Phosphorylation of PPDPF via IL6-JAK2 activates the Wnt/ß-catenin pathway in colorectal cancer.

Inflammation plays an important role in the initiation and progression of colorectal cancer (CRC) and leads to ß-catenin accumulation in colitis-related CRC. However, the mechanism remains largely unknown. Here, pancreatic progenitor cell differentiation and proliferation factor (PPDPF) is found to be upregulated in CRC and significantly correlated with tumor-node-metastasis (TNM) stages and survival time. Knockout of PPDPF in the intestinal epithelium shortens crypts, decreases the number of stem cells, and inhibits the growth of organoids and the occurrence of azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced CRC. Mechanistically, PPDPF is found to interact with Casein kinase 1α (CK1α), thereby disrupting its binding to Axin, disassociating the ß-catenin destruction complex, decreasing the phosphorylation of ß-catenin, and activating the Wnt/ß-catenin pathway. Furthermore, interleukin 6 (IL6)/Janus kinase 2 (JAK2)-mediated inflammatory signals lead to phosphorylation of PPDPF at Tyr16 and Tyr17, stabilizing the protein. In summary, this study demonstrates that PPDPF is a key molecule in CRC carcinogenesis and progression that connects inflammatory signals to the Wnt/ß-catenin signaling pathway, providing a potential novel therapeutic target.

Pantothenate Kinase 1 Inhibits the Progression of Hepatocellular Carcinoma by Negatively Regulating Wnt/ß-catenin Signaling.

Hyperactivation of Wnt/ß-catenin signaling has been reported in hepatocellular carcinoma (HCC). However, the mechanisms underlying the hyperactivation of Wnt/ß-catenin signaling are incompletely understood. In this study, Pantothenate kinase 1 (PANK1) is shown to be a negative regulator of Wnt/ß-catenin signaling. Downregulation of PANK1 in HCC correlates with clinical features. Knockdown of PANK1 promotes the proliferation, growth and invasion of HCC cells, while overexpression of PANK1 inhibits the proliferation, growth, invasion and tumorigenicity of HCC cells. Mechanistically, PANK1 binds to CK1α, exerts protein kinase activity and cooperates with CK1α to phosphorylate N-terminal serine and threonine residues in ß-catenin both in vitro and in vivo. Additionally, the expression levels of PANK1 and ß-catenin can be used to predict the prognosis of HCC. Collectively, the results of this study highlight the crucial roles of PANK1 protein kinase activity in inhibiting Wnt/ß-catenin signaling, suggesting that PANK1 is a potential therapeutic target for HCC.

SIK2 maintains breast cancer stemness by phosphorylating LRP6 and activating Wnt/ß-catenin signaling.

Breast cancer stem cells (BCSCs) are the main drivers of recurrence and metastasis. However, commonly used drugs rarely target BCSCs. Via screenings, we found that Salt-inducible kinase 2 (SIK2) participated in breast cancer (BC) stemness maintenance and zebrafish embryos development. SIK2 was upregulated in recurrence samples. Knockdown of SIK2 expression reduced the proportion of BCSCs and the tumor initiation of BC cells. Mechanistically, SIK2, phosphorylated by CK1α, directly phosphorylated LRP6 in a SIK2 kinase activity-dependent manner, leading to Wnt/ß-catenin signaling pathway activation. ARN-3236 and HG-9-91-01, inhibitors of SIK2, inhibited LRP6 phosphorylation and ß-catenin accumulation and disturbed stemness maintenance. In addition, the SIK2-activated Wnt/ß-catenin signaling led to induction of IDH1 expression, causing metabolic reprogramming in BC cells. These findings demonstrate a novel mechanism whereby Wnt/ß-catenin signaling pathway is regulated by different kinases in response to metabolic requirement of CSCs, and suggest that SIK2 inhibition may potentially be a strategy for eliminating BCSCs.

The Roles of RNA Helicases in DNA Damage Repair and Tumorigenesis Reveal Precision Therapeutic Strategies.

DEAD-box RNA helicases belong to a large group of RNA-processing factors and play vital roles unwinding RNA helices and in ribosomal RNA biogenesis. Emerging evidence indicates that RNA helicases are associated with genome stability, yet the mechanisms behind this association remain poorly understood. In this study, we performed a comprehensive analysis of RNA helicases using multiplatform proteogenomic databases. More than 50% (28/49) of detected RNA helicases were highly expressed in multiple tumor tissues, and more than 60% (17/28) of tumor-associated members were directly involved in DNA damage repair (DDR). Analysis of repair dynamics revealed that these RNA helicases are engaged in an extensively broad range of DDR pathways. Among these factors is DDX21, which was prominently upregulated in colorectal cancer. The high expression of DDX21 gave rise to frequent chromosome exchange and increased genome fragmentation. Mechanistically, aberrantly high expression of DDX21 triggered inappropriate repair processes by delaying homologous recombination repair and increasing replication stress, leading to genome instability and tumorigenesis. Treatment with distinct chemotherapeutic drugs caused higher lethality to cancer cells with genome fragility induced by DDX21, providing a perspective for treatment of tumors with high DDX21 expression. This study revealed the role of RNA helicases in DNA damage and their associations with cancer, which could expand therapeutic strategies and improve precision treatments for cancer patients with high expression of RNA helicases. SIGNIFICANCE: The involvement of the majority of tumor-associated RNA helicases in the DNA damage repair process suggests a new mechanism of tumorigenesis and offers potential alternative therapeutic strategies for cancer.

The Protein Kinase Activity of NME7 Activates Wnt/ß-Catenin Signaling to Promote One-Carbon Metabolism in Hepatocellular Carcinoma.

Metabolic reprogramming by oncogenic signaling is a hallmark of cancer. Hyperactivation of Wnt/ß-catenin signaling has been reported in hepatocellular carcinoma (HCC). However, the mechanisms inducing hyperactivation of Wnt/ß-catenin signaling and strategies for targeting this pathway are incompletely understood. In this study, we find nucleoside diphosphate kinase 7 (NME7) to be a positive regulator of Wnt/ß-catenin signaling. Upregulation of NME7 positively correlated with the clinical features of HCC. Knockdown of NME7 inhibited HCC growth in vitro and in vivo, whereas overexpression of NME7 cooperated with c-Myc to drive tumorigenesis in a mouse model and to promote the growth of tumor-derived organoids. Mechanistically, NME7 bound and phosphorylated serine 9 of GSK3ß to promote ß-catenin activation. Furthermore, MTHFD2, the key enzyme in one-carbon metabolism, was a target gene of ß-catenin and mediated the effects of NME7. Tumor-derived organoids with NME7 overexpression exhibited increased sensitivity to MTHFD2 inhibition. In addition, expression levels of NME7, ß-catenin, and MTHFD2 correlated with each other and with poor prognosis in patients with HCC. Collectively, this study emphasizes the crucial roles of NME7 protein kinase activity in promoting Wnt/ß-catenin signaling and one-carbon metabolism, suggesting NME7 and MTHFD2 as potential therapeutic targets for HCC. SIGNIFICANCE: The identification of NME7 as an activator of Wnt/ß-catenin signaling and MTHFD2 expression in HCC reveals a mechanism regulating one-carbon metabolism and potential therapeutic strategies for treating this disease.

Low Expression of ECT2 Confers Radiation Therapy Resistance Through Transcription Coupled Nucleolar DNA Damage Repair.

PURPOSE: Radioresistance contributes to poor clinical therapeutic efficacy in most cancers. Emerging evidence shows that aberrant DNA damage repair is involved in radioresistance. This study aimed to elucidate the mechanism for radioresistance and explore the precise treatment to sensitize the radioresistant tumors. METHODS AND MATERIALS: Real-time polymerase chain reaction and Western blot were used to confirm the differential expression of epithelial cell transforming 2 (ECT2) in irradiation-resistant and sensitive cell lines. Laser microirradiation was used to examine the ribosome DNA (rDNA) damage response of ECT2. Biotin-identification, in vivo, in vitro binding assay, and dot blotting were used to confirm the interaction of ECT2 and PARP1. The xenograft mouse model and cell survival assay were used to assess the irradiation sensitivity with or without PARP1 inhibitor. RESULTS: We found the expression of ECT2 correlates with sensitivity to radiation therapy in both lung cancer and nasopharyngeal carcinoma. We demonstrated that low expression of ECT2 causes radioresistance, mainly by protecting rDNA in nucleoli from persistent irradiation exposure through transcriptional recovery prevention. ECT2 is recruited to the rDNA damage site in an ataxia-telangiectasia-mutated RNA polymerase I dependent manner. The recruited ECT2 interacts with PARP1 and facilitates the disassociation of PARP1 from rDNA in nucleoli. Thus, ECT2 deficiency results in sustained activation of PARP1, which subsequently inhibits nucleolar transcription and results in a low frequency of rDNA exposure under DNA damage. PARP inhibition synergized with irradiation can sensitize radioresistant tumors with low ECT2 expression. CONCLUSIONS: Our study provides a potential perspective for the application of PARP inhibitor to sensitize low-ECT2 expressing tumors to radiation therapy.

A Ductal-Cell-Related Risk Model Integrating Single-Cell and Bulk Sequencing Data Predicts the Prognosis of Patients With Pancreatic Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly heterogeneous malignancy. Single-cell sequencing (scRNA-seq) technology enables quantitative gene expression measurements that underlie the phenotypic diversity of cells within a tumor. By integrating PDAC scRNA-seq and bulk sequencing data, we aim to extract relevant biological insights into the ductal cell features that lead to different prognoses. Firstly, differentially expressed genes (DEGs) of ductal cells between normal and tumor tissues were identified through scRNA-seq data analysis. The effect of DEGs on PDAC survival was then assessed in the bulk sequencing data. Based on these DEGs (LY6D, EPS8, DDIT4, TNFSF10, RBP4, NPY1R, MYADM, SLC12A2, SPCS3, NBPF15) affecting PDAC survival, a risk score model was developed to classify patients into high-risk and low-risk groups. The results showed that the overall survival was significantly longer in the low-risk group (p < 0.05). The model also revealed reliable predictive power in different subgroups of patients. The high-risk group had a higher tumor mutational burden (TMB) (p < 0.05), with significantly higher mutation frequencies in KRAS and ADAMTS12 (p < 0.05). Meanwhile, the high-risk group had a higher tumor stemness score (p < 0.05). However, there was no significant difference in the immune cell infiltration scores between the two groups. Lastly, drug candidates targeting risk model genes were identified, and seven compounds might act against PDAC through different mechanisms. In conclusion, we have developed a validated survival assessment model, which acted as an independent risk factor for PDAC.

m6A Reader YTHDC2 Promotes Radiotherapy Resistance of Nasopharyngeal Carcinoma via Activating IGF1R/AKT/S6 Signaling Axis.

N6-methyladenosine (m6A) modification has been reported as a critical regulator of gene transcript expression. Although m6A modification plays important roles in tumor development, its role in therapeutic resistance remains unknown. In this study, we aimed to examine the expression level of m6A-modification related proteins and elucidate the effect of m6A-related proteins on radiation response in nasopharyngeal carcinoma (NPC). Among the genes that participated in m6A modification, YTHDC2, a m6A reader, was found to be consistently highly expressed in radioresistant NPC cells. Knocking down of YTHDC2 expression in radioresistant NPC cells improved the therapeutic effect of radiotherapy in vitro and in vivo, whereas overexpression of YTHDC2 in radiosensitive NPC cells exerted an opposite effect. Bioinformatics and mechanistic studies revealed that YTHDC2 could physically bound to insulin-like growth factor 1 receptor (IGF1R) messenger RNA and promoted translation initiation of IGF1R mRNA, which in turn activated the IGF1R-AKT/S6 signaling pathway. Thus, the present study suggests that YTHDC2 promotes radiotherapy resistance of NPC cells by activating the IGF1R/ATK/S6 signaling axis and may serve as a potential therapeutic target in radiosensitization of NPC cells.

CDK11 negatively regulates Wnt/ß-catenin signaling in the endosomal compartment by affecting microtubule stability.

Objectives: Improper activation of Wnt/ß-catenin signaling has been implicated in human diseases. Beyond the well-studied glycogen synthase kinase 3ß (GSK3ß) and casein kinase 1 (CK1), other kinases affecting Wnt/ß-catenin signaling remain to be defined. Methods:To identify the kinases that modulate Wnt/ß-catenin signaling, we applied a kinase small interfering RNA (siRNA) library screen approach. Luciferase assays, immunoblotting, and real-time polymerase chain reaction (PCR) were performed to confirm the regulation of the Wnt/ß-catenin signaling pathway by cyclin-dependent kinase 11 (CDK11) and to investigate the underlying mechanism. Confocal immunofluorescence, coimmunoprecipitation (co-IP), and scratch wound assays were used to demonstrate colocalization, detect protein interactions, and explore the function of CDK11. Results: CDK11 was found to be a significant candidate kinase participating in the negative control of Wnt/ß-catenin signaling. Down-regulation of CDK11 led to the accumulation of Wnt/ß-catenin signaling receptor complexes, in a manner dependent on intact adenomatosis polyposis coli (APC) protein. Further analysis showed that CDK11 modulation of Wnt/ß-catenin signaling engaged the endolysosomal machinery, and CDK11 knockdown enhanced the colocalization of Wnt/ß-catenin signaling receptor complexes with early endosomes and decreased colocalization with lysosomes. Mechanistically, CDK11 was found to function in Wnt/ß-catenin signaling by regulating microtubule stability. Depletion of CDK11 down-regulated acetyl-α-tubulin. Moreover, co-IP assays demonstrated that CDK11 interacts with the α-tubulin deacetylase SIRT2, whereas SIRT2 down-regulation in CDK11-depleted cells reversed the accumulation of Wnt/ß-catenin signaling receptor complexes. CDK11 was found to suppress cell migration through altered Wnt/ß-catenin signaling. Conclusions: CDK11 is a negative modulator of Wnt/ß-catenin signaling that stabilizes microtubules, thus resulting in the dysregulation of receptor complex trafficking from early endosomes to lysosomes.

Heterozygous p53-R280T Mutation Enhances the Oncogenicity of NPC Cells Through Activating PI3K-Akt Signaling Pathway.

A heterozygous point mutation of p53 gene at codon 280 from AGA to ACA (R280T) frequently occurs in nasopharyngeal carcinoma (NPC) cell lines, and about 10% NPC tissues. However, the role of this mutation in the pathogenesis of NPC remains unclear. In this study, we generated p53 knockout (KO) NPC cell lines from CNE2 cells carrying heterozygous p53 R280T (p53-R280T) mutation and C666-1 cells carrying wild-type p53 by CRISPR-Cas9 gene editing system, and found that KO of heterozygous p53-R280T significantly decreased NPC cell proliferation and increased NPC cell apoptosis, whereas KO of wild-type p53 had opposite effects on NPC cell proliferation and apoptosis. Moreover, KO of heterozygous p53-R280T inhibited the anchorage-independent growth and in vivo tumorigenicity of NPC cells. mRNA sequencing of heterozygous p53-R280T KO and control CNE2 cells revealed that heterozygous p53-R280T mutation activated PI3K-Akt signaling pathway. Moreover, blocking of PI3K-Akt signaling pathway abolished heterozygous p53-R280T mutation-promoting NPC cell proliferation and survival. Our data indicate that p53 with heterozygous R280T mutation functions as an oncogene, and promotes the oncogenicity of NPC cells by activating PI3K-Akt signaling pathway.

Downregulation of Fat Mass and Obesity Associated (FTO) Promotes the Progression of Intrahepatic Cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) ranks as the second most malignant type of primary liver cancer with a high degree of incidence and a very poor prognosis. Fat mass and obesity-associated protein (FTO) functions as an eraser of the RNA m6A modification, but its roles in ICC tumorigenesis and development remain unknown. We showed here that the protein level of FTO was downregulated in clinical ICC samples and cell lines and that FTO expression was inversely correlated with the expression of CA19-9 and micro-vessel density (MVD). A Kaplan-Meier survival analysis showed that a low expression of FTO predicted poor prognosis in ICC. in vitro, decreased endogenous expression of FTO obviously reduced apoptosis of ICC cells. Moreover, FTO suppressed the anchorage-independent growth and mobility of ICC cells. Through mining the database, FTO was found to regulate the integrin signaling pathway, inflammation signaling pathway, epidermal growth factor receptor (EGFR) signaling pathway, angiogenesis, and the pyrimidine metabolism pathway. RNA decay assay showed that oncogene TEAD2 mRNA stability was impaired by FTO. In addition, the overexpression of FTO suppressed tumor growth in vivo. In conclusion, our study demonstrated the critical roles of FTO in ICC.

NEK4 kinase regulates EMT to promote lung cancer metastasis.

Epithelial-to-mesenchymal transition (EMT) is a dynamic transitional state from the epithelial to mesenchymal phenotypes. Numerous studies have suggested that EMT and its intermediate states play important roles in tumor invasion and metastasis. To identify novel regulatory molecules of EMT, we screened a siRNA library targeting human 720 kinases in A549 lung adenocarcinoma cells harboring E-cadherin promoter-luciferase reporter vectors. NIMA-related kinase-4 (NEK4) was identified and characterized as a positive regulator of EMT in the screening. Suppression of NEK4 resulted in the inhibition of cell migration and invasion, accompanying with an increased expression of cell adhesion-related proteins such as E-cadherin and ZO1. Furthermore, NEK4 knockdown caused the decreased expression of the transcriptional factor Zeb1 and Smads proteins, which are known to play key roles in EMT regulation. Consistently, overexpression of NEK4 resulted in the decreased expression of E-cadherin and increased expression of Smad3. Using a mouse model with tail vein injection of NEK4 knockdown stable cell line, we found a lower rate of tumor formation and metastasis of the NEK4-knockdown cells in vivo. Thus, this study demonstrates NEK4 as a novel kinase involved in regulation of EMT and suggests that NEK4 may be further explored as a potential therapeutic target for lung cancer metastasis.

Screening for miRNAs and their potential targets in response to TGF-ß1 based on miRNA microarray and comparative proteomics analyses in a mouse GC-1 spg germ cell line.

Transforming growth factor-ß1 (TGF-ß1) is a member of the TGF-ß superfamily that performs a number of cellular functions and shows differential activity at different testicular developmental stages. In the present study, we investigated the effects of exogenous TGF-ß1 on global microRNA (miRNA or miR) expression profiles by miRNA microarray analysis and the alterations in protein profiles by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) in a mouse GC-1 spg germ cell line. A total of 24 differentially expressed miRNAs, including 7 upregulated and 17 downregulated miRNAs were identified. The results obtained by the RT-qPCR analysis of 10 selected differentially expressed miRNAs were in accordance with those obtained by miRNA microarray analysis. In addition, 11 differentially expressed proteins, including 3 upregulated and 8 downregulated proteins were identified through MS-based comparative proteomics analysis. Bioinformatics analysis predicted that peptidyl­prolyl isomerase A (PPIA) and nucleoside diphosphate kinase B (NDKB) are targets of miR-149 and miR-199a-3p, respectively in response to the stimulation of mouse GC-1 spg germ cells with TGF-ß1. RT-qPCR revealed that the expression levels of these miRNAs showed an opposite trend in response to stimulation with TGF-ß1. In conclusion, we identified some important miRNAs and proteins as possible targets involved in TGF-ß1 signaling. Our data suggest the existence of a TGF-ß1­miR­149-PPIA or TGF-ß1-miR-199a-3p-NDKB pathway in GC-1 spg cells. Further studies are warranted to ascertain the role of these miRNAs in spermatogenesis.

SPATA12 and its possible role in DNA damage induced by ultraviolet-C.

Our previous studies indicated that SPATA12, a novel spermatogenesis-associated gene, might be an inhibitor involved in spermatogenesis and tumorigenesis. To obtain a better understanding of the functions of SPATA12, a yeast two-hybrid screening system was used to search for interacting proteins, and chromodomain helicase DNA binding protein 2 (CHD2) was successfully identified. Bimolecular fluorescence complementation (BiFC) and subcellular co-localization assays further suggested a possible interaction between SPATA12 and CHD2 in the nuclei. CHD2 is known to be involved in the later stage of the DNA damage response pathway by influencing the transcriptional activity of p53. Thus, our hypothesis is that SPATA12 might play a role in DNA damage signaling. Western blotting results showed that SPATA12 expression could be induced in ultraviolet-C (UV-C) irradiated cells. Through reporter gene assays and the activator protein-1 (AP-1) decoy oligodeoxynucleotide method, we demonstrated that SPATA12 promoter activity could be up-regulated in response to UV-C radiation exposure and an AP-1 binding site in the SPATA12 promoter may have a role in transcriptional regulation of SPATA12. Using colony formation and host cell reactivation assays, it was demonstrated that SPATA12 might lead to inhibition of cellular proliferation in UV-C-irradiated DNA damage. Furthermore, SPATA12 was transfected into H1299, MCF-7 and HeLa cells, and flow cytometry (FCM) results suggested that there are some biological association between SPATA12 and p53 in UV-C-irradiated DNA damage. In addition, we investigated whether SPATA12 could up-regulate the expression of p53. Taken together, these findings indicate that SPATA12 could be induced under UV-C stress. During DNA damage process, AP-1 involves in the transcriptional up-regulation of SPATA12 in response to UV-C radiation and p53 involves in growth inhibitory effects of SPATA12 on UV-C irradiated cells.

Microarray-based analysis of the gene expression profile in GC-1 spg cells transfected with spermatogenesis associated gene 12.

The unique differentiation mechanisms of spermatogenesis suggest the existence of cell type- and stage-specific molecules. Herein, a microarray-based approach was used to identify changes in the gene expression profile in mouse GC-1 spg germ cells transfected with spermatogenesis associated gene 12 (SPATA12). One hundred and eighty-two upregulated genes and 104 downregulated genes with fold changes of ≥2 or ≤0.5 (P≤0.05) in expression were identified. Ten genes were selected for validation of the microarray results using quantitative RT-PCR. The real-time quantitative RT-PCR results were consistent with that of the microarray. The gene ontology (GO) terms for the biological functions of the differentially expressed genes induced by SPATA12 included binding activity and immune response. Biological pathway analysis identified several related pathways which are associated with immune responses, cell adhesion and the developmental process. In addition, we observed that SPATA12 may interact with the ß-catenin signaling pathway and that SPATA12 may negatively regulate ß-catenin signaling during spermatogenesis.

Transcriptional regulation of human novel gene SPATA12 promoter by AP-1 and HSF.

Human SPATA12 is a spermatogenesis associated gene and is supposed to function as an inhibitor during male germ cell development. SPATA12 is specifically expressed in spermatocytes, spermatids, and spermatozoa of human testis. In order to understand the regulation mechanism of SPATA12 gene expression, we identified and characterized the SPATA12 gene core promoter region and transcription factor binding sites by using reporter gene assays. AP-1 is founded to be a potential transcriptional activator of SPATA12. The promoter activity of SPATA12 was drastically declined after AP-1 binding site mutation or deletion. We also demonstrated that AP-1 combined with Smad3/4 contributes to the transcriptional regulation of SPATA12 in response to TGF-ß1. The expression of SPATA12 could be induced by TGF-ß1 in a dose-dependent manner, suggesting that AP-1 as an activator plays a role in the regulation of SPATA12 promoter. We have also shown that heat shock treatment could activate the expression of SPATA12 and transcription factor HSF binding sites in the SPATA12 promoter might be responsible for this heat-induction. These results suggested that AP-1 and HSF may play an important role in regulating SPATA12 promoter activity.