Expression Of Wee1 Research Articles

Long noncoding RNA LINC01410 promotes the tumorigenesis of neuroblastoma cells by sponging microRNA-506-3p and modulating WEE1.

ObjectiveNeuroblastoma (NBL) is an extra‐cranial solid tumor in children. This study was attempted to investigate the regulatory mechanism of long noncoding RNA LINC01410 (LINC01410) on NBL.MethodsThe expression of LINC01410, miR‐506‐3p, and WEE1 in NBL was evaluated by quantitative real time polymerase chain reaction. The proliferation and colony formation ability of NBL cells were analyzed by MTT and colony formation assay. Flow cytometry assay was executed to measure the apoptosis and cell cycle. Dual‐luciferase reporter assay was used to detect the targeted relationships among LINC01410, miR‐506‐3p, and WEE1. Additionally, the role of LINC01410 on NBL in vivo was evaluated according to a tumor xenograft model.ResultsThe expression of LINC01410 and WEE1 was enhanced and miR‐506‐3p was inhibited in NBL. LINC01410 knockdown attenuated the cell proliferation, colony formation ability, and inhibited tumor growth. Moreover, LINC01410 silencing facilitated the apoptosis and arrested the cell cycle. LINC01410 interacted with miR‐506‐3p to elevate the WEE1 expression in NBL. Additionally, miR‐506‐3p inhibition or WEE1 overexpression weakened the reduction effects of sh‐LINC01410 on cell proliferation, colony formation ability, apoptosis, and cell cycle.ConclusionsKnockdown of LINC01410 inhibited the development of NBL by miR‐506‐3p/WEE1 axis in vitro, which could serve as a potential therapeutic target for NBL therapy.

Exosomal lncRNA UCA1 from cancer-associated fibroblasts enhances chemoresistance in vulvar squamous cell carcinoma cells.

In the current work, we aimed to explore whether Cancer-associated fibroblasts (CAF) exosomes played crucial roles in vulvar squamous cell carcinoma (VSCC) chemoresistance via mediating long noncoding RNAs (lncRNA). The IC50 value and cell apoptosis were assessed by the Cell Counting-8 Kit (CCK-8) assay and flow cytometry, respectively. Western blot analysis was used for the measurement of protein levels. The levels of urothelial cancer-associated 1 (UCA1), miR-103a and WEE1 G2 checkpoint kinase (WEE1) mRNA were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The target relationships among miR-103a, UCA1 and WEE1 were confirmed by dual-luciferase reporter assays. Xenograft model mice were established to observe the impact of exosomal UCA1 on cisplatin (CDDP) resistance in vivo. Our data indicated that CAF enhanced CDDP resistance of VSCC cells in vitro. Extracellular UCA1 was transferred by exosomes derived from CAF. Exosomal UCA1 derived from CAF conferred VSCC cell resistance to CDDP. Moreover, UCA1 functioned as a miR-103a sponge in VSCC cells. The promotion of exosomal UCA1 on VSCC cell resistance to CDDP was mediated by miR-103a. WEE1 was a direct target of miR-103a, and exosomal miR-103a from CAF weakened CDDP resistance of VSCC cells by WEE1. Furthermore, exosomal UCA1 regulated WEE1 expression through sponging miR-103a. Additionally, exosomal UCA1 enhanced tumor growth and CDDP resistance in vivo. Our findings suggested exosomal UCA1 derived from CAF conferred VSCC cell resistance to CDDP in vitro and in vivo at least partly through the miR-103a/WEE1 axis, highlighting a novel therapeutic method for improving the clinical benefits of CDDP chemotherapy in VSCC patients.

Abstract 4084: A DGKA-cJUN-WEE1 signaling axis provides platinum resistance in ovarian cancer

Abstract Although platinum compounds are the first-line treatment for ovarian cancer, the majority of patients relapse and develop resistance to treatment. However, the mechanism underlying resistance is unclear. Here, we report a unique platinum resistance mechanism that is mediated by a lipid messenger, phosphatidic acid (PA), in ovarian cancer. A phospho-proteomics screen revealed that PA, which is predominantly produced by diacylglycerol kinase alpha (DGKA) in ovarian cancer, activates the transcription factor c-JUN. DGKA and PA facilitate JNK recruitment to c-JUN and nuclear localization leading to its activation upon cisplatin exposure. Through a genomic screen, we identified that activated c-JUN enhances expression of WEE1 and consequently prevents cells from aberrant cell cycle progression upon cisplatin exposure. DGKA-cJUN-WEE1 signaling was elevated in tumors derived from ovarian cancer patients who did not respond to platinum-based therapy compared to tumors from patients who responded to treatment. Our study demonstrates how lipid metabolism, kinase signaling, and gene regulatory systems are intertwined to direct cisplatin resistance in ovarian cancer. Citation Format: Jie Li, Chaoyun Pan, Austin C. Boese, Anna Umano, Sumin Kang. A DGKA-cJUN-WEE1 signaling axis provides platinum resistance in ovarian cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4084.

Elevation of FGD5-AS1 contributes to cell progression by improving cisplatin resistance against non-small cell lung cancer cells through regulating miR-140-5p/WEE1 axis.

Non-small cell lung cancer (NSCLC) is a common lung cancer with high mortality worldwide. Cisplatin (DDP) resistance is a huge limitation for NSCLC therapy. FGD5 antisense RNA 1 (FGD5-AS1) was recognized as a significant cancer cell regulator. However, the molecular mechanism of FGD5-AS1 in cisplatin resistance of NSCLC cells is poorly understood. FGD5-AS1 and WEE1 expression were up-regulated in DDP-resistant tumors and cells compared with DDP-sensitive ones. Interestingly, down-regulation of FGD5-AS1 or WEE1 inhibited cell proliferation, migration, invasion, autophagy and stimulated cell apoptosis in NSCLC DDP-resistant cells. What's more, restoration of WEE1 abrogated FGD5-AS1 silencing-induced suppression on cell proliferation, migration, invasion, autophagy and promotion on cell apoptosis in NSCLC DDP-resistant cells. Next, we discovered that FGD5-AS1 was able to enhance WEE1 expression by interacting with miR-140-5p. Furthermore, FGD5-AS1 silencing restrained tumor growth of cisplatin-resistant mice. Overexpression of FGD5-AS1 accelerated cell proliferation, migration, invasion and autophagy by enhancing cisplatin resistance against NSCLC cells through miR-140-5p/WEE1 axis, presenting promising biomarkers for the diagnosis of DDP-resistant NSCLC patients.

Knockdown of long non-coding RNA VIM-AS1 inhibits glioma cell proliferation and migration, and increases the cell apoptosis via modulation of WEE1 targeted by miR-105-5p.

Glioma including glioblastoma is the main type of primary brain tumors worldwide. LncRNAs have participated in glioma formation. This study aims to investigate the underlying mechanism for VIM-AS1/miR-105-5p/WEE1 signaling in glioma. The clinical tumors and adjacent tissues were collected from 24 patients with glioma in the Shang Luo Central Hospital. Then, the clinical samples were subjected to hematoxylin-eosin staining (H&E). VIM-AS1, miR-105-5p, and WEE1 levels were measured using real-time PCR. The protein levels of WEE1, Cyclin A1, PCNA, N-cadherin, Vimentin, and Bcl-2, E-cadherin, and Bax were analyzed using Western blot. The overall survival of glioma patients was evaluated using the Kaplan-Meier analysis. The interaction between VIM-AS1 and miR-105-5p was determined using RIP assay and Dual-Luciferase reporter assay, and the binding between miR-105-5p and WEE1 was also detected by Dual-Luciferase reporter assay. Cell proliferation, colony formation, cell cycle, apoptosis, and migration were confirmed using CCK-8, colony formation assay, flow cytometry, and transwell assay, respectively. VIM-AS1 was elevated in cancer tissues, and high level of VIM-AS1 was positively correlated with poor overall survival. Then, VIM-AS1 could bind to and downregulate miR-105-5p. Furthermore, the knockdown of VIM-AS1 significantly suppressed tumor growth in vivo. The knockdown of VIM-AS1/overexpression of miR-105-5p inhibited glioma cell growth, colony formation, and migration, and enhanced the cell apoptosis by inhibiting expression of Cyclin A1, PCNA, Vimentin, N-cadherin, and Bcl-2, and by increasing the expression of Bax and E-cadherin. Interestingly, the overexpression of VIM-AS1 reversed the tumor-suppressing role of miR-105-5p in glioma cells. Besides, the expression of WEE1 was synergistically regulated by VIM-AS1 and miR-105-5p. Consequently, VIM-AS1 promoted glioma progression via upregulating WEE1 or downregulating miR-105-5p. VIM-AS1/miR-105-5p/WEE1 signaling may be a promising target for glioma treatment.

Antitumor Effect of Cycle Inhibiting Factor Expression in Colon Cancer via Salmonella VNP20009.

Colon cancer is one of the major causes of morbidity and mortality worldwide. Cycle inhibiting factors (Cifs) have been shown to deamidate Nedd8, resulting in cell cycle arrest. To determine the antitumor effect of Cifs on colon cancer by using attenuated Salmonella typhimurium VNP20009. The VNP-SOPE2-cif and VNP-SOPE2-cif-C/A plasmids were transfected into attenuated Salmonella typhimurium VNP20009. The efficiency and specificity of the Cif promoter were validated in colon cancer SW480 cell lines. Western blotting was subsequently performed to evaluate cell cycle regulators, including P21, P27 and Wee1. In vivo, the antitumor effect of VNP20009 was evaluated in a colon cancer xenograft model. Firstly, VNP-SOPE2-cif and VNP-SOPE2-cif-C/A were selectively expressed both in the bacterial and colon cancer cells. Cif expression in SW480 cells via the VNP tumor-targeted expression system induced the accumulation of Wee1, p21 and p27 expression. Moreover, tumor growth was significantly inhibited in the mice with VNP-SOPE2-cif compared to the mice with VNP with the empty construct. These results suggest that Cif gene delivered by VNP20009 is a promising approach for the treatment of colon cancer.

The Wee1 kinase inhibitor MK1775 suppresses cell growth, attenuates stemness and synergises with bortezomib in multiple myeloma.

Multiple myeloma stem-like cells (MMSCs) are responsible for initiation and relapse, though novel treatment paradigms that effectively eradicate MMSCs are yet to be developed. Selective inhibition of the cell cycle regulatory kinase Wee1 by MK1775 is being explored as a potential anti-cancer therapeutic. We report that higher expression of Wee1 is correlated with poor survival in multiple myeloma (MM). The MM models and patient-derived CD138+ plasma cells are particularly sensitive to the growth-inhibitory effects of the Wee1 inhibitor MK1775. MK1775 induces Mus81-Eme1 endonuclease-mediated DNA damage in S-phase cell cycle that results in a blockade of replication and then apoptosis. Furthermore, MK1775 strongly suppresses the features of stemness in vitro, in vivo and in primary CD138+ cells by decreasing ALDH1+ cell fraction and the expression of ALDH1. In addition, co-treatment of MK1775 with bortezomib is synergistic in vitro and in vivo. Bortezomib, although it enhances ALDH1+ cells, when combined with MK1775 abrogates this stimulatory effect on stemness. Considering MM as an invariably incurable malignancy due to the presence of heterogenic myeloma stem-like cells, our study presents inhibition of Wee1 as a promising targeted therapy for MM and provides a compelling rationale to further investigate the activity of MK1775 against myeloma in clinical settings.

Targeting MYC-driven replication stress in medulloblastoma with AZD1775 and gemcitabine.

MYC-driven medulloblastomas are highly aggressive childhood tumors with dismal outcomes and a lack of new treatment paradigms. We identified that targeting replication stress through WEE1 inhibition to suppress the S-phase replication checkpoint, combined with the attenuation of nucleotide synthesis with gemcitabine, is an effective strategy to induce apoptosis in MYC-driven medulloblastoma that could be rapidly translated into early phase clinical trials in children. Attenuation of replication stress is a key component of MYC-driven oncogenesis. Previous studies revealed a vulnerability in MYC medulloblastoma through WEE1 inhibition. Here, we focused on elucidating combinations of agents to synergize with WEE1 inhibition and drive replication stress toward cell death. We first analyzed WEE1 expression in patient tissues by immunohistochemistry. Next, we used high-throughput drug screens to identify agents that would synergize with WEE1 inhibition. Synergy was confirmed by in vitro live cell imaging, ex vivo slice culture models, and in vivo studies using orthotopic and flank xenograft models. WEE1 expression was significantly higher in Group 3 and 4 medulloblastoma patients. The WEE1 inhibitor AZD1775 synergized with inhibitors of nucleotide synthesis, including gemcitabine. AZD1775 with gemcitabine suppressed proliferation and induced apoptosis. Ex vivo modeling demonstrated efficacy in Group 3 medulloblastoma patients, and in vivo modeling confirmed that combining AZD1775 and gemcitabine effectively suppressed tumor growth. Our results identified a potent new synergistic treatment combination for MYC-driven medulloblastoma that warrants exploration in early phase clinical trials.

Quercetin radiosensitizes non-small cell lung cancer cells through the regulation of miR-16-5p/WEE1 axis.

Quercetin, a widely distributed bioflavonoid, plays a role in combating diverse human cancers including non-small cell lung cancer (NSCLC). However, the role of quercetin in reversing the radioresistance of NSCLC cells and its underlying mechanism are far from being elucidated. Radiation-resistant NSCLC cell lines were established. Quantitative real-time PCR (qRT-PCR) was used to detect the expression of miR-16-5p and WEE1 G2 checkpoint kinase (WEE1) mRNA in radiation-resistant cells. After being treated with different concentrations of quercetin and different doses of X-ray, cell proliferation and apoptosis were monitored by CCK-8 assay, colony formation assay, and flow cytometry, respectively. Ultimately, the targeting relationship between miR-16-5p and WEE1 was verified via a dual fluorescent reporter gene assay. The expression of miR-16-5p was down-regulated in radiation-resistant cells, while the expression of WEE1 was up-regulated. Quercetin enhanced the radiosensitivity of NSCLC cells in a dose- and time-dependent manner. Furthermore, quercetin treatment increased the expression of miR-16-5p and decreased the expression of WEE1. The function of quercetin was reversed by miR-16-5p inhibitors or the transfection of WEE1 overexpressing plasmids. In conclusion, quercetin enhanced the radiosensitivity of NSCLC cells via modulating the expression of miR-16-5p and WEE1.

Anticancerous potential of polysaccharides sequentially extracted from Polygonatum cyrtonema Hua in Human cervical cancer Hela cells

The anticancerous effects of PCHPs (HBSS, CHSS, DASS, and CASS) were investigated on Human cervical cancer Hela cells proliferation inhibition, cytotoxicity, caspase-3 activity, cell cycle, and apoptosis. The inhibition rate was expressed as CASS > HBSS > CHSS > DASS, with the maximum inhibition of 74.453 ± 3.399%. Cell cytotoxicity was observed (CASS > CHSS > HBSS > DASS) with the maximum cell death rate of 82.472 ± 3.488%. The caspase-3 activity was induced by CASS > HBSS > DASS > CHSS, with the maximum multiple of 2.954 ± 0.103. CASS induced cell cycle block at the G2/M phase by elevating mRNA expression of CyclinD1, p21, p53 and Wee1, and lowering the expression of Survivin, CHK2, Wee1, CyclinB1, and CDK-1. CASS enhanced the mRNA expression of DR3, DR5, FasL, FADD, PARP, TNF- α, TNF- R1, TRDAA, caspases-8, caspases-10 and the protein expression of FasL and caspases-8, -10 in the death receptor pathway; while, lowered the mRNA expression of antiapoptotic genes (Bcl - 2 and Bcl-xL) and the protein expression of Bcl - 2. The mRNA expression of apoptosis genes (Bak, Cytc, Puma, and caspases-3, -7, -9) and the protein expression of caspases-3, -9 of mitochondria pathway was up regulated which led to cell apoptosis.

WEE1 inhibition induces glutamine addiction in T-cell acute lymphoblastic leukemia

Tcell acute lymphoblastic leukemias (T-ALL) are aggressive and heterogeneous hematologic tumors resulting from the malignant transformation of T-cell progenitors. The major challenges in the treatment of T-ALL are dose-limiting toxicities of chemotherapeutics and drug resistance. Despite important progress in deciphering the genomic landscape of T-ALL, translation of these findings into effective targeted therapies remains largely unsuccessful. New targeted agents with significant antileukemic efficacy and less toxicity are urgently needed. Here we report that the expression of WEE1, a nuclear tyrosine kinase involved in cell cycle G2-M checkpoint signaling, is significantly elevated in T-ALL. Mechanistically, oncogenic MYC directly binds to the WEE1 promoter and activates its transcription. T-ALL cells particularly rely on the elevated WEE1 for cell viability. Pharmacological inhibition of WEE1 elicits global metabolic reprogramming which results in a marked suppression of aerobic glycolysis in T-ALL cells, leading to an increased dependency on glutaminolysis for cell survival. As such, dual targeting of WEE1 and glutaminase (GLS1) induces synergistic lethality in multiple TALL cell lines and shows great efficacy in T-ALL patient-derived xenografts. These findings provide mechanistic insights into the regulation of WEE1 kinase in T-ALL and suggest an additional vulnerability during WEE1 inhibitor treatments. We also highlight a promising combination strategy of dual inhibition of cell cycle kinase and metabolic enzymes for T-ALL therapeutics.

WEE1 inhibition synergizes with CHOP chemotherapy and radiation therapy through induction of premature mitotic entry and DNA damage in diffuse large B-cell lymphoma.

Background:Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease, characterized by high levels of genomic instability and the activation of DNA damage repair pathways. We previously found high expression of the cell cycle regulator WEE1 in DLBCL cell lines. Here, we investigated the combination of the WEE1 inhibitor, AZD1775, with cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP) and radiation therapy (RT), with the aim of improving first-line treatment.Methods:Cell viability experiments were performed to determine synergistic combinations. Levels of DNA damage were established using flow cytometry for γH2AX and protein analysis for DNA damage response proteins CHK1 and CHK2. Flow cytometry analysis for cell cycle and pH3 were performed to determine cell cycle distribution and premature mitotic entry.Results:Treatment with either RT or CHOP led to enhanced sensitivity to AZD1775 in several DLBCL cell lines. Treatment of cells with AZD1775 induced unscheduled mitotic progression, resulting in abnormal cell cycle distribution in combination with RT or CHOP treatment. In addition, a significant increase in DNA damage was observed compared with CHOP or RT alone. Of the single CHOP components, doxorubicin showed the strongest effect together with AZD1775, reducing viability and increasing DNA damage.Conclusion:In conclusion, the combination of RT or CHOP with AZD1775 enhances sensitivity to WEE1 inhibition through unscheduled G2/M progression, leading to increased DNA damage. Based on these results, WEE1 inhibition has great potential together with other G2/M arresting or DNA damaging (chemo) therapeutic compounds and should be further explored in clinical trials.

The impact of propranolol on apoptosis in cutaneous squamous cell carcinomas.

Cutaneous squamous cell carcinoma (cSCC) is a common cancer in Caucasian populations. Treatments registered for high-risk cSCC are still undetermined. Experimental data have demonstrated possibly useful effects of a combined application of beta‑blockers in cancer therapy. The goal of this study was to examine the efficacy of propranolol in the treatment of cSCC and its impact on apoptosis. Thus, we aimed to investigate the apoptotic pathway protein levels and activity in beta‑blocker‑treated cSCC cells. The study was performed on human cSCC cancer cell line culture. One of the cSCC cell lines was treated with propranolol, whereas no treatment was given to the other group. Then, the levels of apoptotic pathway proteins were determined by ELISA test in both groups. The propranolol treatment group exhibited a remarkable difference as compared with the other group. It was found that propranolol treatment enhanced the activity of caspase-3 while the expression of bax, wee1, gadd153, grp78 and AIF decreased bcl-2 which is antiapoptotic protein in cSCC cell lines. Our results suggest that propranolol treatment has anti-cancer properties with an effect on various apoptotic pathways in cSCC. These data are important because propronalol may be involved in future cSCC treatment (Tab. 1, Fig. 3, Ref. 22).

1866 The Role of Microrna-424/503-Wee1 Axis in Ovarian Cancer Stem Like Cells

Study Objective We investigated the role of miR-424/503-WEE1 axis in ovarian cancer and its potential utility as a therapeutic target. Design To determine whether miR 424/503-WEE1 axis is associated with the generation of CSCs, we up/down-regulated miR 424/503-WEE1 axis in ovarian cancer sphere cells. Setting This study was performed in Pusan National University Hospital, Busan, Korea Patients or Participants The human epithelial ovarian cancer cell lines OVCAR3, SKOV3, and OVCAR429 were obtained from the Korean Cell Line Bank. Ovarian cancer cells were maintained in MEM (Life Technologies, Inc., Grand Island, NY, USA) supplemented with 10% fetal bovine serum and 100 μg/ml streptomycin in a humidified 5% CO2 incubator. Interventions In order to assess spheroid formation, approximately 5 × 103 cells were suspended in 10 mL of serum-free DMEM-F12, supplemented with 10 ng/mL basic fibroblast growth factor, 20 ng/mL epidermal growth factor and plated in an ultra-low attachment plate to prevent adherence. Spheres were counted at 14 days after plating. All experiments were performed in triplicate. Measurements and Main Results Firstly, we discovered that miR-424 and miR-503 are lowly expressed in ovarian cancer stem-like cells (CSCs) compared with non-CSCs. Over-expression of miR-424 and miR-503 resulted in decreased cancer colony formation, migration in ovarian cancer cell lines. Moreover, miR-424 and miR-503 over-expression caused inhibition of spheroid formation and the DNA damage repair pathway, while over-expression of Wee1 caused increased spheroid formation and cell survival in ovarian CSCs. Secondly, we found that the stem cell marker NANOG decreased miR-424 and miR-503 transcription in ovarian CSCs. In addition, miR-424 and miR-503 bind to the 3’ UTR of WEE1 to decrease its expression. Finally, we found that atorvastatin treatment increased miR-424 and miR-503 expression while it significantly decreased NANOG and WEE1 expression in ovarian CSCs. Conclusion Our findings indicate that increased WEE1 is an oncogenic marker for ovarian CSCs and is a potential target for CSC-specific therapy.

Isorhamnetin Induces Cell Cycle Arrest and Apoptosis Via Reactive Oxygen Species-Mediated AMP-Activated Protein Kinase Signaling Pathway Activation in Human Bladder Cancer Cells.

Isorhamnetin is an O-methylated flavonol that is predominantly found in the fruits and leaves of various plants, which have been used for traditional herbal remedies. Although several previous studies have reported that this flavonol has diverse health-promoting effects, evidence is still lacking for the underlying molecular mechanism of its anti-cancer efficacy. In this study, we examined the anti-proliferative effect of isorhamnetin on human bladder cancer cells and found that isorhamnetin triggered the gap 2/ mitosis (G2/M) phase cell arrest and apoptosis. Our data showed that isorhamnetin decreased the expression of Wee1 and cyclin B1, but increased the expression of cyclin-dependent kinase (Cdk) inhibitor p21WAF1/CIP1, and increased p21 was bound to Cdk1. In addition, isorhamnetin-induced apoptosis was associated with the increased expression of the Fas/Fas ligand, reduced ratio of B-cell lymphoma 2 (Bcl-2)/Bcl-2 associated X protein (Bax) expression, cytosolic release of cytochrome c, and activation of caspases. Moreover, isorhamnetin inactivated the adenosine 5′-monophosphate-activated protein kinase (AMPK) signaling pathway by diminishing the adenosine triphosphate (ATP) production due to impaired mitochondrial function. Furthermore, isorhamnetin stimulated production of intracellular reactive oxygen species (ROS); however, the interruption of ROS generation using a ROS scavenger led to an escape from isorhamnetin-mediated G2/M arrest and apoptosis. Collectively, this is the first report to show that isorhamnetin inhibited the proliferation of human bladder cancer cells by ROS-dependent arrest of the cell cycle at the G2/M phase and induction of apoptosis. Therefore, our results provide an important basis for the interpretation of the anti-cancer mechanism of isorhamnetin in bladder cancer cells and support the rationale for the need to evaluate more precise molecular mechanisms and in vivo anti-cancer properties.

Sequential combination of bortezomib and WEE1 inhibitor, MK-1775, induced apoptosis in multiple myeloma cell lines

IntroductionMultiple myeloma (MM) remains incurable due to high rates of relapse after various treatment regimens. WEE1 is a cell cycle related gene that regulates the G2/M checkpoint and promotes cell cycle suspension for consequent DNA repair. To date, there are clinical studies for the evaluation of WEE1 inhibitors in the treatment of solid tumors and studies on cell lines of non-MM hematological tumors. ObjectivesTo perform in vitro functional studies to verify the effect of the inhibition of WEE1 on MM cell lines viability and its potential as therapeutic target. Material and methodsWEE1 expression was evaluated in 22 newly diagnosed MM patients and in four MM cell lines, RPMI-8226, U266 and SKO-007 and SK-MM2, by quantitative real-time PCR (qPCR). After treatment with the WEE1 inhibitor (MK-1775), with or without proteasome inhibitor (bortezomib) pretreatment, we assessed cell viability through Prestoblue functional test, microspheres formation in soft agar, and induction of apoptosis and cell cycle alterations by flow cytometry. ResultsAll MM cell lines showed WEE1 expression by qPCR. RPMI-8226 and U266 showed a 50% reduction in cell viability after 24 h of incubation with MK-1775, at concentrations of 5 μM and 20 μM, respectively. SKO-007 showed dose and time dependence to this drug. Combination therapy with bortezomib and MK-1775 abolished the formation of soft agar microspheres in the RPMI-8226 cell line (also responsive to the use of both drugs) and U266, but SKO-007 was resistant to all drugs, isolated and combined. However, treatment of bortezomib followed by MK-1775 (sequential treatment) versus bortezomib alone showed statistically significant impact on cell lines total apoptosis: 88.8% vs 74.1% in RPMI-8222 (confirmed by cell cycle experiments); 92.5% vs 86.6% in U266; and 60.2% 30.9% on SKO-007 (p < 0.05). ConclusionThe sequential combination of bortezomib and WEE1 inhibitor, MK-1775, induced apoptosis in RPMI-8226, U266, and especially SKO-007 cell lines, more efficiently than the use of the same isolated drugs, highlighting its effect in inhibition of proliferation of tumor cells in MM cell lines. Our data suggest that WEE1 can figure as a MM target and that the sequential combination of bortezomib and MK-1775 may be explored in future clinical trials.

Oncogenic splicing abnormalities induced by DEAD-Box Helicase 56 amplification in colorectal cancer.

Alternative splicing, regulated by DEAD‐Box Helicase (DDX) families, plays an important role in cancer. However, the relationship between the DDX family and cancer has not been fully elucidated. In the present study, we identified a candidate oncogene DDX56 on Ch.7p by a bioinformatics approach using The Cancer Genome Atlas (TCGA) dataset of colorectal cancer (CRC). DDX56 expression was measured by RT‐qPCR and immunochemical staining in 108 CRC patients. Clinicopathological and survival analyses were carried out using three CRC datasets. Biological roles of DDX56 were explored by gene set enrichment analysis (GSEA), and cell proliferation in vitro and in vivo, cell cycle assays, and using DDX56‐knockdown or overexpressed CRC cells. RNA sequencing was carried out to elucidate the effect of DDX56 on mRNA splicing. We found that DDX56 expression was positively correlated with the amplification of DDX56 and was upregulated in CRC cells. High DDX56 expression was associated with lymphatic invasion and distant metastasis and was an independent poor prognostic factor. In vitro analysis, in vivo analysis and GSEA showed that DDX56 promoted proliferation ability through regulating the cell cycle. DDX56 knockdown reduced intron retention and tumor suppressor WEE1 expression, which functions as a G2‐M DNA damage checkpoint. We have identified DDX56 as a novel oncogene and prognostic biomarker of CRC that promotes alternative splicing of WEE1.

Wee1 Inhibitor AZD1775 Effectively Inhibits the Malignant Phenotypes of Esophageal Squamous Cell Carcinoma In Vitro and In Vivo.

Esophageal squamous cell carcinoma (ESCC) is a common malignant diagnosed cancer with increasing incidence rate and few treatment options. As a specific small-molecule inhibitor of the Wee1 tyrosine kinase, AZD1775 has previously shown potent antitumor effect on multiple types of cancer in various preclinical studies and clinical trials. However, the expression of Wee1 and the role of AZD1775 in ESCC remain unclear. In the present study, we found that the expression of Wee1 was much higher in ESCC cell lines and clinical samples than that of the corresponding controls. In addition, we demonstrated that AZD1775 exhibited strong inhibitory effect against Wee1 kinase in both tested ESCC cells at nanomolar concentrations. Moreover, AZD1775 effectively suppressed ESCC cell growth and triggered apoptosis via the mitochondrial-dependent signaling pathway. AZD1775 also diminished cell migration and invasion as well as the expression of MMP-2 and MMP-9. Interestingly, knockdown of Wee1 displayed a similar inhibitory effect of AZD1775 on ESCC cells. In addition, there was a synergism between AZD1775 and 5-fluorouracil or cisplatin in inducing cell death. More importantly, the in vivo experiments also demonstrated that AZD1775 potently inhibited ESCC cell growth and metastasis. In summary, our data suggest that the Wee1 inhibitor AZD1775 may be a potential therapeutic agent and warrants a clinical trial for patients with ESCC, even those with metastasis.

Raddeanin A promotes apoptosis and ameliorates 5-fluorouracil resistance in cholangiocarcinoma cells.

BACKGROUNDBile duct cancer is characterized by fast metastasis and invasion and has been regarded as one of the most aggressive tumors due to the absence of effective diagnosis at an early stage. Therefore, it is in the urgent demand to explore novel diagnostic approaches and therapeutic strategies for bile duct cancer to improve patient survival. Raddeanin A (RA) is extracted from the anemone raddeana regel and has been demonstrated to play antitumor roles in various cancers.AIMTo investigate the effects of RA treatment on bile duct cancer cells.METHODSIn this study, four cholangiocarcinoma cell lines (RBE, LIPF155C, LIPF178C, and LICCF) treated with RA were used to test the cell viability. The RA-associated cell functional analysis, 5-fluorouracil (5-Fu) effectiveness as well as cell cycle- and apoptosis-related protein expression were investigated.RESULTSRA reduced cell viability in a dose-dependent pattern in four cell lines, and the migration and colony formation abilities were also impaired by RA in RBE and LIPF155C cell lines. RA sensitized cell lines to 5-Fu treatment and enhanced the effects of 5-Fu in cholangiocarcinoma. Also, RA decreased protein expression of Wee1, while the combinational effect of RA and 5-Fu decreased protein expressions of cyclooxygenase-2, B cell lymphoma 2, and Wee1 but increased protein levels of Bax, cyclin D1, and cyclin E.CONCLUSIONTaken together, the results suggest that RA acts as an anti-cancer agent and enhancer of 5-Fu in bile duct cancer cells via regulating multiple cell cycle and apoptosis-related proteins. This finding provides novel clues to exploring a novel antitumor drug for bile duct cancer.

Enhancement of chemosensitivity by WEE1 inhibition in EGFR-TKIs resistant non-small cell lung cancer

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) is the first-line treatment in non-resectable non-small lung cancer (NSCLC) with EGFR mutation. However, EGFR-TIKs resistance would inevitably develop within 9–14 months after treatment. And, chemotherapy is the main treatment for EGFR-TKIs resistant patients. WEE1 kinase, a G2/M checkpoint regulator, was recently considered as a putative biomarker for the platinum-based chemo-response. The aim of this study is to clarify the relationship between WEE1 kinase and chemosensitivity in EGFR-TKIs resistant NSCLC. WEE1 expression was tested in EGFR-TKIs resistant cell lines (H1299, PC9/G2) and patients’ specimens by western blot, qPCR and immunohistochemistry (IHC). In in vitro experiment, WEE1 expression was higher in EGFR-TKIs resistant than EGFR-TKIs sensitive cell lines and was gradually increased following cisplatin or gemcitabine treatment with the enrichment of G2/M cell cycle phase. And, for patients with acquired Icotinib/Gefitinib resistance, 58.4% (7/12) had increased WEE1 expression compared to its initial expression level. In order to explore the impact of WEE1 on chemo-response, WEE1 knockdown was conducted in EGFR-TKIs resistant H1299 and PC9/G2 cells. MTT and colony formation assay showed that the efficacy of cisplatin and gemcitabine was enhanced in the two cell lines after WEE1 knockdown. And, the IC50 value of cisplatin decreased from 8.64 μg/ml to 3.10 μg/ml or 2.38 μg/ml in H1299 and from 3.66 μg/ml to 0.97 μg/ml or 1.18 μg/ml in PC9/G2 after WEE1 knockdown with two specific shRNAs. This study revealed that WEE1 expression was increased after EGFR-TKIs resistance, and WEE1 knockdown could enhance chemosensitivity in EGFR-TKIs resistant NSCLC. It is suggested the combination of WEE1 inhibitor and chemotherapy might improve the clinical outcome of NSCLC patients with acquired EGFR-TKIs resistance.