Cell Cycle-related Proteins Research Articles

Ipatasertib, an oral AKT inhibitor, exhibits anti-proliferative and anti-tumorigenic effects in pre-clinical studies for endometrioid endometrial cancer: Endometrial Cancer Molecularly Targeted Therapy Consortium

<h3>Objectives:</h3> Ipatasertib (IPAT) is an orally administered selective protein kinase B (AKT) inhibitor that has demonstrated clinical activity in triple-negative breast and metastatic prostate cancer. Given that the AKT/mTOR pathway is altered in the vast majority of endometrioid endometrial cancers (ECs), it is logical that IPAT may also have efficacy in EC. Thus, we aimed to evaluate the anti-proliferative effects of IPAT in human endometrioid cell lines as well as in the <i>LKB1fl/flp53fl/fl</i> genetically engineered mouse model of endometrioid EC. <h3>Methods:</h3> The human endometrioid EC cell lines ECC-1 (PTEN mutant) and HEC-1A (PTEN wild type, PIK3CA mutant, KRAS mutant) were exposed to varying concentrations of IPAT (Genentech). Cell proliferation was assessed by MTT and colony assays. Cell cycle progression was measured by Cellometer. Apoptosis was assessed by cleaved caspase-3 assay. Cellular stress was assessed using tetra-methylrhodamine ester (TMRE) and DCFDA assays. Western immunoblotting determined effects of IPAT on BCL-2, MCL-1, Bip, CDK4, CDK6, cyclin D1, pAKT, pS6, PERK, calnexin, and PDI in both cell <i>lines.LKB1fl/flp53fl/fl</i> mice were used to evaluate the effect of IPAT on endometrial tumor growth. The mice were treated with placebo or IPAT (15mg/kg daily intraperitoneally for 4 weeks) starting 8 weeks after tumor induction via AdCre injection. <h3>Results:</h3> IPAT inhibited cellular proliferation in a dose dependent fashion in both cell lines after 72 hours of treatment. Median IC50 was 0.18µM in ECC-1 and 3.5µM in HEC-1A. IPAT induced the activity of cleaved caspase 3 by 15.6-fold in ECC-1 and 1.41-fold in HEC-1A (p<0.01), while simultaneously reducing expression of the apoptotic proteins MCL-1 and BCL-2. IPAT inhibited cell cycle progression by arrest in G1 phase in both cell lines after 36 hours of treatment and decreased expression of the cell cycle related proteins, CDK4, CDK6, and cyclin D1. DCFDA assay analysis demonstrated that IPAT induced ROS products by 18% and 19% (p<0.01) in the ECC-1 and HEC-1A cell lines, respectively, and increased Bip and PERK protein expression. In addition, IPAT reduced mitochondrial potential by 12% in the HEC-1A cells and 14% in the ECC-1 cells (p<0.01). Treatment of cells with IPAT at 10µM increased phosphorylated-AKT (pAKT) expression and decreased phosphorylated-S6 (p-S6) expression in a time-dependent manner in both cell lines; however, measurable changes in pAKT and pS6 were observed at concentrations as low as 0.1µM. Notably, an increase in phosphorylated-AKT was expected and is indicative of engagement and binding of IPAT with the phospho-AKT complex. Treatment of <i>LKB1fl/flp53fl/fl</i> mice with IPAT was well-tolerated and reduced tumor weight by 52.1% as compared with control mice (p<0.05). <h3>Conclusions:</h3> IPAT significantly inhibited cell proliferation and induced apoptosis and cellular stress via inhibition of the AKT/mTOR pathway in human endometrioid EC cell lines. Furthermore, IPAT reduced tumor growth in a transgenic mouse model of endometrioid EC. These results suggest that IPAT is worthy of further exploration in clinical trials for EC.

Ipatasertib, an oral AKT inhibitor, effectively inhibits cell proliferation and migration, and induces apoptosis in serous endometrial cancer cell lines in vitro: Endometrial Cancer Molecularly Targeted Therapy Consortium

<h3>Objectives:</h3> Ipatasertib (IPAT) is an orally administered selective protein kinase B (AKT) inhibitor that has demonstrated clinical activity in triple-negative breast and metastatic prostate cancer. Given that the AKT/mTOR pathway is altered in the vast majority of endometrioid endometrial cancers (ECs), it is logical that IPAT may also have efficacy in EC. Thus, we aimed to evaluate the anti-proliferative effects of IPAT in human endometrioid cell lines as well as in the <i>LKB1fl/flp53fl/fl</i> genetically engineered mouse model of endometrioid EC. <h3>Methods:</h3> The human endometrioid EC cell lines ECC-1 (PTEN mutant) and HEC-1A (PTEN wild type, PIK3CA mutant, KRAS mutant) were exposed to varying concentrations of IPAT (Genentech). Cell proliferation was assessed by MTT and colony assays. Cell cycle progression was measured by Cellometer. Apoptosis was assessed by cleaved caspase-3 assay. Cellular stress was assessed using tetramethylrhodamine ester (TMRE) and DCFDA assays. Western immunoblotting determined effects of IPAT on BCL-2, MCL-1, Bip, CDK4, CDK6, cyclin D1, pAKT, pS6, PERK, calnexin, and PDI in both cell lines. <i>LKB1fl/flp53fl/fl</i> mice were used to evaluate the effect of IPAT on endometrial tumor growth. The mice were treated with placebo or IPAT (15mg/kg daily intraperitoneally for 4 weeks) starting 8 weeks after tumor induction via AdCre injection. <h3>Results:</h3> IPAT inhibited cellular proliferation in a dose dependent fashion in both cell lines after 72 hours of treatment. Median IC50 was 0.18µM in ECC-1 and 3.5µM in HEC-1A. IPAT induced the activity of cleaved caspase 3 by 15.6-fold in ECC-1 and 1.41-fold in HEC-1A (p<0.01), while simultaneously reducing expression of the apoptotic proteins MCL-1 and BCL-2. IPAT inhibited cell cycle progression by arrest in G1 phase in both cell lines after 36 hours of treatment and decreased expression of the cell cycle related proteins, CDK4, CDK6, and cyclin D1. DCFDA assay analysis demonstrated that IPAT induced ROS products by 18% and 19% (p<0.01) in the ECC-1 and HEC-1A cell lines, respectively, and increased Bip and PERK protein expression. In addition, IPAT reduced mitochondrial potential by 12% in the HEC-1A cells and 14% in the ECC-1 cells (p<0.01). Treatment of cells with IPAT at 10µM increased phosphorylated-AKT (pAKT) expression and decreased phosphorylated-S6 (p-S6) expression in a time-dependent manner in both cell lines; however, measurable changes in pAKT and pS6 were observed at concentrations as low as 0.1µM. Notably, an increase in phosphorylated-AKT was expected and is indicative of engagement and binding of IPAT with the phospho-AKT complex. Treatment of <i>LKB1fl/flp53fl/fl</i> mice with IPAT was well-tolerated and reduced tumor weight by 52.1% as compared with control mice (p<0.05). <h3>Conclusions:</h3> IPAT significantly inhibited cell proliferation and induced apoptosis and cellular stress via inhibition of the AKT/mTOR pathway in human endometrioid EC cell lines. Furthermore, IPAT reduced tumor growth in a transgenic mouse model of endometrioid EC. These results suggest that IPAT is worthy of further exploration in clinical trials for EC.

Effect of MiR-142-3p Targeting HOXA5 on Proliferation, Cycle Arrest and Apoptosis of Acute B Lymphocytic Leukemia Cells

To investigate the effect and molecular mechanism of miR-142-3p to the proliferation, cycle and apoptosis of acute B lymphocytic leukemia (B-ALL) cells by regulating the homeobox gene 5 (HOXA5) expression. Real-time fluorescence quantitative PCR was used to detect the expression levels of miR-142-3p and HOXA5 in human B-ALL cell Nalm6 cell line and human B lymphoblast Hmy2-cir cells. Nalm6 was transfected by using liposome transfection technology, miR-142-3p mimic, pcDNA-HOXA5 overexpression plasmid, miR-142-3p mimic+pcDNA-HOXA5 overexpression plasmid, and control. The binding site of HOXA5 and miR-142-3p was predicted according to microRNA.org, and the targeting relationship between miR-142-3p and HOXA5 gene was detected by double luciferase reporter gene experiment. The effect of miR-142-3p to the proliferation of Nalm6 cells was detected using the Cell Counting Box-8 (CCK-8) method and cell clone formation experiments. Flow cytometry was used to detect the effects of miR-142-3p to cell cycle distribution and apoptosis of Nalm6 cells. The expression levels of cell cycle-related proteins, including G1 /S-specific cyclin-D1 (CyclinD1), Cyclin-dependent kinase 4 (CDK4) and B-cell lymphoma/ leukemia-2 protein (BCL-2), BCL-2 related X protein (Bax), cysteine-aspartate-specific protease (Caspase-3) were detected by Western blot. Compared with Hmy2-cir cells, miR-142-3p showed low expression in Nalm6 cells and HOXA5 showed high expression (P<0.05). MiR-142-3p and HOXA5 3'-UTR showed complementary binding regions, the luciferase activity of miR-142-3p mimic and wild-type HOXA5 3'-UTR was significantly lower than that of miR-142-3p negative control and wild-type HOXA5 3'-UTR (P<0.05). The proliferation of Nalm6 cells and the number of cell clones could be inhibited by miR-142-3p mimic after 48 and 72 hours of transfection (P<0.05), which causing G1 phase arrest of Nalm6 cells and inhibiting the expression of CyclinD1 and CDK4 protein (P<0.01), promoting the apoptosis of Nalm6 cells, and inhibiting the expression of BCL-2 protein, moreover, promoting the expression of Bax and Caspase-3 protein (P<0.05). MiR-142-3p can inhibit the proliferation of Nalm6 cells by targeting down-regulation the expression of HOXA5, arrest the G1 phase of cells, and promote apoptosis of the cells.

San1 deficiency leads to cardiomyopathy due to excessive R-loop-associated DNA damage and cardiomyocyte hypoplasia

R-loops are naturally occurring transcriptional intermediates containing RNA/DNA hybrids. Excessive R-loops cause genomic instability, DNA damage, and replication stress. Senataxin-associated exonuclease (San1) is a protein that interacts with Senataxin (SETX), a helicase resolving R-loops. It remains unknown if R-loops-induced DNA damage plays a role in the heart, especially in the proliferative neonatal cardiomyocytes (CMs). San1−/− mice were generated using the CRISPR/Cas9 technique. The newborn San1−/− mice show no overt phenotype, but their hearts were smaller with larger, yet fewer CMs. CM proliferation was impaired with reduced cell cycle-related transcripts and proteins. S9.6 staining revealed that excessive R-loops accumulated in the nucleus of neonatal San1−/− CMs. Increased γH2AX staining on newborn and adult heart sections exhibited increased DNA damage. Similarly, San1−/− AC16-cardiomyocytes showed cumulative R-loops and DNA damage, leading to the activation of cell cycle checkpoint kinase ATR and PARP1 hyperactivity, arresting G2/M cell-cycle and CM proliferation. Together, the present study uncovers an essential role of San1 in resolving excessive R-loops that lead to DNA damage and repressing CM proliferation, providing new insights into a novel biological function of San1 in the neonatal heart. San1 may serve as a novel therapeutic target for the treatment of hypoplastic cardiac disorders.

Functional and Clinical Characteristics of Cell Adhesion Molecule CADM1 in Cancer.

The cell adhesion molecule CADM1, which participates in cell adhesion and signal transduction, has a regulatory effect on the development of tumors. CADM1 is often involved in malignant tumors of multiple organ systems, such as the respiratory and digestive systems. Upregulated CADM1 promotes tumor cell apoptosis and inhibits malignant proliferation. Along with cell cycle-related proteins, it participates in regulating signaling pathways, such as EMT, STAT3, and AKT, and plays an important role in inhibiting invasion and migration. Considering clinical characteristics, low CADM1 expression is associated with aggressive tumors and poor prognosis. In addition, some long non-coding RNAs (lncRNAs) or miRNAs directly or indirectly act on CADM1 to regulate tumor growth and motility. Interestingly, CADM1 function differs in adult T-cell leukemia/lymphoma (ATLL), and NF-κB is thought to be involved in this process. Taken together, CADM1 could be a potential biomarker for early diagnosis and a target for cancer treatment in future clinical practices.

Demethyleneberberine induces cell cycle arrest and cellular senescence of NSCLC cells via c-Myc/HIF-1α pathway

BackgroundDemethyleneberberine (DMB) is a natural active component of medicinal plant Cortex phellodendri chinensis with favorable bioactivity. However, the role of DMB in suppressing non-small cell lung cancer (NSCLC) remains unknown. PurposeIn this study, we aimed to examine the effect and underlying mechanism of DMB in suppressing NSCLC. MethodsCCK8 assay and colony formation assay were utilized to assess the efficiency of DMB on the viability and colony formation capacity of NSCLC cells. Flow cytometry and β-Galactosidase Staining Kit were utilized to determine the efficiency of DMB on the cell cycle and cellular senescence of NSCLC cells. RT-qPCR and Western blot were used to detect the effect of DMB on cell cycle and cellular senescence related gene and protein expression of NSCLC cells. In vivo tumor model was established to evaluate the anti NSCLC effect of DMB. In addition, RNA-seq analysis was performed to detect the differential gene expression after DMB treatments. ResultsIn this study, we revealed that DMB exhibits efficient inhibitory effect on NSCLC cell proliferation and tumor xenografts growth in vivo. We also demonstrated that DMB could inhibit cell migration by suppressing epithelial-mesenchymal transition (EMT) and trigger cell cycle arrest by down-regulating the expression of cell cycle related genes in NSCLC cells. In addition, DMB treatment efficiently induces cellular senescence of NSCLC cells. From the RNA-seq analysis, we found that DMB accelerates senescence through suppressing HIF-1α expression, which was further elucidated by overexpressing HIF-1α in NSCLC to reduce the inhibitory effect of DMB. Furthermore, we also revealed that DMB decreases the expression of c-Myc, an up-stream protein of HIF-1α. ConclusionsTaken together, we first report that DMB inhibits NSCLC progress through inducing cell cycle arrest and triggering cellular senescence by downregulating c-Myc/HIF-1α pathway.

Antrodia cinnamomea exerts an anti-hepatoma effect by targeting PI3K/AKT-mediated cell cycle progression in vitro and in vivo

Antrodia cinnamomea is extensively used as a traditional medicine to prevention and treatment of liver cancer. However, its comprehensive chemical fingerprint is uncertain, and the mechanisms, especially the potential therapeutic target for anti-hepatocellular carcinoma (HCC) are still unclear. Using UPLC‒Q-TOF/MS, 139 chemical components were identified in A. cinnamomea dropping pills (ACDPs). Based on these chemical components, network pharmacology demonstrated that the targets of active components were significantly enriched in the pathways in cancer, which were closely related with cell proliferation regulation. Next, HCC data was downloaded from Gene Expression Omnibus database (GEO). The Cancer Genome Atlas (TCGA) and DisGeNET were analyzed by bioinformatics, and 79 biomarkers were obtained. Furtherly, nine targets of ACDP active components were revealed, and they were significantly enriched in PI3K/AKT and cell cycle signaling pathways. The affinity between these targets and their corresponding active ingredients was predicted by molecular docking. Finally, in vivo and in vitro experiments showed that ACDPs could reduce the activity of PI3K/AKT signaling pathway and downregulate the expression of cell cycle-related proteins, contributing to the decreased growth of liver cancer. Altogether, PI3K/AKT-cell cycle appears as the significant central node in anti-liver cancer of A. Cinnamomea.

Effect of the phenylpyrrole fungicide fludioxonil on cell proliferation and cardiac differentiation in mouse embryonic stem cells

Fludioxnil is extensively used as a fungicide in agricultural application, but its possible impact on embryonic development is not yet well understood. In this study, the potential effect of fludioxonil on cardiac differentiation was evaluated in mouse embryonic stem cells (mESCs). The water-soluble tetrazolium (WST) and colony formation assays were conducted to confirm the effect of fludioxonil on proliferation of mESCs. The effect of fludioxonil on the ability of mESCs to form mouse embryoid bodies (mEBs) was determined by the hanging drop assay, whereas the ability of cardiomyocyte differentiation in the early stage was evaluated by determining the beating ratio (ratio of the number of contracting cells to the number of attached EBs) of cardiomyocytes. The viability of mESCs was significantly decreased (less than 50 %) at 10−5 M fludioxonil. Results of the colony formation assay revealed suppressed colony formation at 10−5 M fludioxonil (about 50 % at 5 days). Furthermore, the expressions of cell-cycle related proteins, i.e., cyclin D1, cyclin E, p21 and p27, were altered and trending towards inhibiting cell growth. Exposure to fludioxonil also resulted in reduced size of the mEB and induced increasing expression levels of the pluripotency markers Oct4, Sox2 and Nanog. Development of the beating ratio in the process of differentiation to cardiomyocytes derived from mESCs was completely inhibited after exposure to 10−5 M fludioxonil during the early stage of differentiation (day 5), whereas the beating ratio gradually increased after 5-day treatment. Simultaneously, expressions of the cardiomyocyte-related proteins, Gata4, Hand1 and cTnI, were inhibited after exposure to 10−5 M fludioxonil. Taken together, these results imply that fludioxonil may impact on the developmental process of mESCs, particularly the cardiac lineage.

Gallic Acid Hindered Lung Cancer Progression by Inducing Cell Cycle Arrest and Apoptosis in A549 Lung Cancer Cells via PI3K/Akt Pathway.

This study elucidates the anti-cancer potential of gallic acid (GA) as a promising therapeutic agent that exerts its effect by regulating the PI3K/Akt pathway. To prove our research rationale, we used diverse experimental methods such as cell viability assay, colony formation assay, tumor spheroid formation assay, cell cycle analysis, TUNEL assay, Western blot analysis, xenograft mouse model and histological analysis. Treatment with GA inhibited cell proliferation in dose-dependent manner as measured by cell viability assay at 48 h. GA and cisplatin (CDDP) also inhibited colony formation and tumor spheroid formation. In addition, GA and CDDP induced apoptosis, as determined by the distribution of early and late apoptotic cells and DNA fragmentation. Western blot analysis revealed that inhibition of the PI3K/Akt pathway induced upregulation of p53 (tumor suppressor protein), which in turn regulated cell cycle related proteins such as p21, p27, Cyclin D1 and E1, and intrinsic apoptotic proteins such as Bax, Bcl-2 and cleaved caspase-3. The anti-cancer effect of GA was further confirmed in an in vivo mouse model. Intraperitoneal injection with GA for 4 weeks in an A549-derived tumor xenograft model reduced the size of tumor mass. Injection of them downregulated the expression of proliferating cell nuclear antigen and p-Akt, but upregulated the expression of cleaved caspase-3 in tumor tissues. Taken together, these results indicated that GA hindered lung cancer progression by inducing cell cycle arrest and apoptosis, suggesting that GA would be a potential therapeutic agent against non-small cell lung cancer.

Norcantharidin counteracts acquired everolimus resistance in renal cell carcinoma by dual inhibition of mammalian target of rapamycin complex 1 and complex 2 pathways in Vitro.

Everolimus, an oral mammalian target of rapamycin complex 1 (mTORC1) inhibitor, presents a therapeutic option in metastatic renal cell carcinoma (RCC) patients who were intolerant to, or previously failed, immune- and vascular endothelial growth factor-targeted therapies. However, the onset of drug resistance limits its clinical use. One possible mechanism underpinning the resistance is that inhibiting mTORC1 by everolimus results in mTORC2-dependent activation of v-Akt murine thymoma viral oncogene (AKT) and upregulation of hypoxia-inducible transcription factors (HIF). Norcantharidin (NCTD) is a demethylated derivative of cantharidin with antitumor properties which is an active ingredient of the traditional Chinese medicine Mylabris. In this study, everolimus-resistant RCC cells (786-O-R) obtained by chronic everolimus treatment revealed higher level of HIF2α and over-activated mTORC2 pathway and NCTD inhibits cell proliferation in both everolimus-resistant and -sensitive RCC cells by arresting cell cycle in G0/G1 phase and reducing cell cycle-related proteins of C-Myc and cyclin D. Furthermore, NCTD shows synergistic anticancer effects combined with everolimus in everolimus-resistant 786-O-R cells. Mechanically, NCTD repressed both mTORC1 and mTORC2 signaling pathways as well as downstream molecular signaling pathways, such as p-4EBP1, p-AKT, HIF1α and HIF2α. Our findings provide sound evidence that combination of NCTD and everolimus is a potential therapeutic strategy for treating RCC and overcoming everolimus resistance by dual inhibition of mTORC1 and mTORC2.

The mechanisms involved in the resistance of estrogen receptor-positive breast cancer cells to palbociclib are multiple and change over time

PurposeCyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors are widely used for the treatment of advanced estrogen receptor (ER)-positive breast cancer. To develop a treatment strategy for cancers resistant to CDK4/6 inhibitors, here, we established palbociclib-resistant sublines and analyzed their resistance mechanisms.MethodsPalbociclib-resistant sublines were established from T47D and MCF7 cells. Sensitivity to other drugs was assessed via the WST assay. Altered expression/phosphorylation of proteins related to signal transduction and cell cycle regulation was examined using western blotting. Copy number alterations and mutations in the retinoblastoma (RB1) gene were also analyzed.ResultsAlthough an increase in CDK6 and decrease in retinoblastoma protein (Rb) expression/phosphorylation were commonly observed in the resistant sublines, changes in other cell cycle-related proteins were heterogeneous. Upon extended exposure to palbociclib, the expression/phosphorylation of these proteins became altered, and the long-term removal of palbociclib did not restore the Rb expression/phosphorylation patterns. Consistently a copy number decrease, as well as RB1 mutations were detected. Moreover, although the resistant sublines exhibited cross-resistance to abemaciclib, their response to dinaciclib was the same as that of wild-type cells. Of note, the cell line exhibiting increased mTOR phosphorylation also showed a higher sensitivity to everolimus. However, the sensitivity to chemotherapeutic agents was unchanged in palbociclib-resistant sublines.ConclusionER-positive breast cancer cells use multiple molecular mechanisms to survive in the presence of palbociclib, suggesting that targeting activated proteins may be an effective strategy to overcome resistance. Additionally, palbociclib monotherapy induces mutations and copy number alterations in the RB1 gene.

Abstract 1139: Potent SMARCA2 targeted degraders induce genetic synthetic lethality in SMARCA4 deleted cancer

Abstract SWI/SNF complexes play an important role in controlling gene expression by remodeling chromatin. SMARCA2 (BRM) and SMARCA4 (BRG1) are the core subunits of the SWI/SNF complexes which contain ATPase domain and DNA binding bromodomain. SMARCA4 protein expression is lost in some cancers due to damaging mutations (e.g. nonsense, frameshift deletion, splice site mutations) and SMARCA4-deleted cancer cells are highly dependent on its paralog gene SMARCA2 for their survival. Therefore, targeting SMARCA2 in SMARCA4-deleted cancers through the use of selective SMARCA2 degraders induces synthetic lethality. We designed and synthesized a variety of novel SMARCA2 degraders and tested them for degradation potency and selectivity by high-throughput in-cell western blot and probed for both SMARCA2 and SMARCA4 proteins in SMARCA4 WT expressing non-small cell lung cancer (NSCLC) NCI-H520 cells. We identified a unique series of potent SMARCA2 degraders with 20-40 fold greater selectivity for SMARCA2 vs SMARCA4. Global proteomics analysis confirmed that these degraders are highly selective for SMARCA2, and demonstrate minimal degradation of any other protein except PBRM1, another bromodomain VIII family protein. Our data suggest that the greater degradation selectivity observed is a result of preferential SMARCA2 ternary complex formation and/or poly-ubiquitination processing. Global RNA expression analysis shows that treatment with SMARCA2 degraders downregulates gene signatures associated with the cell cycle, cell proliferation, apoptosis and cell adhesion in SMARCA4-deleted NSCLC NCI-H1693 cells. Specifically, we found a significant reduction of gene expression of AXL, SMAD3, MMP2, and KRT80, all of which are important factors for tumor cell signaling and invasion. Additionally, protein set enrichment analysis (PSEA) shows that SMARCA2 degrader-treated NCI-H1693 cells downregulate expression of cell cycle and DNA replication related protein signatures as well as upregulated antigen processing and presentation pathway. A cell line panel analysis for cell viability and clonogenicity demonstrated that our selective SMARCA2 degraders effectively inhibit proliferation of SMARCA4-deleted cancer cells with nanomolar potencies, but not SMARCA4 WT or SMARCA2/4 null cancer cells. Our SMARCA2 degraders also significantly suppressed the growth of patient derived SMARCA4-deleted NSCLC tumor cells in primary 3D culture assay. In summary, consistent with previous studies and genomic perturbation analyses, our potent and selective SMARCA2 degraders induce synthetic lethality in SMARCA4-deleted cancers in vitro. Citation Format: Koichi Ito, Anjana Agarwal, Philip Pitis, Min Wang, Jack Carter, Miles Cowart, Chaofeng Dai, William Gowen-MacDonald, Eliza Elliot, Brian Vidal, Jacob Spruance, Hsin-Yao Tang, Bruce Ruggeri, Liang Lu, Andrew Combs, Hong Lin, Peggy Scherle, Kris Vaddi. Potent SMARCA2 targeted degraders induce genetic synthetic lethality in SMARCA4 deleted cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1139.

Long Non-Coding RNA GABPB1-AS1 Augments Malignancy of Glioma Cells by Sequestering MicroRNA-330 and Reinforcing the ZNF367/Cell Cycle Signaling Pathway.

BackgroundDeregulation of long non-coding RNAs (lncRNAs) is frequently relevant to the malignant phenotypical changes. This study aimed to explore the role of lncRNA GABPB1-AS1 in the malignancy of glioma cells.MethodsAbnormally expressed genes in glioma were analyzed using a GEO GSE2223 dataset. Short hairpin (sh) RNA silencing of GABPB1-AS1 was introduced in glioma cells to explore its correlation with the proliferation, apoptosis, and invasiveness of cancer cells. The target transcripts of GABPB1-AS1 were predicted by bioinformatics analyses. MicroRNA (miR)-330 inhibition was additionally introduced in the glioma cells after GABPB1-AS1 knockdown for rescue experiments. Animal studies were performed by inducing xenograft tumors in nude mice.ResultsGABPB1-AS1 was highly expressed in the glioma tissues and associated with advanced WHO grades. GABPB1-AS1 knockdown reduced proliferation and invasiveness of glioma cells in vitro and in vivo. miR-330 was a target transcript of GABPB1-AS1. miR-330 inhibition restored the malignancy of glioma cells. miR-330 directly bound to ZNF367. ZNF367 was highly expressed in glioma tissues and positively correlated with GABPB1-AS1 expression, and it was relevant to the cell cycle signaling pathway. Downregulation of GABPB1-AS1 reduced the expression of ZNF367 and reduced the levels of cell cycle-related proteins PCNA, CDC20, CDC7 and CCNA1 in cells.ConclusionThis study demonstrated that GABPB1-AS1 competitively bound to miR-330 and de-repressed ZNF367 expression, leading to activation of the cell cycle signaling pathway and the growth and metastasis of glioma cells.

Abstract 2412: Decursin inhibits tumor progression in head and neck squamous cell cancer by down regulating CXCR7 expression

Abstract Background: CX chemokine receptor 7 (CXCR7) is frequently overexpressed in a variety of tumors and plays a significant role in tumor growth and metastasis. Thus, inhibition of CXCR7 is an important potential anticancer treatment strategy. Decursin, a derivative from Angelica gigas Nakai has been reported to have anti-tumor activity in various cancers. However, little is known about its anti-tumor activity and underlying mechanisms of action in head and neck squamous cell cancer (HNSCC). Methods: CXCR7-overexpressing HNSCC cell lines were established in vitro. The effects of decursin on cell growth, motility, migration, and invasion were investigated using CCK-8, wound healing, and transwell assays. In addition, the mechanism by which decusin shows antitumor activity was investigated. Results: Overexpression of CXCR7 significantly increased cell proliferation, migration, invasion, and cell motility compared with control cells. Treatment with decursin decreased membrane expression of CXCR7 in a concentration dependent manner and significantly inhibited tumor progression by suppressing cell growth, migration, invasion, and motility. Decursin was found to induce G0/G1 cell cycle arrest and regulate cell cycle-related proteins such as Cyclin A and CDK2 in HNSCC cells. In addition, decursin inhibited CXCR7-induced STAT3/c-MYC activation. Conclusion: These results demonstrate that CXCR7 promotes cancer progression through the STAT3/c-Myc pathway in HNSCC and that decursin is a natural product that may target CXCR7 for HNSCC treatment. Citation Format: Mina Joo, Solbi Kim, Nayoung Kim, Myung-Won Lee, Heung Jin Jeon, Hyewon Ryu, Ik-Chan Song, Jin-Man Kim, Hyo Jin Lee. Decursin inhibits tumor progression in head and neck squamous cell cancer by down regulating CXCR7 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2412.

Abstract 1961: Cyclin-dependent kinase inhibition in mesothelioma

Abstract Background: Mesothelioma is a cancer that arises out of the pleura or the peritoneum and is considered incurable in the majority of patients afflicted. Abnormalities of the cell cycle pathway are among the most common molecular alterations in mesothelioma. Previously, we have demonstrated that inhibition of CDK4/6 by abemaciclib or palbociclib is effective against mesothelioma in vitro. Increased expression of Cyclin E has been demonstrated to be one mechanism of resistance to CDK4/6 inhibition in other cancers. We hypothesized that select cell cycle-related proteins may compensate for inhibition of one part of the cell cycle machinery and that the CDK2 inhibitor dinaciclib may act synergistically in combination with abemaciclib. Methods: Dinaciclib was evaluated for activity against mesothelioma cells in vitro with regard to cell proliferation and colony formation. In addition, dinaciclib was evaluated in combination with abemaciclib at several concentration combinations in a cell proliferation assay to determine if the combination may be additive or synergistic. Combination indices (CI) were calculated via the Chou-Talalay method. Immunoblotting was used to evaluate for expression of key survival proteins after treatment with abemaciclib. Results: The IC50 of dinaciclib was determined to be 18 nM, 23 nM, 32 nM, and 33 nM in mesothelioma cell lines H2373, H2052, H2452, and H2461, respectively. Colony formation was decreased significantly after 72 hours incubation with 50 nM or 100 nM dinaciclib in Bap1-expressing (35.1 ± 2%; 22.8 ± 2%) and Bap1-null cell lines (14.5 ± 3%; 8.1 ± 2%) versus untreated controls. CI &amp;lt; 1 (denoting synergism) was identified for several combinations of varied concentrations of abemaciclib and dinaciclib for both cell lines evaluated. Further, treatment with abemaciclib for 72 hrs in both Bap1-expressing and Bap 1-null cells at 1x the IC50 resulted in complete loss of FoxM1 expression and pro-survival survivin and significantly decreased the expression of Mcl-1 and XIAP. Conclusions: Dinaciclib is a potent inhibitor of mesothelioma cell growth. The combination of abemaciclib and dinaciclib is synergistic across a spectrum of concentrations in vitro. Further experiments are currently underway to determine which CDKs and related proteins may be more effectively targeted in mesothelioma. As the cell cycle is altered in the vast majority of mesothelioma tumors, select combinations of CDK inhibition may be a feasible future therapeutic strategy against mesothelioma. Citation Format: Marian Kratzke, Betsy Kren, Stephen Porter, George Scaria, Mark A. Klein. Cyclin-dependent kinase inhibition in mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1961.

O2-(2,4-dinitrophenyl) diazeniumdiolate derivative induces G2/M arrest via PTEN-mediated inhibition of PI3K/Akt pathway in hepatocellular carcinoma cells.

The study aimed to investigate whether G2/M arrest caused by O2-(2,4-dinitrophenyl) diazeniumdiolate derivative (JS-K) was related to PTEN-mediated inhibition of PI3K/Akt pathway in hepatocellular carcinoma cells. The cell apoptosis was detected by DAPI staining and Annexin V-FITC/PI dual staining. The cell cycle was analysed by PI staining. The expressions of cell cycle-related proteins, PTEN and PI3K/AKT pathway were measured by Western blot. The rat model of primary hepatic carcinoma was established with diethylnitrosamine to verify the antitumour effects of JS-K. The morphological features of apoptosis were obviously reversed when the cells were pre-treated with bpv(pic), followed by treatment with JS-K. JS-K mediated G2/M arrest and down-regulated expressions of cyclin B1. Meanwhile, it up-regulated the expression of p-Cdk1, p-Chk2 and p-CDC25C while down-regulated that of Cdk1 and CDC25C. Furthermore, JS-K also enhanced the expressions of p21 and p27, PTEN and p53 while decreased the expressions of p-PTEN, PI3K and p-AKT. However, bpv(pic) and Carboxy-PTIO could reverse JS-K-induced G2/M cell arrest and PTEN-mediated inhibition of the PI3K/AKT pathway. The same results were also testified in the rat model of primary hepatic carcinoma. JS-K caused G2/M arrest through PTEN-mediated inhibition of the PI3K/AKT pathway involving Chk2/CDC25C/Cdk1 checkpoint.

Role of Notch pathway in effect of mono-2-ethylhexyl phthalate on the proliferation and cell cycle of SH-SY5Y cell.

Neuroblastoma (NB) is an estrogen-dependent tumor. Mono-2-ethylhexyl phthalate (MEHP) has an estrogen-like effect. However, the effects of MEHP on the progression of NB are not well illustrated. This study was to clarify the effect of Notch pathway on proliferation and cell cycle of SH-SY5Y cell induced by MEHP. The viability of SH-SY5Y and BE2C cells were detected by CCK8; cell cycle and apoptosis were detected by flow cytometry; the protein expression levels of Notch pathway and cell cycle related proteins were detected by Western-blot. Results show that MEHP exposure can promote cell proliferation and altered the cell cycle. MEHP exposure can up-regulate the expression of C-MYC, Cyclin D1, Bcl-2 and affected the Notch pathway. In conclusion, MEHP exposure can promote NB cell proliferation and affect the cell cycle and apoptosis. Notch pathway plays a critical role in accelerating the cell cycle and inhibiting the apoptosis of SH-SY5Y cells caused by MEHP.

Physalin B inhibits cell proliferation and induces apoptosis in undifferentiated human gastric cancer HGC-27 cells.

Physalin B (PB) from Physalis angulata L. (Solanaceae) is a naturally occurring secosteroid with multiple biological activities, including anti-inflammatory and anticancer activity. However, PB's effects and mechanisms in human gastric cancer (GC) cells are not well characterized. The undifferentiated GC cell line HGC-27 and semi-differentiated GC cell line SGC-7901 were treated with PB. Cell counting kit-8 (CCK-8) and colony formation assays were performed to evaluate cell viability. Apoptosis and the cell cycle were assessed by Annexin V/PI and PI/RNase DNA staining assays, respectively, and Western blotting was used to evaluate the expression of a protein. PB significantly inhibited the proliferation of HGC-27 cells in a dose- and time-dependent manner. Moreover, PB induced G0/G1 cycle arrest and caspase-dependent apoptosis of HGC-27 cells. Cleaved caspases 8, 3, and 7, poly(ADP)-ribose polymerase (PARP), and the cyclin-dependent kinase (CDK) inhibitor p-Chk2 was induced by PB in HGC-27 cells, while the cell cycle-related proteins cyclin D1, cyclin D3, CDK4, CDK6, cyclin E, and phosphorylated retinoblastoma tumor suppressor protein (p-Rb) were downregulated in a dose-dependent manner. PB inhibits proliferation via cyclin-dependent kinase and induces caspase-dependent apoptosis in HGC-27 cells, suggesting that PB might be a novel and effective agent for undifferentiated GC therapy.

Incaspitolide A extracted from Carpesium cernuum induces apoptosis in vitro via the PI3K/AKT pathway in benign prostatic hyperplasia.

Benign prostatic hyperplasia (BPH) is a common disease that occurs mainly in older men. The pathogenesis of BPH is complex and patients face a prolonged treatment course, and novel drugs with better selectivity and lower toxicity are required. Incaspitolide A (compound TMJ-12) is a germacrane-type sesquiterpenoid compound extracted from the plant Carpesium carnuum. Extracts of C. carnuum are known to exert suppressive effects on BPH-1 cells. In the present study, we investigated the molecular mechanisms underlying the suppressive effect of TMJ-12 specifically on BPH-1 cells. A cytotoxicity assay indicated that TMJ-12 inhibited BPH-1 cell proliferation, while flow cytometry assays showed that TMJ-12 induced G2/M phase cell cycle arrest and the apoptosis of BPH-1 cells. TMJ-12 was also shown to regulate the expression of several apoptosis- and cell cycle-related proteins, namely Bcl-2, Bax, Bad, Caspase-9, Caspase-3, cyclin-dependent kinase 1 (CDK1), Cyclin B1, CDC25C, and c-Myc, among others. Collapse of the mitochondrial membrane potential (ΔΨm) following exposure to TMJ-12 was detected with the JC-1 staining assay. Further investigation revealed that treatment with TMJ-12 inhibited the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway by increasing the expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Taken together, the results suggest that TMJ-12 prevents BPH-1 cell proliferation via the PI3K/AKT pathway by inducing apoptosis and cell cycle arrest.

MITF Promotes Cell Growth, Migration and Invasion in Clear Cell Renal Cell Carcinoma by Activating the RhoA/YAP Signal Pathway.

Simple SummaryMicrophthalmia-associated transcription factor (MITF) has been reported to play a role in the progression of melanoma and other cancer types. However, the biological role of MITF in clear cell renal cell carcinoma (ccRCC) is largely unknown. In this study, we elucidate the role of MITF in the progression of ccRCC. MITF- and MITF-mediated signaling pathways were investigated in ccRCC cell through MITF knockdown as well as overexpression of MITF in vitro and in vivo. MITF contributed to cell proliferation, migration, invasion and tumor growth in ccRCC through activation of the RhoA/YAP signaling pathways. This study suggests that MITF has potential as a therapeutic target in ccRCC.Microphthalmia-associated transcription factor (MITF) is a basic helix-loop-helix leucine zipper transcription factor involved in the lineage-specific regulation of melanocytes, osteoclasts and mast cells. MITF is also involved in the progression of melanomas and other carcinomas, including the liver, pancreas and lung. However, the role of MITF in clear cell renal cell carcinoma (ccRCC) is largely unknown. This study investigates the functional role of MITF in cancer and the molecular mechanism underlying disease progression in ccRCC. MITF knockdown inhibited cell proliferation and shifted the cell cycle in ccRCC cells. In addition, MITF knockdown reduced wound healing, cell migration and invasion compared with the controls. Conversely, MITF overexpression in SN12C and SNU482 cells increased cell migration and invasion. Overexpression of MITF activated the RhoA/YAP signaling pathway, which regulates cell proliferation and invasion, and increased YAP signaling promoted cell cycle-related protein expression. Additionally, tumor formation was impaired by MITF knockdown and enhanced by MITF overexpression in vivo. In summary, MITF expression was associated with aggressive tumor behavior, and increased the migratory and invasive capabilities of ccRCC cells. These effects were reversed by MITF suppression. These results suggest that MITF is a potential therapeutic target for the treatment of ccRCC.