypes such as acute myeloid leukemia or non-Hodgkin lymphomas still present with significant treatment challenges. A better understanding of molecular drivers of tumor maintenance is needed to improve existing therapies. Thyroid hormone receptor interactor 13 (TRIP13) is an AAA + ATPase that plays an important yet poorly understood role in the regulation of the mammalian cell cycle. The TRIP13 gene is highly expressed in various human tumors including colorectal carcinoma and glioblastoma, and it seems to promote tumorigenesis. Recently, we identified TRIP13 as a gene overexpressed in Peripheral T-cell lymphomas where it plays a critical role in tumor maintenance by regulating the G2-M phase of the cell cycle. However, the biological activities of TRIP13 in non-T cell hematologic malignancies have not been investigated to date. Here we used shRNA-mediated knockdown and a small molecule inhibitor to downregulate TRIP13 in B-cell lymphoid and myeloid lineages. We found genetic downregulation inhibited the proliferation of both B-cell lymphomas and acute myeloid leukemia cell lines in vitro. The pharmacologic inhibition of TRIP13 effectively induced the G0/G1 cell cycle arrest and apoptosis of B-cell lymphomas. Altogether these data demonstrate that TRIP13 is critical for the maintenance of hematologic malignancies of B-cell and myeloid malignancies in vitro. Thus, TRIP13 might be a good target in the treatment of various types of hematologic malignancies.

TRIP13: A promising cancer immunotherapy target.

The tumor microenvironment (TME) facilitates tumor development through intricate intercellular signaling, thereby supporting tumor growth and suppressing the immune response. Thyroid hormone receptor interactor 13 (TRIP13), an AAA+ ATPase, modulates the conformation of client macromolecules, consequently influencing cellular signaling pathways. TRIP13 has been implicated in processes such as proliferation, invasion, migration, and metastasis during tumor progression. Recent studies have revealed that TRIP13 also plays a role in immune response suppression within the TME. Thus, inhibiting these functions of TRIP13 could potentially enhance immune responses and improve the efficacy of immune checkpoint inhibition. This review summarizes the recent research progress of TRIP13 and discusses the potential of targeting TRIP13 to improve immune-based therapies for patients with cancer.

Identification of differentially expressed genes and screening for key genes involved in ovarian cancer prognosis: An integrated bioinformatics and network analysis approach

Objectives: Ovaries are important and essential organs of animals in producing and releasing eggs. Ovarian cancer (OvCa) is one of the most prevalent lethal gynecological malignancies with a lack of distinct biomarkers. Advances in high-throughput genomic data and the continued refinement of bioinformatics tools enable the identification of potential biomarkers. Leveraging these insights, we can employ systems biology approaches to enhance the accuracy of diagnosis and prognosis. Material and Methods: A comparative analysis was conducted between normal and tumor samples, employing bioinformatics software and tools. Differential expression analysis utilized fold-change statistics, while DAVID 6.8 software was used to perform gene ontology enrichment analysis and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses. The protein-protein interaction (PPI) network was constructed differentially expressed genes (DEGs) using Search Tool for the Retrieval of Interacting Genes database, and Cytoscape 3.9.1, along with its Molecular Complex Detection and CytoHubba plugins, facilitated network visualization, analysis, and module detection. Hub gene expression and overall survival were explored through the Kaplan–Meier plotter, while Gene Expression Profiling Interactive Analysis 2 analyzed the tumor stage of OvCa patients. Hub genes protein expression was analyzed using the human protein atlas database through immunostaining results. The NetworkAnalyst program and Cytoscape were employed to analyze and visualize the transcription factor-hub gene associations. Subsequently, single-nucleotide variation, methylation, and pathway activity of hub genes were examined. Validation of hub genes messenger RNA expression was done using quantitative real-time polymerase chain reaction analysis. Results: 607 DEGs, including 248 upregulated and 359 downregulated genes, were identified. The top 20 candidate genes were screened out through PPI network analysis. We discovered that the genes BUB1 Mitotic Checkpoint Serine/Threonine Kinase B (BUB1B), Cyclin A2 (CCNA2), Mitotic Arrest Deficient 2 Like 1 (MAD2L1), Protein Regulator of Cytokinesis 1 (PRC1), Thyroid Hormone Receptor Interactor 13 (TRIP13), and ZW10 Interacting Kinetochore Protein (ZWINT) exhibited significant importance in OvCa prognosis. Conclusion: Six genes, BUB1B, CCNA2, MAD2L1, PRC1, TRIP13, and ZWINT (identified as functional hub genes), are probably playing tumor-promotive roles, except TRIP13. All genes product is functionally related to the cell cycle. These can be targeted in quest of potential therapeutics for OvCa treatment.

Genome-Wide Methylation Profiling of Peripheral T-Cell Lymphomas Identifies TRIP13 as a Critical Driver of Tumor Proliferation and Survival.

Cytosine methylation contributes to the regulation of gene expression and normal hematopoiesis in mammals. It is catalyzed by the family of DNA methyltransferases that include DNMT1, DNMT3A, and DNMT3B. Peripheral T-cell lymphomas (PTCLs) represent aggressive mature T-cell malignancies exhibiting a broad spectrum of clinical features with poor prognosis and inadequately understood molecular pathobiology. To better understand the molecular landscape and identify candidate genes involved in disease maintenance, we profiled DNA methylation and gene expression of PTCLs. We found that the methylation patterns in PTCLs are deregulated and heterogeneous but share 767 hypo- and 567 hypermethylated differentially methylated regions (DMRs) along with 231 genes up- and 91 genes downregulated in all samples, suggesting a potential association with tumor development. We further identified 39 hypomethylated promoters associated with increased gene expression in the majority of PTCLs. This putative oncogenic signature included the TRIP13 (thyroid hormone receptor interactor 13) gene whose genetic and pharmacologic inactivation inhibited the proliferation of T-cell lines by inducing G2-M arrest and apoptosis. Our data thus show that human PTCLs have a significant number of recurrent methylation alterations that may affect the expression of genes critical for proliferation whose targeting might be beneficial in anti-lymphoma treatments.

TRIP13 Activates Glycolysis to Promote Cell Stemness and Strengthen Doxorubicin Resistance of Colorectal Cancer Cells.

Chemotherapy resistance is one of the main causes of clinical chemotherapy failure. Current cancer research explores the drug resistance mechanism and new therapeutic targets. This work aims to elucidate the mechanism of thyroid hormone receptor interactor 13 (TRIP13) affecting doxorubicin (DOX) resistance in colorectal cancer (CRC). Bioinformatics analyses were employed to clarify TRIP13 expression in CRC tissues and predict the correlation of the TRIP13 enrichment pathway with glycolysis-related genes and stemness index mRNAsi. Quantitative real-time polymerase chain reaction and western blot were adopted to analyze the expression of TRIP13 and glycolysis- related genes. Cell Counting Kit-8 was utilized to determine the cell viability and IC50 value. Western blot was employed to measure the expression of stemness-related factors. Cell function assays were performed to detect cells' sphere-forming ability and glycolysis level. Animal models were constructed to determine the effects of TRIP13 expression on CRC tumor growth. TRIP13 was significantly overexpressed in CRC, concentrated in the glycolysis signaling pathway, and positively correlated with stemness index mRNAsi. High expression of TRIP13 facilitated DOX resistance in CRC. Further mechanistic studies revealed that overexpression of TRIP13 could promote cell stemness through glycolysis, which was also confirmed in animal experiments. TRIP13 was highly expressed in CRC, which enhanced the DOX resistance of CRC cells by activating glycolysis to promote cell stemness. These findings offer new insights into the pathogenesis of DOX resistance in CRC and suggest that TRIP13 may be a new target for reversing DOX resistance in CRC.

The prognostic and clinical value of genes associate with immunity and amino acid Metabolism in Lung Adenocarcinoma

BackgroundLung adenocarcinoma (LUAD) is the commonest subtype of primary lung cancer. A comprehensive analysis of the association of immunity with amino acid metabolism in LUAD is critical for understanding the disease. MethodsThe present study examined LUAD and noncancerous cases from the TCGA database. Differentially expressed genes (DEGs) between LUAD and noncancerous tissues were detected by analyzing processed expression profiles. We cross-referenced the up-regulated DEGs with Immune and Amino Acid Metabolism-related genes (I&AAMGs), resulting in Immune and Amino Acid Metabolism related differentially expressed genes (IAAAMRDEGs). The STRING database was employed to analyze PPI on IAAAMRDEGs, obtaining excavated hub genes, whose biological processes, molecular functions and cellular components were examined with GO/KEGG. Potential mechanisms related to LUAD were investigated by GSEA and GSVA. A prognostic model was built by LASSO-COX analysis, taking into consideration risk scores and prognostic factors to determine biomarkers affecting LUAD occurrence and prognosis. ResultsTotally 377 genes were detected at the intersection of upregulated DEGs and I&AAMGs. Analysis of PPI on these 377 IAAAMRDEGs yielded 17 hub genes. A LASSO regression analysis was utilized to assess the prognostic values of the 17 hub genes. Validation using the combined dataset confirmed 4 genes, e.g., polo-like kinase (PLK1), Ribonucleotide Reductase Subunit M2 (RRM2), Thyroid Hormone Receptor Interactor 13 (TRIP13), and Hyaluronan-Mediated Motility Receptor (HHMR). The model's accuracy was further assessed by ROC curve analysis and the COX model. In addition, immunohistochemical staining obtained from the HPA database, revealed enhanced PLK1 expression in LUAD samples. ConclusionLUAD pathogenesis is highly associated with immunity and amino acid metabolism. The PLK1, RRM2, TRIP13, and HMMR genes have prognostic values for LUAD. PLK1 upregulation in LUAD might be involved in tumorigenesis by modulating the cell cycle and represents a potential prognostic factor in clinic.

Knockdown of TRIP13 inhibits gastric cancer stemness and cisplatin resistance by regulating FBXW7/ENO1 axis

Purpose: To explore the role of thyroid hormone receptor interactor 13 (TRIP13) in gastric cancer (GC) and the associated mechanism.
 Methods: TRIP13 expression in GC was found in The Cancer Genome Atlas (TCGA) database and siTRIP13 was transfected in GC cells in order to generate cell lines with low TRIP13 expression. Cell proliferation was determined by colony formation assay, while cell migration and invasion were evaluated by scratch test and Transwell assay, respectively. Sphere formation assay was used to assess cell stemness characteristics whereas half-inhibitory concentration (IC50) value was evaluated by Cell Counting Kit-8 (CCK8). F-box and WD repeat domain containing 7 (FBXW7) and enolase 1 (ENO1) expression were determined by western-blot assay.
 Results: TRIP13 was significantly expressed in GC based on TCGA database (p < 0.001) and high expression predicted poor prognosis in GC patients (p < 0.05). Based on the findings of the cell function assay, si-TRIP13 inhibited the proliferation, migration, invasion and stemness of GC cells (p < 0.001) and decreased the IC50 value. Mechanically, knockdown of TRIP13 decreased ENO1 expression level by stabilizing FBXW7.
 Conclusion: TRIP13 functions as an oncogene in GC and stimulates growth and stemness via FBXW7/ENO1 pathway in GC. Thus, a potential therapeutic target for the treatment of gastric cancer is provided by this study.

TRIP13 Enhances Radioresistance of Lung Adenocarcinoma Cells through the Homologous Recombination Pathway

Radiation therapy is one of the most common treatments for non-small cell lung cancer (NSCLC). However, the insensitivity of some tumor cells to radiation is one of the major reasons for the poor efficacy of radiotherapy and the poor prognosis of patients, and exploring the underlying mechanisms behind radioresistance is the key to solving this clinical challenge. This study aimed to identify the molecules associated with radioresistance in lung adenocarcinoma (LUAD), identified thyroid hormone receptor interactor 13 (TRIP13) as the main target initially, and explored whether TRIP13 is related to radioresistance in LUAD and the specific mechanism, with the aim of providing theoretical basis and potential targets for the combination therapy of LUAD patients receiving radiotherapy in the clinic. Three datasets, GSE18842, GSE19188 and GSE33532, were selected from the Gene Expression Omnibus (GEO) database and screened for differentially expressed genes (|log FC|>1.5, P<0.05) in each of the three datasets using the R 4.1.3 software, and then Venn diagram was used to find out the differentially expressed genes common to the three datasets. The screened differential genes were then subjected to protein-protein interaction (PPI) analysis and module analysis with the help of STRING online tool and Cytoscape software, and survival prognosis analysis was performed for each gene with the help of Kaplan-Meier Plotter database, and the TRIP13 gene was identified as the main molecule for subsequent studies. Subsequently, the human LUAD cell line H292 was irradiated with multiple X-rays using a sub-lethal dose irradiation method to construct a radioresistant cell line, H292DR. The radioresistance of H292DR cells was verified using cell counting kit-8 (CCK-8) assay and clone formation assay. The expression levels of TRIP13 in H292 and H292DR cells were measured by Western blot. Small interfering RNA (siRNA) was used to silence the expression of TRIP13 in H292DR cells and Western blot assay was performed. The clone formation ability and migration ability of H292DR cells were observed after TRIP13 silencing, followed by the detection of changes in the expression levels of proteins closely related to homologous recombination, such as ataxia telangiectasia mutated (ATM) protein. Screening of multiple GEO datasets, validation of external datasets and survival analysis revealed that TRIP13 was highly expressed in LUAD and was associated with poor prognosis in LUAD patients who had received radiation therapy. And the results of gene set enrichment analysis (GSEA) of TRIP13 suggested that TRIP13 might be closely associated with LUAD radioresistance by promoting homologous recombination repair after radiation therapy. Experimentally, TRIP13 expression was found to be upregulated in H292DR, and silencing of TRIP13 was able to increase the sensitivity of H292DR cells to radiation. TRIP13 is associated with poor prognosis in LUAD patients treated with radiation, possibly by promoting a homologous recombination repair pathway to mediate resistance of LUAD cells to radiation.

Abstract PR007: The combination of TRIP13 and Aurora kinase A inhibition caused cell cycle specific DNA damage and death in Rb-deficient cancers

Abstract Many diverse, common, and lethal cancer types have inactivation of the retinoblastoma (RB1, Rb) tumor suppressor pathway including cancers caused by the human papillomavirus (HPV), which causes &amp;gt;5% of all cancers worldwide. Despite therapeutic advances, advanced Rb-deficient cancers are still lethal with therapy-related adverse effects. Moreover, there are no therapies that uniquely target HPV-driven or Rb-deficient cancers. Therefore, biomarker-selected, targeted therapy for Rb-deficient cancers represents both a substantial unmet need and a significant translational knowledge gap. Rb-deficient cancer cells depend on Aurora A kinases to bypass the spindle assembly checkpoint (SAC) and survive due to overexpression of the mitotic checkpoint protein Mad2. Thyroid hormone receptor interactor 13 (TRIP13) catalyzes the ATP-dependent conversion of Mad2 from its active to inactive form, which turns off the SAC. Our lab discovered that the combination of Aurora A inhibition with alisertib plus TRIP13 depletion induced extensive apoptosis selectively in Rb-deficient cancer cells. Moreover, our results demonstrated that the synthetic lethality between Aurora A and TRIP13 reflects mitotic catastrophe. The combination led to highly abnormal mitoses in Rb-deficient cancer cells, evidenced by unaligned, dispersed chromosomes, mitotic spindle defects, and G2/M arrest. Moreover, live cell imaging showed that alisertib treatment increased the duration of first mitoses and induced mitotic cell death, which was even more pronounced in second mitotic events. The combination resulted in both prolonged mitotic arrest and enhanced lethality, indicated by increased mitotic cell death after the first mitoses and interphase cell death following the first mitotic arrest. Furthermore, using degron tagging to rapidly degrade TRIP13, we showed that TRIP13 depletion led to transient mitotic arrest in cells that were in late G2, supporting the role of TRIP13 in regulating the SAC and mitotic progression. Since prolonged mitotic arrest is known to cause DNA damage and both Aurora A and TRIP13 are known to be involved in the DNA damage response, we also tested the effect of the combination treatment on DNA damage. Treating Rb-deficient cancer cells with the combination resulted in robust increases in gamma-H2AX, pTIF1 beta (KAP1, TRIM28), and DNA PKcs. The lethal effects of the combination required mitotic entry as concurrent CDK1 inhibition with RO-3306 prevented apoptosis and DNA damage induced by the combination. The selective effect of the combination in Rb-deficient cancer cells may have a wide therapeutic index resulting in less toxic and more effective therapy. Additionally, targeting mitosis may contribute to a cancer-specific effect due to cancer cells’ high proliferation rate. Our study suggests that targeting TRIP13 and Aurora A led to DNA damage and death specifically during mitosis in Rb-deficient cells. This work has potential to establish a foundation for future clinical trials and may change current clinical practice paradigms. Citation Format: Lacin Yapindi, Soma Ghosh, Tuhina Mazumdar, Faye M. Johnson. The combination of TRIP13 and Aurora kinase A inhibition caused cell cycle specific DNA damage and death in Rb-deficient cancers [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr PR007.

Suppression of TRIP13 Induces Metabolic Changes and Potentiates Ferroptosis in Multiple Myeloma

Introduction Despite the remarkable success of combination treatments, multiple myeloma (MM) still poses significant challenges, with a high percentage of patients experiencing relapse over time. Additional metabolic alterations are found in relapsed and refractory MM; however, their impacts have not been fully elucidated. Expression of thyroid hormone receptor interactor 13 (TRIP13) has been identified by Gene Expression Profile (GEP)-70 to correlate with poor prognosis in MM. TRIP13 is an AAA ATPase that regulates the spindle assembly checkpoint in mitosis. Based on GEP data from 34 paired baseline and relapse samples, TRIP13 continuously increased from baseline to first and to second relapse. Overexpression of TRIP13 induces bortezomib resistance. In this study, we aimed to investigate how TRIP13 might regulate MM cell metabolism and to use this information to develop a novel combination strategy to overcome MM resistance. Methods Knockout (KO) of TRIP13 in OPM2 and MM1.S cells was generated by dual gRNAs CRISPR-Cas9 editing deleting a fragment between exon 4 and exon 5. Cell growth curves and response to bortezomib were recorded in the control and TRIP13-KO cells. Metabolomics was performed to reveal cellular metabolic alterations. Seahorse analysis was used to test the mitochondrial stress and glycolysis stress. To investigate whether TRIP13 KO promotes ferroptosis, an intracellular iron-dependent cell death characterized by the accumulation of lipid peroxides, we applied C11-BODIY staining after treatment with Erastin, a ferroptosis-inducer. Expression levels of cyclooxygenase 2 (encoded by PTGS2, a hallmark of ferroptosis), cystine transporter (xCT), and intracellular glutathione peroxidase 4 (GPX4) were analyzed. In addition, the protein levels of iron metabolic molecules, including iron importer transferrin receptor protein 1 (TFRC), iron exporter ferroportin 1 (FPN1), ferritin heavy chain 1 (FTH1), and nuclear receptor coactivator 4 (NCOA4) were assessed. To study whether TRIP13 KO delays MM progression in an immune-competent MM mouse model, Vκ12653 murine MM cells were genetically modified by CRISPR-mediated TRIP13-KO and then injected into C57BL/6 mice. Mouse survival was monitored. Results The cell-doubling rate of TRIP13-KO cells was decreased by 50% compared to control cells. After 24 hours of treatment with 5 nM bortezomib, cell viability of TRIP13-KO cells decreased by 31% compared with control cells. The metabolomic studies revealed that TRIP13-KO OPM2 and MM1.S cells both had significantly decreased intracellular L-serine (FC = 0.57) but increased pyruvate (FC = 1.79), citric acid (FC = 1.76), and isocitric acid (FC = 1.64), indicating TRIP13 may regulate serine metabolism, mitochondrial function, and cellular redox balance. Seahorse analysis indicated that TRIP13-KO MM cells showed decreased glycolysis (decreased by 30%, p &amp;lt; 0.001) and glycolytic capacity (decreased by 33%, p &amp;lt; 0.001) while oxidative phosphorylation showed little change. Moreover, lipid peroxidation and PTGS2 were increased in TRIP13-KO cells after Erastin treatment, indicating that these cells were more vulnerable to ferroptosis. Mechanistically, inhibition of TRIP13 decreased GPX4, FPN1, FTH1, and increased NCOA4, while overexpression of TRIP13 showed the opposite changes, suggesting that targeting TRIP13 sensitizes MM cells to ferroptosis by impairing cellular redox balance and increasing the labile-iron pool. Lastly, TRIP13-KO significantly prolonged mouse survival in the Vκ12653 mouse model (median survival: TRIP13-KO 93 days vs. Control 60.5 days, p &amp;lt; 0.001). Conclusion Our study revealed that TRIP13 plays a critical role in regulating MM cell metabolism. By suppressing TRIP13, we observed increased sensitivity of MM cells to bortezomib, leading to enhanced ferroptosis-mediated cell death and prolonged mouse survival. These findings strongly suggest that targeting TRIP13 could be a promising approach in MM therapy. Moving forward, our future investigations will concentrate on unraveling the precise molecular mechanisms underlying TRIP13-mediated alterations in cell metabolism. Additionally, we plan to explore the potential synergistic effects of TRIP13 inhibition in combination with metabolic targeted treatments, such as venetoclax, to further enhance therapeutic outcomes in MM.

Targeting TRIP13 for overcoming anticancer drug resistance (Review).

Cancer is one of the greatest dangers to human wellbeing and survival. A key barrier to effective cancer therapy is development of resistance to anti‑cancer medications. In cancer cells, the AAA+ ATPase family member thyroid hormone receptor interactor 13 (TRIP13) is key in promoting treatment resistance. Nonetheless, knowledge of the molecular processes underlying TRIP13‑based resistance to anticancer therapies is lacking. The present study evaluated the function of TRIP13 expression in anticancer drug resistance and potential methods to overcome this resistance. Additionally, the underlying mechanisms by which TRIP13 promotes resistance to anticancer drugs were explored, including induction of mitotic checkpoint complex surveillance system malfunction, promotion of DNA repair, the enhancement of autophagy and the prevention of immunological clearance. The effects of combination treatment, which include a TRIP13 inhibitor in addition to other inhibitors, were discussed. The present study evaluated the literature on TRIP13 as a possible target and its association with anticancer drug resistance, which may facilitate improvements in current anticancer therapeutic options.

Ribosomal protein S3 mediates drug resistance of proteasome inhibitor: potential therapeutic application in multiple myeloma.

Multiple myeloma (MM) remains incurable due to drug resistance. Ribosomal protein S3 (RPS3) has been identified as a non-Rel subunit of NF-κB. However, the detailed biological roles of RPS3 remain unclear. Here, we report for the first time that RPS3 is necessary for MM survival and drug resistance. RPS3 was highly expressed in MM, and knockout of RPS3 in MM inhibited cell growth and induced cell apoptosis both in vitro and in vivo. Overexpression of RPS3 mediated the proteasome inhibitor resistance of MM and shortened the survival of MM tumor-bearing animals. Moreover, our present study found an interaction between RPS3 and the thyroid hormone receptor interactor 13 (TRIP13), an oncogene related to MM tumorigenesis and drug resistance. We demonstrated that the phosphorylation of RPS3 was mediated by TRIP13 via PKCδ, which played an important role in activating the canonical NF-κB signaling and inducing cell survival and drug resistance in MM. Notably, the inhibition of NF-κB signaling by the small-molecule inhibitor targeting TRIP13, DCZ0415, was capable of triggering synergistic cytotoxicity when combined with bortezomib in drug-resistant MM. This study identifies RPS3 as a novel biomarker and therapeutic target in MM.

Abstract 4969: Enhancing the efficacy of small molecule inhibitor of TRIP13 in prostate cancer using thymoquinone

Abstract Prostate cancer (PCa) is one of the leading causes of cancer-related death in men worldwide. Chemotherapy is effective in the early management of PCa; long-term use activates drug efflux pumps, resulting in resistance and recurrence. In this context, finding the genetic changes causing PCa development could significantly alter therapeutic results. The approach has been to search for specific molecular targets for the selective elimination of cancer cells. Small molecule inhibitor drugs have successfully targeted cancer cell growth and metastasis promotion. Still, small molecule inhibitors bind to multiple molecular targets, including cell surface receptors and other intracellular proteins, thus increasing the risk of toxicity and having a short life span. Therefore, it is crucial to identify new anti-cancer agents that work with small molecule inhibitors to target cancer cells while preventing normal cells. It has been reported that overexpression of the Thyroid hormone receptor interactor 13 (TRIP13) gene, a critical mitosis regulator, plays a role in many cancers. Our analysis using UALCAN (ualcan.path.uab.edu) showed overexpression of TRIP13 in PCa and it plays a role in PCa biology. When TRIP13 is overexpressed, it phosphorylates EGFR and activates a downstream pathway, making cancer cells more aggressive. Recently, DCZ0415, a small molecule TRIP13 inhibitor, was reported to prevent cell proliferation and invasion in several malignancies. Nevertheless, the therapeutic targeting of TRIP13 driving PCa is not well understood. In this study, we demonstrated Thymoquinone (TQ), a natural compound with multifaceted chemo-preventive potential and anti-cancer agent, could reinforce the effect of DCZ0415 in PCa cell lines (DU145 and PC3). PCa cells were treated with TQ or DCZ0415 alone or combined, followed by cell viability (MTT assay) to determine the optimal IC50 values. In a western blot, RT-qPCR analysis, and flow cytometry assays, the combined drug treatment was most effective in blocking TRIP13, inhibiting cell proliferation, and inducing apoptosis. Furthermore, this study determines the combination can upregulate the expression of cell cycle inhibitor (p21WAF1/CIP1), which, in turn, inhibits the expression of CDK4/Cyclin D1 complex, resulting the cell cycle arrest. In addition, TQ, in combination, downregulates the activation of EGFR-dependent PI3K/Akt-1/ERK1/2 and epithelial-mesenchymal transition (EMT) pathways in both cell lines. In conclusion, these findings show thymoquinone's potential to improve DCZ0415 effectiveness and suggest a new promising anti-cancer strategy for treating patients with prostate cancer. Citation Format: Santosh K. Singh, Manoj K. Mishra, Sooryanarayana Varambally, Rajesh Singh. Enhancing the efficacy of small molecule inhibitor of TRIP13 in prostate cancer using thymoquinone. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4969.

TRIP13 overexpression promotes gefitinib resistance in non‑small cell lung cancer via regulating autophagy and phosphorylation of the EGFR signaling pathway.

Non‑small cell lung cancer (NSCLC) accounts for the majority of lung cancers and remains the most common cause of cancer‑related death. Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR‑TKIs) have been used as first‑line treatment for patients with NSCLC showing EGFR mutations. Unfortunately, drug resistance is a crucial barrier affecting the treatment of patients with NSCLC. Thyroid hormone receptor interactor 13 (TRIP13) is an ATPase that is overexpressed in numerous tumors and is involved in drug resistance. However, whether TRIP13 plays a role in regulating sensitivity to EGFR‑TKIs in NSCLC remains unknown. TRIP13 expression was evaluated in gefitinib‑sensitive (HCC827) and ‑resistant (HCC827GR and H1975) cell lines. The effect of TRIP13 on gefitinib sensitivity was assessed using the MTS assay. The expression of TRIP13 was upregulated or knocked down to determine its effect on cell growth, colony formation, apoptosis and autophagy. Additionally, the regulatory mechanism of TRIP13 on EGFR and its downstream pathways in NSCLC cells were examined using western blotting, immunofluorescence and co‑immunoprecipitation assays. The expression levels of TRIP13 were significantly higher in gefitinib‑resistant than in gefitinib‑sensitive NSCLC cells. TRIP13 upregulation enhanced cell proliferation and colony formation while reducing the apoptosis of gefitinib‑resistant NSCLC cells, suggesting that TRIP13 may facilitate gefitinib resistance in NSCLC cells. In addition, TRIP13 improved autophagy to desensitize gefitinib in NSCLC cells. Furthermore, TRIP13 interacted with EGFR and induced its phosphorylation and downstream pathways in NSCLC cells. The present study demonstrated that TRIP13 overexpression promotes gefitinib resistance in NSCLC by regulating autophagy and activating the EGFR signaling pathway. Thus, TRIP13 could be used as a biomarker and therapeutic target for gefitinib resistance in NSCLC.

CRISPR screening reveals gleason score and castration resistance related oncodriver ring finger protein 19 A (RNF19A) in prostate cancer.

Prostate cancer (PCa) is one of the most lethal causes of cancer-related death in male. It is characterized by chromosomal instability and disturbed signaling transduction. E3 ubiquitin ligases are well-recognized as mediators leading to genomic alterations and malignant phenotypes. There is a lack of systematic study on novel oncodrivers with genomic and clinical significance in PCa. In this study we used clustered regularly interspaced short palindromic repeats (CRISPR) system to screen 656 E3 ubiquitin ligases as oncodrivers or tumor repressors in PCa cells. We identified 51 significantly changed genes, and conducted genomic and clinical analysis on these genes. It was found that the Ring Finger Protein 19A (RNF19A) was a novel oncodriver in PCa. RNF19A was frequently amplified and highly expressed in PCa and other cancer types. Clinically, higher RNF19A expression correlated with advanced Gleason Score and predicted castration resistance. Mechanistically, transcriptomics, quantitative and ubiquitination proteomic analysis showed that RNF19A ubiquitylated Thyroid Hormone Receptor Interactor 13 (TRIP13) and was transcriptionally activated by androgen receptor (AR) and Hypoxia Inducible Factor 1 Subunit Alpha (HIF1A). This study uncovers the genomic and clinical significance of a oncodriver RNF19A in PCa. The results of this study indicate that targeting AR/HIF1A-RNF19A-TRIP13 signaling axis could be an alternative option for PCa diagnosis and therapy.

TRIP13/FLNA Complex Promotes Tumor Progression and Is Associated with Unfavorable Outcomes in Melanoma.

Cutaneous melanoma is a high-grade malignant tumor originating from skin melanocytes with high risk of recurrence and metastasis. Further study on the mechanism of melanoma development is urgently needed. Here, we performed a bioinformatic analysis to identify critical genes in melanoma using public datasets in the Gene Expression Omnibus database. Among these differentially expressed genes, thyroid hormone receptor interactor 13 (TRIP13) has been reported to exert an important role in the development of various tumors, while its role in melanoma remains unclear. We selected TRIP13 as a candidate gene for further study. TRIP13 expression in clinical specimens was evaluated by immunohistochemistry, and its association with patient prognosis was analyzed by the Kaplan-Meier method and log-rank test. MV3 and A2058 melanoma cells were transfected with lentiviral vector to overexpress or knockdown TRIP13 expression level, and then, its biological function was studied using a series of in vitro and in vivo assays. RNA sequencing, co-immunoprecipitation, and mass spectrometry were used to identify the underlying mechanism of TRIP13. The results of this study exhibited that TRIP13 expression was upregulated in melanoma tissue compared with normal tissues, and high levels of TRIP13 were closely correlated with poor prognoses of melanoma patients. Elevated TRIP13 promoted the invasion and migration of melanoma cells in vitro and enhanced lung metastasis in vivo, without an influence on tumor growth. Importantly, elevated TRIP13 promoted the epithelial-mesenchymal transition (EMT) of melanoma cells, indicating a higher metastatic potential of these cells. Mechanically, TRIP13 physically interacted with filamin A (FLNA) and then activated the PI3K/AKT pathway to transcriptional activation of EMT-related genes. The present study revealed that TRIP13 is a novel prognostic biomarker and potential therapeutic target for melanoma treatment.

TRIP13 Induces Nedaplatin Resistance in Esophageal Squamous Cell Carcinoma by Enhancing Repair of DNA Damage and Inhibiting Apoptosis.

Thyroid hormone receptor interactor 13 (TRIP13) plays a crucial role in poor prognosis and chemotherapy resistance of cancer patients. This present study is aimed at investigating the role of high expression of TRIP13 inducing nedaplatin (NDP) resistance in esophageal squamous cell carcinoma (ESCC) cells. High expression of TRIP13 promoted the proliferation and migration of ESCC cells performed by MTS assay, colony formation assay, wound healing assay, and transwell assay. High TRIP13 expression induced NDP resistance to ESCC based on the cell proliferation promoting/inhibition rate and cell migration promoting/inhibition rate analysis, flow cytometry assay of apoptotic subpopulations with a combination of Annexin V-FITC and propidium iodide, and Western blot analysis downregulating cleaved PARP, γH2A.X, cleaved caspase-3, and Bax and upregulating Bcl-2 expression. This study indicated that high expression of TRIP13 promoted proliferation and migration of ESCC cells and induced NDP resistance via enhancing repair of DNA damage and inhibiting apoptosis. This will provide a preliminary reference for the clinical use of NDP in ESCC treatment.

TRIP13, identified as a hub gene of tumor progression, is the target of microRNA-4693-5p and a potential therapeutic target for colorectal cancer

Colorectal cancer (CRC) is one of the digestive tract malignancies whose early symptoms are not obvious. This study aimed to identify novel targets for CRC therapy, especially early-stage CRC, by reanalyzing the publicly available GEO and TCGA databases. Thyroid hormone receptor interactor 13 (TRIP13) correlated with tumor progression and prognosis of patients after several rounds of analysis, including weighted gene correlation network analysis (WGCNA), and further chosen for experimental validation in cancer cell lines and patient samples. We identified that mRNA and protein levels of TRIP13 increased in CRC cells and tumor tissues with tumor progression. miR-4693-5p was significantly downregulated in CRC tumor tissues and bound to the 3′ untranslated region (3′UTR) of TRIP13, downregulating TRIP13 expression. DCZ0415, a small molecule inhibitor targeting TRIP13, induced anti-tumor activity in vitro and in vivo. DCZ0415 markedly suppressed CRC cell proliferation, migration, and tumor growth, promoted cell apoptosis, and resulted in the arrest of the cell cycle. Our research suggests that TRIP13 might play a crucial role in CRC progression and could be a potential target for CRC therapy.

Inducing Synergistic DNA Damage by TRIP13 and PARP1 Inhibitors Provides a Potential Treatment for Hepatocellular Carcinoma.

Thyroid hormone receptor interactor 13 (TRIP13), an AAA-ATPase, participates in the development of many cancers. This study explores the function of TRIP13 and synergistic effects of TRIP13 and PARP1 inhibitors in hepatocellular carcinoma (HCC). The dose-dependent effects of TRIP13 and PARP1 inhibitors on HCC cells proliferation or migration were investigated by the CCK-8 and Transwell assays. Using siRNA or lentivirus to knock down TRIP13, we tested HCC cell and tumor growth in vitro and in vivo. The DNA damage caused by TRIP13 and PARP1 inhibitors was measured by the phosphorylation of H2AX, one of the DNA damage biomarkers. The phosphorylation of H2AX was increased after treatment with DCZ0415 or TRIP13 knockdown. Combining DCZ0415 with PARP1 inhibitor, Olaparib induced synergistic anti-HCC activity. We also found that the overexpression of TRIP13 is significantly associated with early recurrent HCC and poor survival. Up-regulation of TRIP13 in HCC was regulated by transcription factor SP1. In conclusion, our study demonstrated that DCZ0415 targeting TRIP13 impaired non-homologous end-joining repair to inhibit HCC progression and had a synergistic effect with PARP1 inhibitor Olaparib in HCC, suggesting a potential treatment of HCC.

Phosphorylation of TRIP13 at Y56 induces radiation resistance but sensitizes head and neck cancer to cetuximab

Radiation therapy, a mainstay of treatment for head and neck cancer, is not always curative due to the development of treatment resistance; additionally, multi-institutional trials have questioned the efficacy of concurrent radiation with cetuximab, the epidermal growth factor receptor (EGFR) inhibitor. We unraveled a mechanism for radiation resistance; that is, radiation induces EGFR, which phosphorylates TRIP13 (thyroid hormone receptor interactor 13) on tyrosine 56. Phosphorylated (phospho-)TRIP13 promotes non-homologous end joining (NHEJ) repair to induce radiation resistance. NHEJ is the main repair pathway for radiation-induced DNA damage. Tumors expressing high TRIP13 do not respond to radiation but are sensitive to cetuximab or cetuximab combined with radiation. Suppression of phosphorylation of TRIP13 at Y56 abrogates these effects. These findings show that EGFR-mediated phosphorylation of TRIP13 at Y56 is a vital mechanism of radiation resistance. Notably, TRIP13-pY56 could be used to predict the response to radiation or cetuximab and could be explored as an actionable target.