Expression Of Cyclin Research Articles

Tazarotene-induced gene 1 interacts with Polo-like kinase 2 and inhibits cell proliferation in HCT116 colorectal cancer cells.

Tazarotene-induced gene 1 (TIG1) is considered to be a tumor suppressor gene that is highly expressed in normal or well-differentiated colon tissues, while downregulation of TIG1 expression occurs in poorly differentiated colorectal cancer (CRC) tissues. However, it is still unclear how TIG1 regulates the tumorigenesis of CRC. Polo-like kinases (Plks) are believed to play an important role in regulating the cell cycle. The performance of PLK2 in CRC is negatively correlated with the differentiation status of CRC tissues. Here, we found that PLK2 can induce the growth of CRC cells and that TIG1 can prevent PLK2 from promoting the proliferation of CRC cells. We also found that the expression of PLK2 in CRC cells was associated with low levels of Fbxw7 protein and increased expression of cyclin E1. When TIG1 was coexpressed with PLK2, the changes in Fbxw7/cyclin E1 levels induced by PLK2 were reversed. In contrast, silencing TIG1 promoted the proliferation of CRC, and when PLK2 was also silenced, the proliferation of CRC cells induced by TIG1 silencing was significantly inhibited. The above research results suggest that TIG1 can regulate the tumorigenesis of CRC by regulating the activity of PLK2.

Effects of water-soluble components of atmospheric particulates from rare earth mining areas in China on lung cancer cell cycle

BackgroundThis study aims to investigate the effects of water soluble particulate matter (WSPM) on the viability and protein expression profile of human lung adenocarcinoma cell A549 in the Bayou Obo rare earth mining area, and explore the influence of WSPM on the A549 cell cycle.ResultsIt was found that WSPM can inhibit the viability of A549 cells and induce cell arrest in the G2/M phase. Compared with controls, exposure to WSPM10 and WSPM2.5 induced 134 and 116 proteins to be differentially expressed in A549 cells, respectively. In addition, 33 and 31 differentially expressed proteins were further confirmed, and was consistent with the proteomic analysis. The most prominent enrichment in ribosome-associated proteins were presented. When RPL6, RPL13, or RPL18A gene expression was inhibited, A549 cells were arrested in the G1 phase, affecting the expression of Cyclin D1, p21, RB1, Cyclin A2, Cyclin B1, CDC25A, CDK2, CHEK2 and E2F1. Furthermore, the La3+, Ce3+, Nd3+ and F- in WSPM also inhibited the viability of A549 cells. After 24 h of exposure to 2 mM of NaF, A549 cells were also arrested in the G2/M phase, while the other three compounds did not have this effect. These four compounds affected the cell cycle regulatory factors in A549 cells, mainly focusing on effecting the expression of CDK2, CDK4, RB1, ATM, TP53 and MDM2 genes. These results are consistent with the those from WSPM exposure.ConclusionsThese results revealed that WSPM from rare earth mines decreased the viability of A549 cells, and induced cell cycle G2/M phase arrest, and even apoptosis, which may be independent of the NF-κB/MYD88 pathway, and be perceived by the TLR4 receptor. The dysfunction of the cell cycle is correlated to the down-expression of ribosomal proteins (RPs). However, it is not the direct reason for the A549 cell arrest in the G2/M phase. La3+, Ce3+, and F- are probably the main toxic substances in WSPM, and may be regulate the A549 cell cycle by affecting the expression of genes, such as MDM2, RB1, ATM, TP53, E2F1, CDK2 and CDK4. These results indicate the importance for further research into the relationship between APM and lung cancer.

Effect of CDK1 Interferes with the Regulation of PLK1, Aurora B and TRF1 on the Proliferation of Leukemia Cells

To investigate the effect of CDK1 interference regulation of PLK1, Aurora B and TRF1 on the proliferation of leukemia cells. The human myelogenous leukemia cell line HL-60 was selected as the research object, and the effect of TRF1 expression and its changes on cell proliferation and cycle was investigated by regulating intracellular CDK1 expression. The objects were divided into 5 groups, including control group, shRNA-NC group, CDK1-shRNA group, pcDNA group and pcDNA-CDK1 group. RT-PCR was used to detect the CDK1 expression of cells in each group; colony formation was used to detect the proliferation of the cells. Western blot was used to detect the expression of CDK1, PLK1, Aurora B, TRF1, and cyclin p53, p27, cyclinA. The phosphorylation level of PLK1, Aurora B and the expression of TRF1 in the CDK1-shRNA group were significantly down-regulated as compared with those in the control group (P<0.05). Compared with the control group, the cells in CDK1-shRNA group showed lower clone formation rate, the increasing of cycle-associated proteins p53 and p27 and the decreasing of cyclinA expression (P<0.05). It was shown that interfered CDK1 expression could inhibit the proliferation of HL-60 cells and prolong the time that they enter mitosis, thereby extending the cell cycle. Compared with the control group, the overexpressed CDK1 in the pcDNA-CDK1 group made the phosphorylation level of PLK1, Aurora B, and TRF1 expression increase significantly (P<0.05), also the colony formation rate (P<0.05). The cycle-related proteins p53 and p27 was down-regulated, while cyclinA expression was up-regulate significantly (P<0.05). The results indicted that overexpressed CDK1 could stimulate adverse reactions, thereby promoting the proliferation of HL-60 cells and shortening the cell cycle. Knocking out CDK1 can inhibit the phosphorylation of PLK1 and Aurora B and negatively regulate TRF1, thereby inhibiting the proliferation of leukemia cells.

Set7/9 controls proliferation and genotoxic drug resistance of NSCLC cells

The SET domain containing lysine-specific methyltransferase, Set7/9, covalently attaches methyl moieties to a variety of histone and non-histone substrates. Among the substrates of Set7/9 are: p53, NF-kB, PARP1, E2F1, and other transcription factors that regulate many vital processes in the cell. Through the post-translational regulation of these critical master-regulators Set7/9 is involved in regulation of cell proliferation, cancer progression, and DNA damage response. Noteworthy, the role of Set7/9 in tumorigenesis is contradictory and apparently depends on the cellular context. In this study, we investigated the effect of Set7/9 on tumorigenic characteristics of lung cancer cells. We showed that CRISPR/Cas9-mediated knock-out of Set7/9 in A549 and its shRNA-mediated knock-down in H1299 NSCLC cell lines both augment the proliferation rate of tumor cells compared to the matching wild-type cells. Mechanistically, ablation of Set7/9 increased the expression of cyclin A2 and D1 genes thereby promoting the accumulation of cells in S phase. Furthermore, knockout of Set7/9 decreased the expression of E-cadherin, whose product is critical for cell-cell interactions. Accordingly, this led to the increased migration of lung cancer cells.Finally, both ablation or pharmacological inhibition of Set7/9 enzymatic methyltransferase activity by the selective inhibitor (R)-PFI-2 sensitized NSCLC cells to genotoxic drug, doxorubicin. This effect was also recapitulated on patients-derived NSCLC cell lines. Taken together, our results suggest that Set7/9 plays anti-proliferative and DNA damage-protective roles in NSCLC cells and hence represents an attractive target for anti-cancer chemotherapy.

Local ablation of gastric cancer by reconstituted apolipoprotein B lipoparticles carrying epigenetic drugs

Epigenetic inhibitors have shown anticancer effects. Combination chemotherapy with epigenetic inhibitors has shown high effectiveness in gastric cancer clinical trials, but severe side effect and local progression are the causes of treatment failure. Therefore, we sought to develop an acidity-sensitive drug delivery system to release drugs locally to diminish unfavorable outcome of gastric cancer. In this study, we showed that, as compared with single agents, combination treatment with the demethylating agent 5′-aza-2′-deoxycytidine and HDAC inhibitors Trichostatin A or LBH589 decreased cell survival, blocked cell cycle by reducing number of S-phase cells and expression of cyclins, increased cell apoptosis by inducing expression of Bim and cleaved Caspase 3, and reexpressed tumor suppressor genes more effectively in MGCC3I cells. As a carrier, reconstituted apolipoprotein B lipoparticles (rABLs) could release drugs in acidic environments. Orally administrated embedded drugs not only showed inhibitory effects on gastric tumor growth in a syngeneic orthotopic mouse model, but also reduced the hepatic and renal toxicity. In conclusion, we have established rABL-based nanoparticles embedded epigenetic inhibitors for local treatment of gastric cancer, which have good therapeutic effects but do not cause severe side effects.

High Glucose Induced Upregulation of Cyclin a Associating with a Short Survival of Patients with Cholangiocarcinoma: A Potential Target for Treatment of Patients with Diabetes Mellitus

Diabetes mellitus (DM) is associated with an increased risk and progression of cholangiocarcinoma (CCA). High glucose underlying the association between DM and CCA by modulating the intracellular signaling has been demonstrated. However, the effects of DM and hyperglycemia on cell cycle machineries and progression of CCA remain elucidated. CCA cells, KKU-213A and KKU-213B were cultured in normal (NG, 5.6 mM) or high glucose (HG, 25 mM) resembling euglycemia and hyperglycemia. Western blotting was used to determine expressions of cell cycle machineries in CCA cells. The expression of cyclin A in CCA tissues from patients with or without hyperglycemia was determined by immunohistochemistry. Pan-cyclin dependent kinases (CDKs) inhibitor and silencing of cyclin A expression were investigated as a possible modality targeting CCA treatment in patients with DM. High glucose induced expression of cell cycle machinery proteins in both CCA cells. Among these, cyclin A was consistently and significantly upregulated. Nuclear cyclin A was significantly increased in tumor tissues from CCA patients with hyperglycemia and was significantly associated with post-operative survival of shorter than 5 mo. Silencing cyclin A expression sensitized CCA cells to pan-CDKs inhibitor, suggesting the combined treatment as an alternative approach for treatment of CCA patients with DM.

MRNP granule proteins Fmrp and Dcp1a differentially regulate mRNP complexes to contribute to control of muscle stem cell quiescence and activation

BackgroundDuring skeletal muscle regeneration, satellite stem cells use distinct pathways to repair damaged myofibers or to self-renew by returning to quiescence. Cellular/mitotic quiescence employs mechanisms that promote a poised or primed state, including altered RNA turnover and translational repression. Here, we investigate the role of mRNP granule proteins Fragile X Mental Retardation Protein (Fmrp) and Decapping protein 1a (Dcp1a) in muscle stem cell quiescence and differentiation.MethodsUsing isolated single muscle fibers from adult mice, we established differential enrichment of mRNP granule proteins including Fmrp and Dcp1a in muscle stem cells vs. myofibers. We investigated muscle tissue homeostasis in adult Fmr1-/- mice, analyzing myofiber cross-sectional area in vivo and satellite cell proliferation ex vivo. We explored the molecular mechanisms of Dcp1a and Fmrp function in quiescence, proliferation and differentiation in a C2C12 culture model. Here, we used polysome profiling, imaging and RNA/protein expression analysis to establish the abundance and assembly status of mRNP granule proteins in different cellular states, and the phenotype of knockdown cells.ResultsQuiescent muscle satellite cells are enriched for puncta containing the translational repressor Fmrp, but not the mRNA decay factor Dcp1a. MuSC isolated from Fmr1-/- mice exhibit defective proliferation, and mature myofibers show reduced cross-sectional area, suggesting a role for Fmrp in muscle homeostasis. Expression and organization of Fmrp and Dcp1a varies during primary MuSC activation on myofibers, with Fmrp puncta prominent in quiescence, but Dcp1a puncta appearing during activation/proliferation. This reciprocal expression of Fmrp and Dcp1a puncta is recapitulated in a C2C12 culture model of quiescence and activation: consistent with its role as a translational repressor, Fmrp is enriched in non-translating mRNP complexes abundant in quiescent myoblasts; Dcp1a puncta are lost in quiescence, suggesting stabilized and repressed transcripts. The function of each protein differs during proliferation; whereas Fmrp knockdown led to decreased proliferation and lower cyclin expression, Dcp1a knockdown led to increased cell proliferation and higher cyclin expression. However, knockdown of either Fmrp or Dcp1a led to compromised differentiation. We also observed cross-regulation of decay versus storage mRNP granules; knockdown of Fmrp enhances accumulation of Dcp1a puncta, whereas knockdown of Dcp1a leads to increased Fmrp in puncta.ConclusionsTaken together, our results provide evidence that the balance of mRNA turnover versus utilization is specific for distinct cellular states.

Novel thiazolidine derivatives as potent selective pro-apoptotic agents

A series of 2-arylthiazolidine-4-carboxylic acid amide derivatives were synthesized and their cytotoxic activity against three cancer cell lines (PC-3, SKOV3 and MDA-MB231) was evaluated. Various structural modifications were tried including modifications of the length of the amide chain and modifications of the 2-aryl part using disubstituted phenyl and thiophene derivatives. The structure activity relationship was evaluated based on the in vitro biological evaluation against the above mentioned three cancer cell lines. The most selective compounds towards cancer cells were further evaluated against DLD-1, NCI-H520, Du145, MCF-7 and NCI-N87 cancer cells. The dodecyl amide having the 4-bromothienyl as the 2-aryl, compound 2e, exhibited the highest selectivity for cancer cells vs non-tumor cells. Mechanistic studies of the anticancer effect of compound 2e in prostate cancer PC-3 and colorectal cancer DLD-1 cells revealed that 2e could prevent the cell cycle in the G0/G1 phase by up-regulating the expression of p21 and reducing the expression of CDK2 and cyclin E. It increased the pro-apoptotic protein Bax and cleaved caspase 3, and down-regulated the expression of anti-apoptotic protein Bcl-2 to induce apoptosis. In addition, 2e also downregulated AKT, N-cadherin, and vimentin proteins expression giving indication that 2e inhibit the PI3K/AKT pathway to regulate cell cycle arrest and induce apoptosis, and can regulate the expression of epithelial–mesenchymal transition-related proteins.

3',4'-Dihydroxyflavonol Modulates the Cell Cycle in Cancer Cells: Implication as a Potential Combination Drug in Osteosarcoma.

New agents are demanded to increase the therapeutic options for osteosarcoma (OS). Although OS is the most common bone cancer in children and adolescents, it is considered a rare disorder. Therefore, finding adjuvant drugs has potential to advance therapy for this disease. In this study, 3′,4′-dihydroxyflavonol (DiOHF) was investigated to assess the effects in OS cellular models in combination with doxorubicin (Dox). MG-63 and U2OS human OS cells were exposed to DiOHF and Dox and tested for cell viability and growth. To elucidate the inhibitory effects of DiOHF, additional studies were conducted to assess apoptosis and cell cycle distribution, gene expression quantification of cell cycle regulators, and cytokinesis-block cytome assay to determine nuclear division rate. DiOHF decreased OS cell growth and viability in a concentration-dependent manner. Its combination with Dox enabled Dox dose reduction in both cell lines, with synergistic interactions in U2OS cells. Although no significant apoptotic effects were detected at low concentrations, cytostatic effects were demonstrated in both cell lines. Incubation with DiOHF altered cell cycle dynamics and resulted in differential cyclin and cyclin-dependent kinase expression. Overall, this study presents an antiproliferative action of DiOHF in OS combination therapy via modulation of the cell cycle and nuclear division.

Abstract 1017: Arsenic trioxide reduces the expression of E2F1, cyclin E, and phosphorylation of PI3K signaling molecules in acute leukemia cells

Abstract Arsenic trioxide (ATO) has been used for the treatment of acute promyelocytic leukemia (APL). Although ATO modulates cell cycle progression and apoptosis in APL cells, its exact mechanism of action remains elusive. In this research, we investigated its effects on E2F1, cyclin E, p53, pRb and PI3K signaling molecules by western blotting, immunocytochemistry and/or confocal imaging. We found that ATO inhibited the proliferation of APL cells through down-regulation of E2F1 and cyclin E expression, and stimulation of pRb. It also reduced the interaction of pRb and E2F1with binding to the E2F1 promoter, by stimulating pRb association. ATO also effected the phosphorylation of pRb at S608 and T373 residues and association of E2F1, pRb, and p53, simultaneously. However, in p53-knockdown NB4 cells, ATO did not significantly reduce E2F1 and cyclin E expression. Our findings demonstrate that ATO inhibits APL cell growth through reduced expression of E2F1, cyclin E, and stimulation of pRb. It also effected both interaction and association of E2F1, pRb and p53 by phosphorylation of pRb at T373 and S608 residues and reduced phosphorylation of PI3K signaling molecules. This novel mode of action of ATO in APL cells may be useful for designing new APL drugs. Citation Format: Paul B. Tchounwou, Sanjay Kumar. Arsenic trioxide reduces the expression of E2F1, cyclin E, and phosphorylation of PI3K signaling molecules in acute leukemia cells [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 1017.

Abstract 1386: The role of MDM2 inhibition in the radiosensitization of ER+ breast cancers

Abstract Background​: Radiation therapy (RT) is standard in the treatment of many women with breast cancer (BC). Despite this, women with estrogen receptor positive (ER+) BC respond heterogeneously to RT. Radiosensitization methods for aggressive ER+ disease are needed. We performed a radiosensitizer screen paired with transcriptomic and proteomic data from ER+ models treated +/-RT to identify potential mediators of RT resistance. Methods​: Clonogenic survival assays were used to determine RT sensitivity of 21 BCC lines as well as radiosensitization with drug treatment. IC50 values were determined for 130 clinical compounds and correlation coefficients were calculated using IC50 values and SF-2Gy. Microarray and RPPA data was used for differential gene/protein expression and pathway analysis. AlamarBlue was used to determine IC-50 values of the MDM2 inhibitor AMG-232. Western blot analysis of Cleaved PARP and Annexin V staining for FLOW was used to measure apoptosis and Cyclins A, E, B and p-Histone H3 and flow cytometry to measure cell cycle progression. yH2AX immunofluorescence was used to measure dsDNA breaks. Results​: A MDM2 inhibitor (JNJ-26854165) was nominated as an effective drug in treatment for RT-resistant BC cell lines (R2​ = 0.43, p-value &amp;lt;0.01) in our novel radiosensitizer screen. Differential gene expression and pathway analysis in multiple non-overlapping ER+ BC cell lines treated +/-RT identified apoptosis, cell cycle, and p53 signaling as the top pathways induced in ER+ cell lines by RT. Within these MDM2 was significantly overexpressed after RT+ compared to RT- in ER+ p53 wild-type (WT) cells. In p53 mutant (MT) cell lines, however, MDM2 was not differentially expressed. This suggests MDM2 may mediate radioresistance in a p53 dependent manner. Cell growth in the p53​ ​WT cell lines MCF-7 and ZR-75-1 was inhibited by AMG-232, an MDM2 inhibitor (IC-50 values of 554nM and 264nM). p53 MT ER+ cell lines were not sensitive to MDM2 inhibition with this drug (IC-50&amp;gt; 10uM). Clonogenic survival assays demonstrated that at sub-IC50 doses MDM2 inhibition leads to radiosensitization in p53 WT ER+ cell lines (MCF-7 rER: 1.17-2.13; ZR751 rER: 1.30-1.65), however, p53 MT ER+ cells were not radiosensitized (T47D rER: 0.94-1.11; CAMA-1 rER: 0.88-0.95). AMG-232 and RT combined led to an increase in apoptosis compared to RT alone in ER+ p53 WT cells but not p53 MT cells. Combination treatment led to differential cyclin and p-Histone H3 expression in p53 WT cells but not p53 MT cells. ​G1 cell cycle arrest was a secondary effect of MDM2 inhibition and radiation. Experiments investigating the role of dsDNA breaks in radiosensitization are ongoing. Conclusions​: Our novel radiosensitizer screen identifies MDM2 as a potential mediator of radioresistance in ER+ BC in a p53-dependent manner and suggests that MDM2 targeting concurrent with RT may represent a tractable clinical strategy in women with locally advanced ER+, p53 WT BC. Citation Format: Cassandra Lynne Ritter, Benjamin C. Chandler, Andrea M. Pesch, Anna R. Michmerhuizen, Nicole Hirsh, Tanner Ward, Amanda Zhang, Mattia Cremona, Lori J. Pierce, Bryan Hennessy, Corey W. Speers. The role of MDM2 inhibition in the radiosensitization of ER+ breast cancers [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 1386.

Abstract 946: A novel monoclonal antibody targeting cell-surface plectin has potent antitumor activity in ovarian cancer models

Abstract Background: Cell surface plectin (CSP) is an abundant bioavailable target exclusively found on the surface of ovarian epithelial cancers while remaining entirely intracellular in pre-malignant tissue. Under current treatment options, 80% of women, even if remission is achieved, will experience recurrence of disease demonstrating an urgent need for more effective therapeutics. Herein, we investigate the anticancer effects of a CSP-targeting monoclonal antibody (ZB131) in in-vitro and in-vivo models of ovarian cancer (OC) as well as elucidate key signaling pathways it modulates. We demonstrate that treatment with ZB131 is efficacious both as a monotherapy and in combination with chemotherapy. Methods: A first-in-class CSP-targeting monoclonal antibody, ZB131, was developed and characterized for specificity and affinity via ELISA and kinetic binding assays. In-vitro efficacy of ZB131 was determined by Cell Titer-Glo assay and flow cytometry, impact on cell-cycle by flow cytometry, and on migration by scratch assay using multiple OC cell lines. ROS accumulation was evaluated by DCFDA assay. Phosphorylation changes of signaling kinases were measured by LiCor. In-vivo validation was performed in an OC xenograft model. The expression of proliferative markers was evaluated by IHC staining of tumor sections. Treatment toxicity was assessed from serum chemistry tests and histological evaluation of major organs. Similar in-vivo and in-vitro techniques were used to characterize ZB131's effect in combination with cisplatin. Results: ZB131 demonstrates high affinity (0.4±0.1nM) and specificity for both murine and human CSP. Treatment with ZB131 decreased cell proliferation, migration, induced G0/G1 growth arrest, and increased ROS accumulation followed by cell death in OC cell lines. ZB131 induced two coordinated events, decreased phosphorylation of cell proliferation cascades (AKT, PRAS40, ERK1/2, and GSK3alpha/beta) and increased phosphorylation of stress signaling pathways (JNK, c-Jun, p38, MSK1/2, and AMPK). In vivo, ZB131 therapy caused a 50% tumor growth reduction as well as decreased expression of cyclin D and Ki67 with no toxic side effects. Impressively, ZB131 synergizes with cisplatin resulting in a greater than 50-fold decrease in IC50 in-vitro and showed sustained tumor growth reduction with greater than 85% tumor necrosis over the course of treatment in-vivo. Conclusion: CSP is a novel highly abundant therapeutic target exclusively localized to the cell surface of ovarian, colorectal, lung, and pancreatic cancers which collectively account for more than half of all estimated cancer deaths. Our findings illustrate that CSP-targeting with ZB131 induces cytotoxicity by activating stress pathways and dampening proliferation and survival pathways. This study serves as the basis for informing a novel treatment strategy for CSP-positive tumors. Citation Format: Samantha Melinda Perez, Julien Dimastromatteo, Kimberly A. Kelly. A novel monoclonal antibody targeting cell-surface plectin has potent antitumor activity in ovarian cancer models [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 946.

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.

The Mechanisms of PIM Kinase Inhibitor, SGI-1776 Free Base, in Treating Myeloma-Associated Bone Disease

Background and Purpose: The PIM kinase family, which includes PIM1, 2 and 3 and is expressed in a variety of solid and hematological malignancies, has an essential role in cell growth and survival. Multiple myeloma remains an incurable hematologic cancer which always companions with osteolytic bone disease. In this work, we investigated the mechanisms of a PIM kinase inhibitor, SGI-1776 free base, in anti-myeloma-associated osteolytic bone disease in vitro and in vivo.Experiment Design: CCK-8 and flow cytometry were used to assess the cytotoxicity of SGI-1776 free base on myeloma cell lines (MM.1s, CAG, ARP-1, 8226, H929, LP-1, OPM2) . The effect on RANKL-induced osteoclasts was assessed in osteoclastogenic and osteoblastic cultures. Western blot and immunofluorescence were used to investigate the mechanisms of SGI-1776 free base. SiRNAs were used to mimic the inhibition of PIM kinase by the drug. IVIS in vivo imaging system, ELISA (human Igk, CTX-I, PINP) and micro-CT (parameters such as trabecular numbers and trabecular space) were used to surveillance the tumor burden and examine the bone destruction in murine models.Results: SGI-1776 free base induced apoptosis and cell cycle arrest in the G1 phase in myeloma cells mediated by decreasing the phosphorylation of PIM kinase targets C-Myc, Bad, 4EBP1 and H3. Also, the expression of Mcl-1, C-Myc, cyclin D2 and cyclin E1 were reduced, and the inhibition of NF-kB and STAT3 pathways also observed. The IC50 varies from 2 to 6 uM in different cells. Expression of apoptosis related proteins Cleaved Caspase-3, 7, 8, 9 and PARP-1 were increased. Importantly, SGI-1776 free base could overcome the protective effect of bone marrow stromal cells and osteoclasts on myeloma cells. Knocking down PIM kinase (PIM-1, 2, and 3) by SiRNAs slowed down the proliferation rates of myeloma cells (8226 and MM.1s), indicating the pro-growth impact of PIM kinase on myeloma. Notably, SGI-1776 free base inhibited osteoclast formation and resorption though decreasing PIM-1, c-fos, NFATc1, Cathepsin K and DUSP-1. Calcium coated slide assay, TRAP staining and F-acting ring formation also proved the inhibition effect. Also, phosphorylation levels of Erk, p-38, p-65, AKT and AMPK were reduced in the cells treated with SGI-1776 free base. Knocking down PIM-1 impaired the F-acting ring formation and reduced OC numbers compared with controls, and STAT3 pathway was inhibited as well. ALP staining, ALP activity and alizarin red staining showed SGI-1776 would not suppress osteoblasts formation and its mineralization. Finally, SGI-1776 free base reduced the tumor burden in subcutaneous and disseminated myeloma murine models and prevented myeloma associated bone loss with serum PINP (bone formation) and trabecular numbers increased while serum monoclonal Igk (tumor burden), CTX-I (bone resorption) and trabecular space diminished compared with the vehicle group. Moreover, SGI-1776 free base could directly reduce the RANLK-induced rapid bone loss in non-tumor bearing mouse model.Conclusion: Our investigation reveals the mechanisms of SGI-1776 free base in treating myeloma associated bone disease. The drug has a dual effect of anti-myeloma and bone protection. These results support the clinical use of this drug in multiple myeloma. DisclosuresNo relevant conflicts of interest to declare.

Curcumin inhibits adverse psychological stress-induced proliferation and invasion of glioma cells via down-regulating the ERK/MAPK pathway.

Curcumin is a natural polyphenol extracted from the rhizome of Curcuma that has an important antitumour effect, but its effect on adverse psychological stress‐induced tumour proliferation and invasion has not been reported to date. Here, we found that curcumin not only inhibited the growth of xenografts in chronically stressed nude mice, but also decreased the expression of matrix metalloproteinase (MMP)‐2/9 and CD147 in tumour tissues. Exogenous norepinephrine (NE) was used to stimulate glioma cells to simulate the stress environment in vitro, and it was found that curcumin inhibited the NE‐induced proliferation and invasion of glioma cells in a dose‐dependent manner. Further research found that the effects of NE on glioma cells could lead to the activation of the mitogen‐activated protein kinase (MAPK) signalling pathway through β‐adrenergic receptor, while curcumin suppressed the level of extracellular signal–regulated kinase (ERK)1/2 phosphorylation. In addition, blocking ERK1/2 expression with U0126 resulted in the down‐regulated expression of CD147, which further led to the decreased expression of MMP‐2 and MMP‐9. Curcumin could also inhibit the expression of cyclin D1/CDK4/6 and anti‐apoptotic protein Bcl‐2/Bcl‐XL induced by NE, and induced cell cycle changes and increased apoptosis. Therefore, curcumin may be a potential candidate drug for preventing and treating the progression of glioma induced by adverse psychological stress.

6-Methylsulfinylhexyl Isothiocyanate Inhibits Cell Cycle Progression in Quiescent Jb6 Cells Stimulated with Epidermal Growth Factor

Objective: The effects of 6-MSITC on cell cycle progression were investigated in quiescent mouse epidermal JB6 cells.&#x0D; Background: 6-Methylsulfinylhexyl isothiocyanate (6-MSITC) derived from wasabi (Wasabia japonica) has been reported to prevent tumor development in vivo.&#x0D; Material and methods: Treatment with epidermal growth factor (EGF) to quiescent JB6 cells, which were serum-starved for 36 h, promoted cell cycle progression from the G0/G1 phase to the S phase. Effects of pretreatment with 6-MSITC on cell cycle progression were estimated by flowcytometry and real-time RT-PCR.&#x0D; Results: Pretreatment with 6-MSITC at 0.25-1.0 μg/ml prior to the growth stimulation with EGF significantly inhibited cell cycle progression. Pretreatment with 6-MSITC inhibited the gene expression of DNA synthesis-related proteins cyclin A2, dumbbell former 4, and proliferating cell nuclear antigen.&#x0D; Conclusion: These results showed that 6-MSITC inhibits cell cycle progression in quiescent cells, accompanied by the inhibition of gene expression of DNA synthesis proteins.

Mechanosensitive expression of lamellipodin promotes intracellular stiffness, cyclin expression and cell proliferation.

Cell cycle control is a key aspect of numerous physiological and pathological processes. The contribution of biophysical cues, such as stiffness or elasticity of the underlying extracellular matrix (ECM), is critically important in regulating cell cycle progression and proliferation. Indeed, increased ECM stiffness causes aberrant cell cycle progression and proliferation. However, the molecular mechanisms that control these stiffness-mediated cellular responses remain unclear. Here, we address this gap and show good evidence that lamellipodin (symbol RAPH1), previously known as a critical regulator of cell migration, stimulates ECM stiffness-mediated cyclin expression and intracellular stiffening in mouse embryonic fibroblasts. We observed that increased ECM stiffness upregulates lamellipodin expression. This is mediated by an integrin-dependent FAK-Cas-Rac signaling module and supports stiffness-mediated lamellipodin induction. Mechanistically, we find that lamellipodin overexpression increased, and lamellipodin knockdown reduced, stiffness-induced cell cyclin expression and cell proliferation, and intracellular stiffness. Overall, these results suggest that lamellipodin levels may be critical for regulating cell proliferation. This article has an associated First Person interview with the first author of the paper.

First person – Joseph A. Brazzo III

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Joseph A. Brazzo III is first author on ‘ Mechanosensitive expression of lamellipodin promotes intracellular stiffness, cyclin expression and cell proliferation’, published in JCS. Joseph is an MD–PhD student in the lab of Yongho Bae at the Jacobs School of Medicine and Biomedical Sciences, State University of New York University at Buffalo, NY, USA, investigating how extracellular matrix stiffness affects cell functions and behaviors, and dissecting the cellular pathways and molecular mechanisms that mediate mechanotransduction in health and disease.

Inhibition of TMPRSS4 mediated epithelial-mesenchymal transition is critically involved in antimetastatic effect of melatonin in colorectal cancers.

In the current study, the underlying anti-metastatic mechanism of melatonin contained in some edible plants was explored in association with transmembrane protease serine 4 (TMPRSS4) mediated metastasis and epithelial-mesenchymal transition (EMT) signaling in human HCT15 and SW620 colorectal cancer cells. Here, TMPRSS4 was highly expressed in HCT15, but was weakly expressed in SW620 cells. Melatonin exerted weak cytotoxicity, decreased invasion, adhesion, and migration, and attenuated the expression of TMPRSS4, cyclin E, pro-urokinase-type plasminogen activator (pro-uPA), p-signal transducer and activator of transcription 3 (p-STAT3), p-focal adhesion kinase (p-FAK), Snail and increased the expression of E-cadherin, p27, pp38 and p-Jun N-terminal kinases (p-JNK) in HCT15 cells. Conversely, overexpression of TMPRSS4 reduced the ability of melatonin to activate E-cadherin and reduce Snail. Furthermore, even in SW620 cells transfected with TMPRSS4-overexpression plasmid, melatonin effectively suppressed invasion and migration along with decreased expression of Snail, cyclin A, cyclin E, pro-uPA and p-FAK and increased expression of E-cadherin and p27. Overall, these findings provide evidence that melatonin suppresses metastasis in colon cancer cells via inhibition of TMPRSS4 mediated EMT.

PRMT5 Promotes Cyclin E1 and Cell Cycle Progression in CD4 Th1 Cells and Correlates With EAE Severity.

Multiple Sclerosis (MS) is a debilitating central nervous system disorder associated with inflammatory T cells. Activation and expansion of inflammatory T cells is thought to be behind MS relapses and influence disease severity. Protein arginine N-methyltransferase 5 (PRMT5) is a T cell activation-induced enzyme that symmetrically dimethylates proteins and promotes T cell proliferation. However, the mechanism behind PRMT5-mediated control of T cell proliferation and whether PRMT5 contributes to diseases severity is unclear. Here, we evaluated the role of PRMT5 on cyclin/cdk pairs and cell cycle progression, as well as PRMT5’s link to disease severity in an animal model of relapsing-remitting MS. Treatment of T helper 1 (mTh1) cells with the selective PRMT5 inhibitor, HLCL65, arrested activation-induced T cell proliferation at the G1 stage of the cell cycle, suggesting PRMT5 promotes cell cycle progression in CD4+ T cells. The Cyclin E1/Cdk2 pair promoting G1/S progression was also decreased after PRMT5 inhibition, as was the phosphorylation of retinoblastoma. In the SJL mouse relapsing-remitting model of MS, the highest PRMT5 expression in central nervous system-infiltrating cells corresponded to peak and relapse timepoints. PRMT5 expression also positively correlated with increasing CD4 Th cell composition, disease severity and Cyclin E1 expression. These data indicate that PRMT5 promotes G1/S cell cycle progression and suggest that this effect influences disease severity and/or progression in the animal model of MS. Modulating PRMT5 levels may be useful for controlling T cell expansion in T cell-mediated diseases including MS.