Checkpoint Kinase 1 Expression Research Articles

Suppression of cyclin-dependent kinase 1 synergizes with checkpoint kinase 2 inhibitor to promote death of non-small cell lung cancer cells with radiotherapeutic resistance

ObjectiveRadiotherapy resistance poses a prevalent clinical challenge in non-small cell lung cancer (NSCLC), necessitating the development of ongoing treatment protocols following the emergence of radiotherapy resistance, as well as the identification of radiotherapy sensitization drugs. The objective of this study was to exclusively investigate therapeutic targets associated with radiotherapy resistance in NSCLC, thereby expanding the range of treatment alternatives available to patients grappling with radiotherapy resistance. MethodsVarious databases were employed to screen for therapeutic targets associated with radiotherapeutic resistance. The impact of administering inhibitors for cyclin-dependent kinase 1 (CDK1) and checkpoint kinase 2 (CHEK2) on cell proliferation and apoptosis in radioresistant NSCLC cell lines was assessed. ResultsThe potential of CDK1 and CHEK2 kinase as a novel biomarker and therapeutic target in radioresistant NSCLC cells is evident. The up-regulation of CDK1 and CHEK2 expression in NSCLC tissues, as well as their activation in NSCLC cells exposed to X-irradiation, leads to radiotherapeutic resistance. Additionally, the heightened expression of CDK1 and CHEK2 may contribute to an increased score of cancer stem cells (CSCs) in NSCLC patients. The suppression of CDK1 in combination with a CHEK2 inhibitor synergistically promotes the death of NSCLC cells with radiotherapeutic resistance. Notably, the sensitivity of radioresistant NSCLC cell lines to the CHEK2 inhibitor (PV-1019) is enhanced through the pharmacological or genetic inhibition of CDK1. ConclusionsThe current study provides new perspectives on the synergistic effects of CDK1 and CHEK2 inhibitors in inducing apoptosis in NSCLC cells that have acquired resistance to radiotherapy.

Exploration of the carcinogenetic and immune role of CHK1 in human cancer.

Background: Previous study indicated that CHK1 was important for repairing DNA damage and cell cycle regulation. Aims: To investigate the association of Checkpoint kinase 1 (CHK1) expression with clinicopathological features, prognosis, and immune infiltration in cancer. Methods: Several databases were searched for relevant publications to conduct a meta-analysis to reveal the association between CHK1 and clinicopathological features of cancer. TIMER and GEPIA datasets were utilized to explore the differential expression of CHK1 of tumors. GEPIA and Kaplan-Meier Plotter databases were adopted to detect the prognostic significance of CHK1 in tumor. TIMER and cBioPortal databases were used for the analysis regarding immune infiltration and mutation respectively. Results: We found that CHK1 expression was significantly associated with low differentiation (OR=3.94, 95% CI: 2.73-5.67, P<0.05), advanced stage (OR=3.20, 95% CI: 2.30-4.44, P<0.05), vascular infiltration (OR=3.24, 95% CI: 2.18-4.82, P<0.05) and lymph node metastasis (OR=3.55, 95% CI: 2.62-4.82, P<0.05) of various cancers. CHK1 was highly expressed in multiple cancers and was related to clinical stage, survival, immune infiltration in pan-cancers. Conclusions: Our study found that CHK1 was significantly related to prognosis and immunological status in various cancers, suggesting that CHK1 may serve as a useful biomarker for prognosis and immune infiltration in cancer.

CHEK2 is a potential prognostic biomarker associated with immune infiltration in clear cell renal cell carcinoma

Checkpoint kinase 2 (CHEK2) plays a crucial role in responding to DNA damage and is linked to diverse cancer types. However, its significance in the prediction of prognosis and impacts on the immune status of clear cell renal cell carcinoma (ccRCC) remains unclear. This study aimed to identify the role of CHEK2 in prognosis and immune microenvironment of ccRCC. We analyzed transcriptome and clinicopathological data from the cancer genome atlas (TCGA) database and conducted functional enrichment analysis to explore molecular mechanisms. The relationship between CHEK2 and immune infiltration was evaluated, and drug sensitivity analysis was performed using the CellMiner database. The results showed that CHEK2 was an independent predictor of ccRCC prognosis and was closely associated with immune-related processes. Additionally, high expression of CHEK2 was linked to resistance to certain targeted drugs. These findings suggest that CHEK2 could serve as a biomarker for ccRCC, providing insights into tumor immune microenvironment alterations and immunotherapeutic response. Further investigation is needed to fully understand the potential of CHEK2 as a prognostic predictor and therapeutic target for ccRCC.

Overexpression of CHK1 and CHK2 in pediatric patients of B-acute lymphoblastic leukemia

Background: Despite breakthroughs in the development of chemotherapy drugs to treat pediatric B-acute lymphoblastic leukemia (B-ALL), the relapse rate remains a major therapeutic challenge, requiring more detailed characterization of molecular elements underlying disease development and resistance to treatment. Checkpoint kinase 1 (CHK1) and checkpoint kinase 2 (CHK2) are two critical mediators of the DNA damage response (DDR) mechanism that activate the downstream components responsible for DNA repair, cell cycle regulation, and apoptosis. It has been shown that altered expression of CHK1 and CHK2 in various tumor entities promotes tumorigenesis and disease progression.&#x0D; Materials and Methods: In this case-control study, we evaluated the relative expression status of CHK1 and CHK2 genes in pediatric B-ALL patients at diagnosis (n=20), during complete remission (n=23) and relapse phase (n=10), as well as 20 peripheral blood samples from healthy children as a normal control group. The mRNA expression levels of CHK1 and CHK2 were determined by the Real-time PCR method. Data were compared using the Mann–Whitney U test for the relative expression level of target mRNA in different phases of B-ALL. Data were presented as median and statistical significance was described as a P-value less than 0.05.&#x0D; Results: Our results revealed that CHK1 expression increased in newly diagnosed patients than in healthy individuals (p ≤ 0.001). Relapsed patients had higher CHK1 expression than the newly diagnosed (p ≤ 0.05) and complete remission (p ≤ 0.001) counterparts. CHK2 was overexpressed in all phases of the diseases (p ≤ 0.001) without any significant alteration among the studied groups.&#x0D; Conclusion: Given the CHK1 ability to endow cancer cells with a survival advantage upon chemotherapy, the present study suggests it as a potentially promising target in the fight against B-ALL.

MiR-669b-5p inhibits the Alzheimer's disease development via regulation of CHEK2 in Neuro2a APPSwe/Δ9 cells.

DNA damage of neurons is accumulated in Alzheimer's disease (AD). DNA damage-activated Checkpoint kinase 2 (CHEK2) is evaluated in Aβ-treated Neuro2a APPSwe/Δ9 cells, and the miR-669b-5p was specifically down-regulated. However, the underlying molecular mechanism between CHEK2 and miR-669b-5p in Neuro2a APPSwe/Δ9 cells remains unclear. This research discovers that in A-treated Neuro2a APPSwe/Δ9 cells, CHEK2 expression and miR-669b-5p expression were inversely correlated. In addition, miR-669b-5p mimics increased cell survival and proliferation in Neuro2a APPSwe/Δ9 cells while decreasing the production of inflammatory cytokines and cell death. Furthermore, it is observed that CHEK2 was a miR-669b-5p downstream target gene and that CHEK2 restored the miR-669b-5p's functions. According to this research, miR-669b-5p is a potential therapy for Alzheimer's patients since it slows the advancement of the disease.

Endothelial activation and fibrotic changes are impeded by laminar flow-induced CHK1-SENP2 activity through mechanisms distinct from endothelial-to-mesenchymal cell transition.

The deSUMOylase sentrin-specific isopeptidase 2 (SENP2) plays a crucial role in atheroprotection. However, the phosphorylation of SENP2 at T368 under disturbed flow (D-flow) conditions hinders its nuclear function and promotes endothelial cell (EC) activation. SUMOylation has been implicated in D-flow-induced endothelial-to-mesenchymal transition (endoMT), but the precise role of SENP2 in counteracting this process remains unclear. We developed a phospho-specific SENP2 S344 antibody and generated knock-in (KI) mice with a phospho-site mutation of SENP2 S344A using CRISPR/Cas9 technology. We then investigated the effects of SENP2 S344 phosphorylation under two distinct flow patterns and during hypercholesteremia (HC)-mediated EC activation. Our findings demonstrate that laminar flow (L-flow) induces phosphorylation of SENP2 at S344 through the activation of checkpoint kinase 1 (CHK1), leading to the inhibition of ERK5 and p53 SUMOylation and subsequent suppression of EC activation. We observed a significant increase in lipid-laden lesions in both the aortic arch (under D-flow) and descending aorta (under L-flow) of female hypercholesterolemic SENP2 S344A KI mice. In male hypercholesterolemic SENP2 S344A KI mice, larger lipid-laden lesions were only observed in the aortic arch area, suggesting a weaker HC-mediated atherogenesis in male mice compared to females. Ionizing radiation (IR) reduced CHK1 expression and SENP2 S344 phosphorylation, attenuating the pro-atherosclerotic effects observed in female SENP2 S344A KI mice after bone marrow transplantation (BMT), particularly in L-flow areas. The phospho-site mutation SENP2 S344A upregulates processes associated with EC activation, including inflammation, migration, and proliferation. Additionally, fibrotic changes and up-regulated expression of EC marker genes were observed. Apoptosis was augmented in ECs derived from the lungs of SENP2 S344A KI mice, primarily through the inhibition of ERK5-mediated expression of DNA damage-induced apoptosis suppressor (DDIAS). In this study, we have revealed a novel mechanism underlying the suppressive effects of L-flow on EC inflammation, migration, proliferation, apoptosis, and fibrotic changes through promoting CHK1-induced SENP2 S344 phosphorylation. The phospho-site mutation SENP2 S344A responds to L-flow through a distinct mechanism, which involves the upregulation of both mesenchymal and EC marker genes.

BRD7 suppresses tumor chemosensitivity to CHK1 inhibitors by inhibiting USP1-mediated deubiquitination of CHK1

Checkpoint kinase 1 (CHK1), a key effector in the cellular response to DNA lesions, is a crucial component of all cell cycle checkpoints. Recent reports have revealed that CHK1 is highly expressed in numerous cancer types in the clinical settings. However, the mechanisms underlying the regulation of CHK1 expression in tumor cells remain unclear. Here, we report that CHK1 is negatively regulated by the bromodomain-containing protein 7 (BRD7). Specifically, BRD7 silencing increased CHK1 (but not CHK2) expression at both mRNA and protein levels, in a p53-independent manner in multiple tumor cell lines. Furthermore, BRD7 silencing stabilized CHK1 via reducing its ubiquitination. Mechanistically, BRD7 knockdown not only increased the levels of USP1, a deubiquitinase for CHK1, but also promoted the interaction between CHK1 and USP1, subsequently enhancing the de-ubiquitination of CHK1. USP1 knockdown abrogated BRD7 silencing-induced CHK1 induction. Biologically, the increased expression of CHK1 in tumor cells caused by BRD7 silencing significantly increased cell sensitivity to CHK1 inhibitors by enhancing tumor cell apoptosis, and this effect was reversed by the simultaneous knockdown of CHK1 or USP1. Taken together, our findings suggest that BRD7 is a potential genetic or drug target that may help to improve the efficacy of chemotherapeutic drugs targeting CHK1 in combinatorial therapy.

Inhibition of Checkpoint Kinase 1 (CHK1) Upregulates Interferon Regulatory Factor 1 (IRF1) to Promote Apoptosis and Activate Anti-Tumor Immunity via MICA in Hepatocellular Carcinoma (HCC).

CHK1 is considered a key cell cycle checkpoint kinase in DNA damage response (DDR) pathway to communicate with several signaling pathways involved in the tumor microenvironment (TME) in numerous cancers. However, the mechanism of CHK1 signaling regulating TME in hepatocellular carcinoma (HCC) remains unclear. CHK1 expression in HCC tissue was determined by IHC staining assay. DNA damage and apoptosis in HCC cells induced by cisplatin or CHK1 inhibition were detected by WB and flow cytometry. The interaction of CHK1 and IRF1 was analyzed by single-cell RNA-sequence, WB, and immunoprecipitation assay. The mechanism of IRF1 regulating MICA was investigated by ChIP-qPCR. CHK1 expression is upregulated in human HCC tumors compared to the background liver. High CHK1 mRNA level predicts advanced tumor stage and worse prognosis. Cisplatin and CHK1 inhibition augment cellular DNA damage and apoptosis. Overexpressed CHK1 suppresses IRF1 expression through proteolysis. Furthermore, single-cell RNA-sequence analyses confirmed that MICA expression positively correlated with IRF1 in HCC cells. Immunoprecipitation assay showed the binding between CHK1 and IRF1. Cisplatin and CHK1 inhibition upregulate MICA expression through IRF1-mediated transcriptional effects. A novel specific cis-acting IRF response element was identified at -1756 bp in the MICA promoter region that bound IRF1 to induce MICA gene transcription. MICA may increase NK cell and CD8+T cell infiltration in HCC. DNA damage regulates the interaction of CHK1 and IRF1 to activate anti-tumor immunity via the IRF1-MICA pathway in HCC.

Expressional and functional characteristics of checkpoint kinase 1 as a prognostic biomarker in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common pathological subtype of liver cancer and is the third leading cause of cancer death worldwide. Checkpoint kinase 1 (CHEK1), an essential serine/threonine kinase that regulates the cell cycle, is reported to be associated with carcinogenesis. However, the biological role and clinical significance of CHEK1 in HCC are still incompletely known. In this research, CHEK1 messenger RNA (mRNA) levels in various liver hepatocellular carcinoma (LIHC) cohorts from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases were evaluated. The Kaplan-Meier database was applied to identify the correlation between survival time and CHEK1 expression in patients with HCC. Gene set enrichment analysis (GSEA) was performed to explore the potential mechanism of CHEK1 in HCC, and NetworkAnalyst v. 3.0 (https://www.networkanalyst.ca/) was used to construct the regulatory networks of CHEK1 in HCC. Discriminant Regulon Expression Analysis (DoRothEA) was used to detect the activity of transcriptional factors (TFs) in gene-enriched cells (EC) with CHEK1 coexpression. In vitro experiments were conducted to investigate the effects of CHEK1 on the biological function of HCC cells. The CHEK1 mRNA level was overexpressed in HCC, and increased CHEK1 expression correlated with poor survival outcomes. The homo sapiens-microRNA-195 (hsa-miR-195) may have contributed to the upregulation of CHEK1 in HCC. GSEA and NetworkAnalyst v. 3.0 showed that CHEK1 played a crucial part in tumor proliferation of HCC and may be regulated by TF E2F1. DoRothEA showed increased transcriptional activity of E2F1 in gene-EC with CHEK1 coexpression. Moreover, experiments of cell function showed that the knockdown of CHEK1 weakened the aggressive behavior and proliferation of HCC cells. Overexpression of E2F1 increased the proliferation and invasion of HCC cells in vitro, while the silencing of CHEK1 dampened cell invasion induced by E2F1 overexpression. These results identified the prognostic significance and expression characteristics of CHEK1 in HCC through bioinformatics analysis and experimental verification. This lays the foundation for further research on the diagnosis and treatment of HCC.

Checkpoint Kinase 1 (CHK1) Functions as Both a Diagnostic Marker and a Regulator of Epithelial-to-Mesenchymal Transition (EMT) in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is more difficult to treat and has a higher mortality rate than other subtypes. Although hormone receptor-targeted therapy is an effective treatment to increase survival rate in breast cancer patients, it is not suitable for TNBC patients. To address the issues, differentially expressed genes (DEGs) in TNBC patients from the Gene Expression Omnibus (GEO) database were analyzed. A total of 170 genes were obtained from three Genomic Spatial Events (GSEs) using the intersection of each GSE dataset and 61 DEGs were identified after validation with the gene enrichment analysis. We combined this with the degree scores from the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and protein-protein interaction (PPI) network, of which 7 genes were correlated with survival rate. Finally, a proteomics database revealed that only the CHK1 protein level was differently expressed in basal-like compared with other subtypes. We demonstrated that CHK1 expression was higher in TNBC cell lines compared with non-TNBC cell lines, and CHK1 promotes epithelial to mesenchymal transition (EMT) as well as migration and invasion ability. Our study provides new insight into the TNBC subnetwork that may be useful in the prognosis and treatment of TNBC patients.

Abstract 15362: Checkpoint Kinase 1-Associated Senp2 S344 Phosphorylation Under Laminar Flow Attenuates Endothelial-Mesenchymal Transition and Atherogenesis

Aims: To investigate the role of Checkpoint Kinase 1 (CHK1)-associated SENP2 S344 phosphorylation in laminar flow (LF)-regulated Endothelial-Mesenchymal Transition (EndMT) inhibition and the underlying mechanisms. Methods: We generated deSUMOylation enzyme sentrin-specific isopeptidase 2 (SENP2) S343A (equivalent to human SENP2 S344) knock-in (KI) and endothelial cell (EC)-specific SENP2 knock-out (SENP2 EKO) mice to detect EC activation, EndMT, and atherogenesis in vitro and in vivo . We also performed next-generation RNA sequencing (RNA-seq) analysis in ECs derived from these mice. Results: LF, but not disturbed flow (DF), triggered SENP2 S344 phosphorylation through CHK1. The decreased ERK5- and p53-SUMOylation by LF was abolished in ECs derived from SENP2 S343A KI mice. Both EC apoptosis and VCAM-1 expression were upregulated in SENP2 S344A KI mice. The increased atherogenesis in SENP2 S344A KI was regulated by vascular, but not hematopoietic, cells based on bone marrow transplantation (BMT) study. CHK1 expression was decreased by ionizing radiation (IR), leading to an enhanced ERK5- and p53-SUMOylation as well as EC inflammation. RNA-seq analysis revealed the involvement of SENP2 S344 phosphorylation in changes of fibrotic phenotypes and EndMT-related genes. Further, SENP2 S344A point mutation prevented LF-regulated TGFbR1 SUMOylation inhibition in vitro, as well as hypercholesterolemia-induced EndMT in vivo. Discussions: These data revealed the unique role of CHK1-associated SENP2 S344 phosphorylation in not only LF-regulated EndMT inhibition but also IR-associated EndMT activation and the subsequent atherogenesis. Maintaining CHK1-associated SENP2 S344 phosphorylation can be an ideal approach to mitigate EndMT, which is important in tumor radio-resistance as well as IR-associated cardiovascular disease.

Thiosemicarbazones Can Act Synergistically with Anthracyclines to Downregulate CHEK1 Expression and Induce DNA Damage in Cell Lines Derived from Pediatric Solid Tumors.

Anticancer therapy by anthracyclines often leads to the development of multidrug resistance (MDR), with subsequent treatment failure. Thiosemicarbazones have been previously suggested as suitable anthracycline partners due to their ability to overcome drug resistance through dual Pgp-dependent cytotoxicity-inducing effects. Here, we focused on combining anthracyclines (doxorubicin, daunorubicin, and mitoxantrone) and two thiosemicarbazones (DpC and Dp44mT) for treating cell types derived from the most frequent pediatric solid tumors. Our results showed synergistic effects for all combinations of treatments in all tested cell types. Nevertheless, further experiments revealed that this synergism was independent of Pgp expression but rather resulted from impaired DNA repair control leading to cell death via mitotic catastrophe. The downregulation of checkpoint kinase 1 (CHEK1) expression by thiosemicarbazones and the ability of both types of agents to induce double-strand breaks in DNA may explain the Pgp-independent synergism between anthracyclines and thiosemicarbazones. Moreover, the concomitant application of these agents was found to be the most efficient approach, achieving the strongest synergistic effect with lower concentrations of these drugs. Overall, our study identified a new mechanism that offers an avenue for combining thiosemicarbazones with anthracyclines to treat tumors regardless the Pgp status.

Circ_0011292 knockdown mitigates progression and drug resistance in PTX-resistant non-small-cell lung cancer cells by regulating miR-433-3p/CHEK1 axis.

BackgroundNon‐small‐cell lung cancer (NSCLC) is one of the most common malignant tumors on earth. Circular RNAs have been disclosed to be vital regulators in the chemoresistance and development of diverse cancers, including NSCLC. Here, we attempted to explore the function of circ_0011292 in paclitaxel (PTX)‐resistant NSCLC cells.MethodsQuantitative real‐time polymerase chain reaction or western blot was performed to detect the expression of circ_0011292, microRNA‐433‐3p (miR‐433‐3p), and checkpoint kinase 1 (CHEK1). Ribonuclease R (RNase R) assay was performed to assess the stability of circ_0011292. Cell Counting Kit‐8 assay was conducted to evaluate the half maximal inhibitory concentration of PTX and cell viability. Cell proliferation was monitored by Edu incorporation and colony formation assays. Cell cycle and apoptosis were detected by flow cytometry. Transwell assay was implemented to assess cell migration and invasion. Western blot assay was utilized to determine the protein levels. Dual‐luciferase reporter assay was carried out to verify the targeted interaction between miR‐433‐3p and circ_0011292 or CHEK1. Xenograft tumor model was constructed for determining the effect of circ_0011292 in NSCLC growth in vivo.ResultsCirc_0011292 was upregulated in PTX‐resistant NSCLC tissues and cells. Circ_0011292 or CHEK1 knockdown enhanced PTX sensitivity and cell apoptosis, and repressed cell proliferation, migration, and invasion in PTX‐resistant NSCLC cells. Mechanistically, circ_0011292 was a sponge of miR‐433‐3p and miR‐433‐3p directly targeted CHEK1. Meanwhile, silencing miR‐433‐3p or overexpressing CHEK1 respectively abrogated the impacts of circ_0011292 deletion or miR‐433‐3p introduction on PTX resistance and cell progression in PTX‐resistant NSCLC cells in vitro. Moreover, circ_0011292 could positively modulate CHEK1 expression through sponging miR‐433‐3p. In addition, circ_0011292 knockdown retarded tumor growth of NSCLC in vivo.ConclusionCirc_0011292 could accelerate PTX resistance and cell malignant progression of NSCLC cells partially through the regulation of circ_0011292/miR‐433‐3p/CHEK1 axis.

Lipid raft-disrupting miltefosine preferentially induces the death of colorectal cancer stem-like cells.

BackgroundLipid rafts (LRs), cholesterol‐enriched microdomains on cell membranes, are increasingly viewed as signalling platforms governing critical facets of cancer progression. The phenotype of cancer stem‐like cells (CSCs) presents significant hurdles for successful cancer treatment, and the expression of several CSC markers is associated with LR integrity. However, LR implications in CSCs remain unclear.MethodsThis study evaluated the biological and molecular functions of LRs in colorectal cancer (CRC) by using an LR‐disrupting alkylphospholipid (APL) drug, miltefosine. The mechanistic role of miltefosine in CSC inhibition was examined through normal or tumour intestinal mouse organoid, human CRC cell, CRC xenograft and miltefosine treatment gene expression profile analyses.ResultsMiltefosine suppresses CSC populations and their self‐renewal activities in CRC cells, a CSC‐targeting effect leading to irreversible disruption of tumour‐initiating potential in vivo. Mechanistically, miltefosine reduced the expression of a set of genes, leading to stem cell death. Among them, miltefosine transcriptionally inhibited checkpoint kinase 1 (CHEK1), indicating that LR integrity is essential for CHEK1 expression regulation. In isolated CD44high CSCs, we found that CSCs exhibited stronger therapy resistance than non‐CSC counterparts by preventing cell death through CHEK1‐mediated cell cycle checkpoints. However, inhibition of the LR/CHEK1 axis by miltefosine released cell cycle checkpoints, forcing CSCs to enter inappropriate mitosis with accumulated DNA damage and resulting in catastrophic cell death.ConclusionOur findings underscore the therapeutic potential of LR‐targeting APLs for CRC treatment that overcomes the therapy‐resistant phenotype of CSCs, highlighting the importance of the LR/CHEK1 axis as a novel mechanism of APLs.

Preclinical efficacy of prexasertib in acute lymphoblastic leukemia

Preclinical efficacy of prexasertib in acute lymphoblastic leukemia

GDC-0575, a CHK1 Inhibitor, Impairs the Development of Colitis and Colitis-Associated Cancer by Inhibiting CCR2+ Macrophage Infiltration in Mice.

BackgroundCheckpoint kinase 1 (CHK1) plays an important role in DNA damage response and cell cycle progression. Thus, targeting CHK1 is an efficient strategy for cancer therapy.PurposeThe present study aimed to investigate the potential therapeutic effects of GDC-0575, a CHK1-specific inhibitor, in colitis-associated cancer (CAC) and colitis.MethodsWe established a DSS-induced acute colitis model and an azoxymethane/dextran sodium sulfate (DSS)-induced CAC model using mice and tested the effect of GDC-0575 on them. Flow cytometry and immunofluorescence were employed to investigate the infiltration of immune cells, and inflammatory cytokine expression in the colon of mice with CAC or colitis was investigated using ELISA and qPCR. We also investigated the correlation between CHK1 and CCL2/CCR2 in human colorectal cancer (CRC) tissues.ResultsAdministration of GDC-0575 significantly inhibited CHK1 expression in the colon and dramatically impaired the development of CAC and colitis in mice. Moreover, the inhibition of CHK1 expression resulted in efficient inhibition of infiltration by iNOS-positive macrophages, but had no significant effect on CD4 T cells, CD8 T cells, and myeloid-derived suppressor cells (MDSCs). Significant downregulation of TNF-α, IL-6, and IL-1β and dramatic upregulation of IL-10 were observed in the colons of both mice with CAC and colitis treated with GDC-0575. CCL2 expression was also downregulated by GDC-0575 in both mice with CAC and colitis; this was followed by the inhibition of CCR2+ macrophage infiltration in the colon. Furthermore, we report a positive correlation between CHK1 expression and CCL2/CCR2 expression in the malignant tissues of patients with CRC.ConclusionTaken together, we infer that GDC-0575 impairs the development of CAC and colitis by regulating cytokine expression and inhibiting CCR2+ macrophage infiltration in mice colon.

In search for biomarkers and potential drug targets for uterine serous endometrial cancer

ObjectiveSerous endometrial cancer (USC) is a challenging malignancy associated with metastasis, recurrence and poor outcome. To identify clinically relevant prognostic biomarkers, we focused on a panel of proteins selected after a comprehensive literature review, for tumour profiling of a homogeneous cohort of USC patients.MethodsProtein levels and localization were assessed by immunohistochemistry analysis in 36 hysterectomy samples. Tissue sections were stained with the following antibodies: Aurora A, phospho (T288) Aurora A, BRCA1, CHK1, CIP2A, Cyclin B1, Cyclin E, E2F-1, phospho (S364) E2F-1, FBXW7, FOXM1, phospho (S9) GSK3Beta, PLK1, phospho (T210) PLK1, PPP2R1B, p73, RAD51. Each marker was evaluated as a continuously-scaled variable for association with disease progression and death, using Cox proportional hazards models. The sample consisted of 36 patients with USC, half with stage III or IV disease.ResultsResults showed that higher CHK1 (Checkpoint kinase 1) expression was associated with a decreased risk of progression and death, after adjusting for stage. Interestingly, analysis of a TCGA data set of 109 USC patients corroborates our results showing a favourable prognostic role of CHEK1 after adjusting for stage. Higher FBXW7 (F-box and WD repeat domain containing 7) expression and higher cytoplasmic expression of PPP2R1B (Protein Phosphatase 2 A, Scaffold Subunit Abeta) were each associated with a decreased risk of progression, after adjusting for stage.ConclusionsIn conclusion, results from the present study identify new clinically relevant biomarkers and potential drug targets for uterine serous endometrial cancer.

The Expression and Therapeutic Potential of Checkpoint Kinase 2 in Laryngeal Squamous Cell Carcinoma.

IntroductionLaryngeal squamous cell carcinoma (LSCC) is the most common histological subtype of laryngeal cancer. The involved molecular mechanisms and suitable therapeutic targets for LSCC still need to be further investigated. Checkpoint kinase 2 (CHK2) participates in several cellular physiology pathways and plays a role in tumor progression. However, the roles of CHK2 in LSCC remain unclear.MethodsmRNA expression data were obtained from The Cancer Genome Atlas (TCGA) database, and bioinformatic analysis was performed. Western blot and immunohistochemical analyses were conducted to detect protein expression. MTS assays were performed to examine cell growth of LSCC-derived cell lines.ResultsIn the present study, we found that both active form of CHK2 and total CHK2 protein expressions were up-regulated in LSCC tissues. Positive expression of CHK2 was closely associated with advanced clinical features and poor prognosis. Moreover, potential CHK2-involving bioprocesses and signaling pathways were analyzed. In addition, repressed proliferation of LSCC cells was induced by CHK2 inhibitor.DiscussionTaken together, our findings elucidated that CHK2 may act as an oncogenic factor in LSCC, suggesting a potential target for clinical treatment.

Constitutive CHK1 Expression Drives a pSTAT3-CIP2A Circuit that Promotes Glioblastoma Cell Survival and Growth.

High-constitutive activity of the DNA damage response protein checkpoint kinase 1 (CHK1) has been shown in glioblastoma (GBM) cell lines and in tissue sections. However, whether constitutive activation and overexpression of CHK1 in GBM plays a functional role in tumorigenesis or has prognostic significance is not known. We interrogated multiple glioma patient cohorts for expression levels of CHK1 and the oncogene cancerous inhibitor of protein phosphatase 2A (CIP2A), a known target of high-CHK1 activity, and examined the relationship between these two proteins in GBM. Expression levels of CHK1 and CIP2A were independent predictors for reduced overall survival across multiple glioma patient cohorts. Using siRNA and pharmacologic inhibitors we evaluated the impact of their depletion using both in vitro and in vivo models and sought a mechanistic explanation for high CIP2A in the presence of high-CHK1 levels in GBM and show that; (i) CHK1 and pSTAT3 positively regulate CIP2A gene expression; (ii) pSTAT3 and CIP2A form a recursively wired transcriptional circuit; and (iii) perturbing CIP2A expression induces GBM cell senescence and retards tumor growth in vitro and in vivo. Taken together, we have identified an oncogenic transcriptional circuit in GBM that can be destabilized by targeting CIP2A. IMPLICATIONS: High expression of CIP2A in gliomas is maintained by a CHK1-dependent pSTAT3-CIP2A recursive loop; interrupting CIP2A induces cell senescence and slows GBM growth adding impetus to the development of CIP2A as an anticancer drug target.

High CHK2 protein expression is a strong and independent prognostic feature in ERG negative prostate cancer

Checkpoint kinase 2 (CHK2) is a serine-threonine kinase with a role in DNA repair, cell cycle arrest or apoptosis in response to DNA damage. Both reduced and increased CHK2 expression has been described in different tumour types with impact on patient prognosis. To evaluate prevalence and significance of altered CHK2 expression in prostate cancer, a tissue microarray containing 17,747 tumours was analysed by immunohistochemistry. Nuclear CHK2 immunostaining was absent or weak in benign prostate epithelium but often more prominent in cancers. CHK2 immunostaining was considered weak in 38.8%, moderate in 33.6% and strong in 11.2% of prostate cancers. High CHK2 expression was strongly associated with TMPRSS2:ERG fusions (p<0.0001). Subgroup analysis of ERG positive and negative cancers revealed that high CHK2 staining was significantly linked to advanced tumour stage, high Gleason score, positive nodal status, positive surgical margin, high preoperative PSA (p<0.0001 each) and early prostate-specific antigen (PSA) recurrence (p=0.0001) in the subset of ERG negative cancers, while most of these associations were absent in ERG positive cancers. In ERG negative cancers, high CHK2 expression was an independent predictor of patient prognosis, even if parameters were included that were only available postoperatively. High CHK2 expression was also linked to presence of chromosomal deletions, high level of androgen receptor expression, positive p53 immunostaining, and high Ki-67 labelling index. These provide further invivo evidence for previously described functional interactions. In summary, high CHK2 expression is linked to adverse tumour features and independently predicts early biochemical recurrence in ERG negative prostate cancer. CHK2 measurement, either alone or in combination, might be of clinical utility in this prostate cancer subgroup.