Reduced Cell Cycle Progression Research Articles

CTRP13-Mediated Effects on Endothelial Cell Function and Their Potential Role in Obesity.

Obesity, a major component of cardiometabolic syndrome, contributes to the imbalance between pro- and anti-atherosclerotic factors via dysregulation of adipocytokine secretion. Among these adipocytokines, the C1q/TNF-related proteins (CTRPs) play a role in the modulation of atherosclerosis development and progression. Here, we investigated the vascular effects of CTRP13. CTRP13 is not only expressed in adipose tissue but also in vessels/endothelial cells (ECs) of mice, rats, and humans. Obese individuals (mice, rats, and humans) showed higher vascular CTRP13 expression. Human Umbilical Vein Endothelial Cells (HUVECs), cultured in the presence of serum from obese mice, mimicked this obesity-associated effect on CTRP13 protein expression. Similarly, high glucose conditions and TNF-alpha, but not insulin, resulted in a strong increase in CTRP13 in these cells. Recombinant CTRP13 induced a reduction in EC proliferation via AMPK. In addition, CTRP13 reduced cell cycle progression and increased p53 phosphorylation and p21 protein expression, but reduced Rb phosphorylation, with the effects largely depending on alpha-2 AMPK as suggested by adenoviral overexpression of dominant-negative (DN) or wild-type (WT) alpha 1/alpha 2 AMPK. The present study demonstrates that CTRP13 expression is induced in ECs under diabetic conditions and that CTRP13 possesses significant vaso-modulatory properties which may have an impact on vascular disease progression in patients.

Abstract LB171: First-in-class inhibitors of the tRNA methyltransferase METTL1 for the treatment of cancer

Abstract METTL1 is an RNA methyltransferase that catalyzes N7-methylation on guanine at position 46 (m7G46) in a subset of transfer RNAs (tRNAs). These tRNAs are stabilized by m7G46, and this increases the translation of mRNAs containing the cognate codons. METTL1 is often over-expressed in cancer tissue, and its gene locus is frequently amplified in specific tumor types. Recent studies have shown that reducing METTL1 expression leads to tumor growth inhibition highlighting this enzyme as a potential novel target for anti-cancer drugs. We optimized small molecule inhibitors of METTL1 from a HTS hit using structure-guided medicinal chemistry. Representative compounds of two distinct chemical series exhibit potent in vitro METTL1 inhibition at low nanomolar concentrations while displaying exquisite selectivity over other RNA and protein methyltransferases. The effects of inhibiting METTL1 were measured by cellular mechanistic assays and modification-induced misincorporation tRNA sequencing (mim-tRNAseq). As expected, pharmacological inhibition led to decreased m7G46 methylation, lower levels of a subgroup of tRNAs and reduced cancer cell proliferation.The efficacy of METTL1 inhibition in a panel of cancer cell lines representing a wide range of solid and hematologic tumor types was determined. Cell proliferation was impaired in a subset of cell lines originating from various cancer types, including lymphoma, sarcoma, esophageal squamous cell carcinoma, glioblastoma, colorectal and lung adenocarcinoma. Importantly, viability was not affected in non-transformed cell lines, indicating a highly selective mechanism of action. The phenotypic consequences of METTL1 inhibition in selected cell lines were further investigated by flow cytometry and western blotting. Cell lines sensitive to METTL1 inhibition displayed reduced cell cycle progression and lowered expression of cell cycle regulators. RNA sequencing and proteome analyses identified molecular changes at both transcript and protein level. METTL1 inhibition led to induction of ATF4 expression and activation of the integrated stress response, as well as global changes to the translation machinery. Studies on cell-line derived xenograft or syngeneic in vivo mouse models were conducted to assess tumor growth inhibition after oral or intraperitoneal administration of the potent METTL1 small molecule inhibitor STM9005. Inhibition of METTL1 by this compound reduced tumor growth in both immune-deficient and immune-competent mouse models, effects accompanied by dose-dependent changes in pharmacodynamic biomarkers. Here, we describe the discovery of first-in-class inhibitors of METTL1 tRNA methyltransferase. To our knowledge, our data provide the first evidence that pharmacological inhibition of a tRNA methyltransferase reduces tumor growth in vivo. Citation Format: Alexandra Sapetschnig, Beth Thomas, Eliza Yankova, Harry Fischl, Aleksandra Azevedo, Sarah Bucknell, Richard Fosbeary, Sapphire Sawyer, Sian Evans, Carmen Livi, Byron Andrews, Jack Rogan, Natalie Webster, Matthew Fyfe, Konstantinos Tzelepis, Oliver Rausch. First-in-class inhibitors of the tRNA methyltransferase METTL1 for the treatment of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB171.

Xanthohumol hinders invasion and cell cycle progression in cancer cells through targeting MMP2, MMP9, FAK and P53 genes in three-dimensional breast and lung cancer cells culture

BackgroundDespite recent advances in the treatment of lung and breast cancer, the mortality with these two types of cancer is high. Xanthohumol (XN) is known as a bioactive compound that shows an anticancer effect on cancer cells. Here, we intended to investigate the anticancer effects of XN on the breast and lung cancer cell lines, using the three-dimensional (3D) cell culture.MethodsXN was isolated from Humulus lupulus using Preparative-Thin Layer Chromatography (P-TLC) method and its authenticity was documented through Fourier Transform Infrared spectroscopy (FT-IR) and Hydrogen Nuclear Magnetic Resonance (H-NMR) methods. The spheroids of the breast (MCF-7) and lung (A549) cancer cell lines were prepared by the Hanging Drop (HD) method. Subsequently, the IC50s of XN were determined using the MTT assay in 2D and 3D cultures. Apoptosis was evaluated by Annexin V/PI flow cytometry and NFκB1/2, BAX, BCL2, and SURVIVIN expressions. Cell cycle progression was determined by P21, and P53 expressions as well as PI flow cytometry assays. Multidrug resistance was investigated through examining the expression of MDR1 and ABCG2. The invasion was examined by MMP2, MMP9, and FAK expression and F-actin labeling with Phalloidin-iFluor.ResultsWhile the IC50s for the XN treatment were 1.9 µM and 4.74 µM in 2D cultures, these values were 12.37 µM and 31.17 µM in 3D cultures of MCF-7 and A549 cells, respectively. XN induced apoptosis in MCF-7 and A549 cell lines. Furthermore, XN treatment reduced cell cycle progression, multidrug resistance, and invasion at the molecular and/or cellular levels.ConclusionsAccording to our results of XN treatment in 3D conditions, this bioactive compound can be introduced as an adjuvant anti-cancer agent for breast and lung cancer.

Abstract 4928: Gedatolisib, a well-tolerated pan-PI3K/mTOR inhibitor, exhibits potent therapeutic effects on endometrial cancer models regardless of their PI3K pathway mutational status

Abstract Background: The PI3K, AKT, and mTOR (PAM) pathway is one of the most commonly activated oncogenic pathways in gynecological cancers. Loss of PTEN function and PIK3CA activating mutations are especially frequent in endometrial cancer (EC). Targeting the PAM pathway, in combination with other cooperative oncogenic pathways (e.g., the estrogen pathway), is a promising EC treatment strategy. Currently, everolimus, an mTORC1 inhibitor, in combination with letrozole, an aromatase inhibitor, is one of the available hormonal therapy options for patients with advanced endometrioid EC. Most PAM inhibitors (PAM-i) selectively spare (or weakly inhibit) one or more key PAM pathway components, which can lead to drug resistance. To minimize drug resistance, a more comprehensive inhibition of the PI3K isoforms and downstream mTOR complexes may be required. We hypothesized that gedatolisib, which potently inhibits all Class I PI3K isoforms, mTORC1, and mTORC2, can potentially be an effective and well tolerated therapy for patients with EC. Methods: A panel of endometrial, ovarian and cervical cancer cell lines with different PI3K pathway mutational status were assayed for their sensitivity to gedatolisib and other PAM inhibitors (PI3K-α: alpelisib, pan-PI3K: copanlisib; AKT: capivasertib; mTORC1: everolimus; pan-PI3K/mTOR: gedatolisib). Cell viability, cell proliferation, and flow cytometry analytical assays were conducted. Xenograft studies evaluating gedatolisib in EC cell lines were also performed. Results: Gedatolisib strongly inhibited PAM pathway activation, and reduced cell cycle progression and cell viability in the endometrial, ovarian, and cervical cancer cell lines. Compared to the other PAM-i, Gedatolisib exhibited superior anti-proliferative potency and efficacy in almost all the cell lines tested, regardless of the PI3K pathway mutational status or cancer type. In EC xenografts, gedatolisib substantively inhibited tumor growth irrespective of the cell line models’ PI3K pathway mutational status. Conclusions: We demonstrated that, gedatolisib induces superior anti-proliferative activity compared to the other PAM pathway inhibitors, regardless of the PI3K pathway mutational status of the cancer cell lines evaluated. Gedatolisib as a single agent and in combination with other therapies has previously reported promising preliminary clinical efficacy and safety in various solid tumor types, including EC. In summary, nonclinical and preliminary clinical data supports the development of gedatolisib in combination with hormonal therapy for treatment of patients with EC. Citation Format: Stefano Rossetti, Aaron Broege, Ian MacNeil, Ben Rich, Jhomary Molden, Brian Sullivan, Lance Laing. Gedatolisib, a well-tolerated pan-PI3K/mTOR inhibitor, exhibits potent therapeutic effects on endometrial cancer models regardless of their PI3K pathway mutational status. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4928.

PURINE-DERIVED PHENYLHYDROXAMATE COMPOUNDS INDUCES CELL DEATH IN HEMATOLOGICAL NEOPLASM MODELS

The histone acetylation process is the best-studied type of post-translational modification, playing a key role in chromatin remodeling regulated by the activity of two enzymes: histone acetyltransferases and histone deacetylases (HDAC). High levels of HDAC have been observed in patients with hematological and solid cancers and are associated with poor prognosis. The present study aimed to evaluate the cellular effects of three novel potential HDAC inhibitors in hematological neoplasm models. A panel containing myeloid (OCI-AML3, Kasumi 1, HL60, THP1, MOLM13, MV4-11, U937, NB4, NB4-R2, K562, KU812, SET2, and HEL) and lymphoid (Jurkat, CEM, Namalwa, NALM6, Daudi, Raji, SUP-B15, REH, U266, MM1.S, MM1.R, and Karpas 422) neoplasm cells were used. Peripheral blood mononuclear cells (normal leukocytes) from healthy donors were used for toxicity evaluation (n = 4). Three novel purine-derived phenylhydroxamate with potential HDAC inhibitor activity (named here as 82, 83, and 84) were used. Vorinostat was used as a reference drug. Cell viability was assessed by MTT assay, apoptosis by annexin V/propidium iodide labeling and flow cytometry (FC), clonogenicity by autonomous colony formation, cell cycle by propidium iodide and FC, and protein expression by Western Blot. IC 50 values were obtained by non-linear regression. Statistical analysis was performed by ANOVA and Bonferroni post-test. A p < 0.05 was considered significant. All compounds tested reduced cell viability at submicromolar concentrations being compound 82 more potent than the others exhibiting less concentration necessary to reduce cell viability by 50% (IC 50 ranged from 0.09 to 2.04 μM). THP1, MOLM13, NB4-R2, and HEL leukemia cells were more sensitive to these compounds and were selected for the next experiments. In normal leukocytes, vorinostat and compound 82 did not reduce cell viability at the same extension as observed for leukemia cells, indicating a selective antineoplastic activity. All compounds induced time- and dose-dependent manner apoptosis in THP1, MOLM13, NB4-R2, and HEL cells (all p < 0.05). Vorinostat and compound 82 strongly reduced colony formation, being compound 82 more efficient, especially in MOLM13 and NB4-R2 cells (all p < 0.05). Low concentration of vorinostat and compound 82 lead to reduced cell cycle progression (all p < 0.05). THP1, MOLM13, NB4-R2, and HEL cells were exposed to vorinostat or compound 82 and the expression of proteins involved in apoptosis and HDAC activity was modulated. Our results indicate that the three purine-derived phenylhydroxamate tested presented antineoplastic effects being a future candidate to compose the list of HDAC inhibitors in the antineoplastic arsenal. CNPq, CAPES, and FAPESP.

Pharmacological fatty acid synthase inhibitors differently affect the malignant phenotype of oral cancer cells.

ObjectiveFatty acid synthase levels are associated with aggressiveness, prognosis, and risk of metastasis in oral squamous cell carcinomas. This enzyme contains seven catalytic domains and its inhibition by synthetic or natural drugs has antineoplastic properties such as C75, which is a synthetic inhibitor of the β- ketoacyl synthase domain, the antibiotic triclosan, ligand of the enoyl reductase domain, and the antiobesity drug orlistat, which inhibits the thioesterase domain. Here, we sought to investigate and compare the in vitro effects of C75, triclosan, and orlistat on malignant phenotypes of the cell line SCC-9: proliferation, cell cycle, apoptosis, adhesion, migration, and invasion. DesignHalf-maximal inhibitory concentration (IC50) was determined using cell viability assays. Cell death and cell cycle progression were analyzed by Annexin V-PE/7-ADD-PerCP labeling and propidium iodide staining, respectively. Cell migration and invasion were assayed by transwells assays and cell adhesion using collagen and fibronectin. ResultsC75 showed the lowest IC50 and higher inhibition of lipid droplets at low concentrations and reduced cell motility. Triclosan showed the intermediate IC50 value, excellent reduction of lipid bodies at the IC50 when compared with C75 and orlistat. Also, triclosan reduced cell cycle progression, adhesion, migration, and invasion of SCC-9 and induced the highest levels of apoptosis. Orlistat promoted cell cycle arrest, but showed the lowest induction of apoptosis and did not affected invasion and adhesion of SCC-9. ConclusionAltogether, despite the particular effects of the analyzed fatty acid synthase inhibitors, triclosan showed to better interfere in tumorigenic phenotypes of SCC-9 cells.

Effects of metabolizable protein concentration, amino acid profile, and fiber source on the messenger RNA expression of skeletal muscle in peripartum dairy cows

After parturition, dairy cows mobilize AA from skeletal muscle to meet metabolizable protein (MP) requirements. High mobilization may compromise cow health and longer-term milk production. Postpartum diets with higher MP concentrations, improved AA profiles, or MP increased at the expense of forages rather than nonforage fiber sources may attenuate muscle catabolism; however, the molecular mechanisms responsible need investigation. We evaluated mRNA expression in the longissimus dorsi of cows fed postpartum diets differing in MP concentration, AA profile, and fiber source. From 0 to 25 d after parturition, 40 multiparous cows received the following diets: (1) 13% deficient in MP (D-MP), (2) adequate in MP using primarily soy protein (A-MP), (3) adequate in MP using blends of proteins and individual AA to improve the AA profile (Blend), or (4) similar to Blend except additional protein replaced forage (Blend-fNDF). Biopsies were taken approximately -5, 7, and 25 d relative to parturition. Greater dietary MP concentration (D-MP vs. A-MP and Blend) decreased expression of genes related to protein synthesis (MTOR, RPS6KB1) and degradation (FOXO1), inflammation (IFNG, TLR4), and endoplasmic reticulum (ER) stress (HSPA5, DDIT) and increased genes associated with lipogenesis (PPARG) and glucose oxidation (LDH, MB). In Blend versus A-MP (i.e., effect of AA profile), expression related to apoptosis (CASP8) and inflammation (TNFA) decreased and genes associated with cell cycle progression (E2F1) and fast-twitch glycolytic muscle fiber type (MYH4) increased. Less forage (Blend-fNDF vs. Blend) decreased genes associated with lipogenesis (PPARG, ACACA) and ER stress (BCL2, DDIT3, EIF2AK3, PPP1R15A) and increased genes associated with inflammation (TNF), inhibition of myogenesis (MSTN), and autophagy (PEBP1). In summary and based on mRNA expression, increasing MP supply may attenuate muscle turnover and ER stress. However, an unbalanced AA supply reduced cell cycle progression and protein synthesis. Lower energy supplies may reduce cell growth and cause autophagy.

Identification of DHX9 as a cell cycle regulated nucleolar recruitment factor for CIZ1

CIP1-interacting zinc finger protein 1 (CIZ1) is a nuclear matrix associated protein that facilitates a number of nuclear functions including initiation of DNA replication, epigenetic maintenance and associates with the inactive X-chromosome. Here, to gain more insight into the protein networks that underpin this diverse functionality, molecular panning and mass spectrometry are used to identify protein interaction partners of CIZ1, and CIZ1 replication domain (CIZ1-RD). STRING analysis of CIZ1 interaction partners identified 2 functional clusters: ribosomal subunits and nucleolar proteins including the DEAD box helicases, DHX9, DDX5 and DDX17. DHX9 shares common functions with CIZ1, including interaction with XIST long-non-coding RNA, epigenetic maintenance and regulation of DNA replication. Functional characterisation of the CIZ1-DHX9 complex showed that CIZ1-DHX9 interact in vitro and dynamically colocalise within the nucleolus from early to mid S-phase. CIZ1-DHX9 nucleolar colocalisation is dependent upon RNA polymerase I activity and is abolished by depletion of DHX9. In addition, depletion of DHX9 reduced cell cycle progression from G1 to S-phase in mouse fibroblasts. The data suggest that DHX9-CIZ1 are required for efficient cell cycle progression at the G1/S transition and that nucleolar recruitment is integral to their mechanism of action.

RETRACTED ARTICLE: The POU2F1/miR-4490/USP22 axis regulates cell proliferation and metastasis in gastric cancer

PurposeGrowing evidence indicates that aberrant expression of microRNAs contributes to tumor development. However, the biological role of microRNA-4490 (miR-4490) in gastric cancer (GC) remains to be clarified.MethodsTo explore the function of miR-4490 in GC, we performed colony formation, EdU incorporation, qRT-PCR, Western blotting, in situ hybridization (ISH), immunohistochemistry (IHC), flow cytometry, ChIP and dual-luciferase reporter assays. In addition, the growth, migration and invasion capacities of GC cells were evaluated.ResultsWe found that miR-4490 was significantly downregulated in primary GC samples and in GC-derived cell lines compared with normal controls, and that this expression level was negatively correlated with GC malignancy. Exogenous miR-4490 expression not only reduced cell cycle progression and proliferation, but also significantly inhibited GC cell migration, invasion and epithelial-mesenchymal transition (EMT) in vitro. Mechanistically, we found that miR-4490 directly targets USP22, which mediates inhibition of GC cell proliferation and EMT-induced metastasis in vitro and in vivo. Moreover, we found through luciferase and ChIP assays that transcription factor POU2F1 can directly bind to POU2F1 binding sites within the miR-4490 and USP22 promoters and, by doing so, modulate their transcription. Spearman’s correlation analysis revealed a positive correlation between USP22 and POU2F1 expression and negative correlations between miR-4490 and USP22 as well as miR-4490 and POU2F1 expression in primary GC tissues.ConclusionBased on our results we conclude that miR-4490 acts as a tumor suppressor, and that the POU2F1/miR-4490/USP22 axis plays an important role in the regulation of growth, invasion and EMT of GC cells.

SMARCB1 Acts as a Quiescent Gatekeeper for Cell Cycle and Immune Response in Human Cells.

Switch/sucrose non-fermentable (SWI/SNF)-related matrix-associated actin-dependent regulator of chromatin (SMARC) subfamily B member 1 (SMARCB1) is a core subunit of the switch/sucrose non-fermentable (SWI/SNF) complex, one of the adenosine triphosphate (ATP)-dependent chromatin remodeler complexes. The unique role of SMARCB1 has been reported in various cellular contexts. Here, we focused on the general role of the ubiquitous expression of SMARCB1 in a normal cell state. We selected ARPE19 (human primary retinal pigment epithelium) and IMR90 (from human fetal lung fibroblasts) cell lines as they have completely different contexts. Furthermore, although these cell lines have been immortalized, they are relatively close to normal human cells. The loss of SMARCB1 in ARPE19 and IMR90 cells reduced cell cycle progression via the upregulation of P21. Transcriptome analysis followed by SMARCB1 knockdown in both cell lines revealed that SMARCB1 was not only involved in cell maintenance but also conferred immunomodulation. Of note, SMARCB1 bound to interleukin (IL) 6 promoter in a steady state and dissociated in an active immune response state, suggesting that SMARCB1 was a direct repressor of IL6, which was further confirmed via loss- and gain-of-function studies. Taken together, we demonstrated that SMARCB1 is a critical gatekeeper molecule of the cell cycle and immune response.

Dietary Compound Isoliquiritigenin, an Antioxidant from Licorice, Suppresses Triple-Negative Breast Tumor Growth via Apoptotic Death Program Activation in Cell and Xenograft Animal Models.

Patients with triple-negative breast cancer have few therapeutic strategy options. In this study, we investigated the effect of isoliquiritigenin (ISL) on the proliferation of triple-negative breast cancer cells. We found that treatment with ISL inhibited triple-negative breast cancer cell line (MDA-MB-231) cell growth and increased cytotoxicity. ISL reduced cell cycle progression through the reduction of cyclin D1 protein expression and increased the sub-G1 phase population. The ISL-induced apoptotic cell population was observed by flow cytometry analysis. The expression of Bcl-2 protein was reduced by ISL treatment, whereas the Bax protein level increased; subsequently, the downstream signaling molecules caspase-3 and poly ADP-ribose polymerase (PARP) were activated. Moreover, ISL reduced the expression of total and phosphorylated mammalian target of rapamycin (mTOR), ULK1, and cathepsin B, whereas the expression of autophagic-associated proteins p62, Beclin1, and LC3 was increased. The decreased cathepsin B cause the p62 accumulation to induce caspase-8 mediated apoptosis. In vivo studies further showed that preventive treatment with ISL could inhibit breast cancer growth and induce apoptotic and autophagic-mediated apoptosis cell death. Taken together, ISL exerts an effect on the inhibition of triple-negative MDA-MB-231 breast cancer cell growth through autophagy-mediated apoptosis. Therefore, future studies of ISL as a supplement or alternative therapeutic agent for clinical trials against breast cancer are warranted.

VtRNA2-1/nc886 Produces a Small RNA That Contributes to Its Tumor Suppression Action through the microRNA Pathway in Prostate Cancer.

vtRNA2-1 is a vault RNA initially classified as microRNA precursor hsa-mir-886 and recently proposed as “nc886”, a new type of non-coding RNA involved in cancer progression acting as an oncogene and tumor suppressor gene in different tissues. We have shown that vtRNA2-1/nc886 is epigenetically repressed in neoplastic cells, increasing cell proliferation and invasion in prostate tissue. Here we investigate the ability of vtRNA2-1/nc886 to produce small-RNAs and their biological effect in prostate cells. The interrogation of public small-RNA transcriptomes of prostate and other tissues uncovered two small RNAs, snc886-3p and snc886-5p, derived from vtRNA2-1/nc886 (previously hsa-miR-886-3p and hsa-miR-886-5p). Re-analysis of PAR-CLIP and knockout of microRNA biogenesis enzymes data showed that these small RNAs are products of DICER, independent of DROSHA, and associate with Argonaute proteins, satisfying microRNA attributes. In addition, the overexpression of snc886-3p provokes the downregulation of mRNAs bearing sequences complementary to its “seed” in their 3′-UTRs. Microarray and in vitro functional assays in DU145, LNCaP and PC3 cell lines revealed that snc886-3p reduced cell cycle progression and increases apoptosis, like its precursor vtRNA2-1/nc886. Finally, we found a list of direct candidate targets genes of snc886-3p upregulated and associated with disease condition and progression in PRAD-TCGA data. Overall, our findings suggest that vtRNA2-1/nc886 and its processed product snc886-3p are synthesized in prostate cells, exerting a tumor suppressor action.

Liposomal Delivery of miR-34b-5p Induced Cancer Cell Death in Thyroid Carcinoma.

This study aims to determine the functional roles of microRNA-34b-5p (miR-34b) in the suppression of anaplastic thyroid carcinoma. We used hydration-of-freeze-dried-matrix (HFDM) formulated liposomes (liposome-loaded miR-34b) for effective delivery of miR-34b to anaplastic thyroid carcinoma in vitro and in vivo. Real time polymerase chain was used to determine the level of miR-34b. Immunocytochemistry, Western blot and ELISA were carried out to determine the effect of this manipulation on VEGF-A expression. In addition, an in vivo xenotransplantation mouse model was used to investigate the functional roles of overexpression of miR-34b in the carcinoma. In anaplastic thyroid carcinoma cells, miR-34b expression was low and significant overexpression (p < 0.05) was noted following transfection with liposome-loaded miR-34b. The miR-34b overexpressed thyroid carcinoma cell lines showed reduction in VEGF-A protein expression, decreased cell proliferation, decreased wound healing, reduced cell cycle progression and increased apoptosis (p < 0.05). In in vivo experiments, when compared to control groups, smaller tumours formed upon intravenous administration of liposome-loaded miR-34b. To conclude, the current study confirmed the tumour suppressor properties of miR-34b via VEGF-A regulation in anaplastic thyroid carcinoma. In addition, delivery of miR-34b using cationic liposome could be a useful therapeutic strategy for targeting therapy in the carcinoma.

Effects of MALAT1 on proliferation and apo- ptosis of human non-small cell lung cancer A549 cells in vitro and tumor xenograft growth in vivo by modulating autophagy.

To explore the ability of MALAT1 to influence non-small cell lung cancer (NSCLC) A549 cells in vitro and tumor xenograft growth in vivo by modulating autophagy. LncRNA MALAT-1 in normal HBE cells and human NSCLC cells was measured. A549 cells were treated with si-MALAT-1, negative control and si-MALAT-1 + rapamycin. The mRNA levels of MALAT-1, P62 and LC3 was determined by the qRT-PCR and the protein levels of autophagy-related proteins by the western blotting. The CCK8 assay was performed for cell proliferation, the scratch test for cell migration, the Transwell assay for cell invasion, and the flow cytometry for cell cycle and apoptosis. Tumor xenograft in nude mice is performed to test tumorigenesis of the transfected A549 cells. The expression level of MALAT-1 in A549, SPC-A-1 and NCI-H460 cells was increased compared to HBE cells. And A549 with a high expression level of MALAT-1 were selected for cell transfection. si-MALAT-1 decreased cell proliferation, migration, invasion, and LC3-II/LC3-I ratio, reduced cell cycle progression, and increased cell apoptosis and P62 protein expression. No significant difference was found between A549 cells and A549 cells transfected with si-MALAT-1 + RAPA, A549 cells transfected with NC and A549 cells transfected with si-MALAT-1 + RAPA. Nude mice injected with A549 cells transfected with si-MALAT-1 had smallest tumor on size and weight among other nude mice. Downregulation of MALAT1 may promote apoptosis and suppress proliferation, migration and invasion of human NSCLC A549 cells by inhibiting autophagy, thereby suppressing the development of NSCLC.

Transcriptional analyses provide new insight into the late-stage immune response of a diseased Caribbean coral.

Increasing global temperatures due to climate change have resulted in respective increases in the severity and frequency of epizootics around the globe. Corals in particular have faced rapid declines due to disease outbreaks. Understanding immune responses and associated potential life-history trade-offs is therefore a priority. In the autumn of 2011, a novel disease of octocorals of the genus Eunicea was first documented in the Florida Keys. Termed Eunicea Black Disease (EBD), the disease is easily identified by the dark appearance of affected tissue, caused by a strong melanization response on the part of the host. In order to better understand the response of corals to EBD, we conducted full transcriptome analysis of 3 healthy and 3 diseased specimens of Eunicea calyculata collected from offshore southeast Florida. Differential expression and protein analyses revealed a strong, diverse immune response to EBD characterized by phagocytosis, adhesion and melanization on the part of the host. Furthermore, coexpression network analyses suggested this might come at the cost of reduced cell cycle progression and growth. This is in accordance with past histological studies of naturally infected hard corals, suggesting that potential trade-offs during infection may affect post-outbreak recovery of reef ecosystems by reducing both organismal growth and fecundity. Our findings highlight the importance of considering factors beyond mortality when estimating effects of disease outbreaks on ecosystems.

Bach2 Promotes B Cell Receptor-Induced Proliferation of B Lymphocytes and Represses Cyclin-Dependent Kinase Inhibitors.

BTB and CNC homology 2 (Bach2) is a transcriptional repressor that is required for the formation of the germinal center (GC) and reactions, including class switch recombination and somatic hypermutation of Ig genes in B cells, within the GC. Although BCR-induced proliferation is essential for GC reactions, the function of Bach2 in regulating B cell proliferation has not been elucidated. In this study, we demonstrate that Bach2 is required to sustain high levels of B cell proliferation in response to BCR signaling. Following BCR engagement in vitro, B cells from Bach2-deficient (Bach2-/-) mice showed lower incorporation of BrdU and reduced cell cycle progression compared with wild-type cells. Bach2-/- B cells also underwent increased apoptosis, as evidenced by an elevated frequency of sub-G1 cells and early apoptotic cells. Transcriptome analysis of BCR-engaged B cells from Bach2-/- mice revealed reduced expression of the antiapoptotic gene Bcl2l1 encoding Bcl-xL and elevated expression of cyclin-dependent kinase inhibitor (CKI) family genes, including Cdkn1a, Cdkn2a, and Cdkn2b Reconstitution of Bcl-xL expression partially rescued the proliferation defect of Bach2-/- B cells. Chromatin immunoprecipitation experiments showed that Bach2 bound to the CKI family genes, indicating that these genes are direct repression targets of Bach2. These findings identify Bach2 as a requisite factor for sustaining high levels of BCR-induced proliferation, survival, and cell cycle progression, and it promotes expression of Bcl-xL and repression of CKI genes. BCR-induced proliferation defects may contribute to the impaired GC formation observed in Bach2-/- mice.

Abstract 4157: Co-blockade of mTORC1, ERBB and estrogen receptor signalling pathways in endocrine resistance breast cancer: Combating tumor plasticity

Abstract Introduction The majority of breast cancers (BC) are estrogen (E) receptor positive (ER+). Endocrine therapies target E stimulation of tumour growth but resistance remains problematic, often a result of enhanced crosstalk between ER and growth factor pathways. Previously we reported the antiproliferative efficacy of combining everolimus (RAD001, mTORC1 inhibitor) with endocrine therapy in resistance models, but potential routes of escape from treatment via ERBB2/3 signalling were observed. We hypothesised that combined targeting of three cellular nodes (ER, ERBB and mTORC1) may target tumour rewiring and provide enhanced long-term clinical utility in endocrine resistant BCs. Methods Several ER+ BC lines adapted to long term E deprivation (LTED), modelling relapse to an aromatase inhibitor (AI), were treated in vitro with a combination of RAD001 and neratinib (pan ERBB inhibitor) in the presence or absence of estradiol (E2), tamoxifen or fulvestrant. Effects on proliferation, cell signalling, cell cycle and transcription were assessed. Additionally, an in vivo model of AI resistance was treated with monotherapies or combinations to evaluate efficacy in delaying tumour progression. Results All cell lines showed dose dependent decreases in proliferation in response to RAD001 (IC50 0.6-50nM without E2; 1-10nM with E2). Neratinib showed a wide range of IC50 values in the presence of E2 (300-1000nM). In the absence of E2, wild type (wt) cell lines showed IC50 values in excess of 1800nM with hormetic response curves, whilst in the LTED IC50 values ranged between 400-900nM. Combination of either agent with endocrine therapy caused a concentration dependent decrease in proliferation in both wt and LTED lines but the maximum effect was observed with a triple combination of RAD001, neratinib and endocrine therapy. Expression of pS6 was suppressed by RAD001 in all cells tested, whilst neratinib caused a cell specific reduction in expression of ERBB family proteins. Upregulation of pAKT was observed in all cell lines upon treatment with RAD001. Combination of RAD001 with neratinib suppressed the upregulation of pAKT and reduced cell cycle progression. In the absence of E2, RAD001 reduced ER mediated transcription and recruitment of ER and CREB binding protein to the TFF1 promoter, contrasting with neratinib, which caused a marked increase. In vivo study using LTED tumour xenografts showed the triple combination of RAD001, neratinib and fulvestrant was the most effective at reducing tumour volume (89%, p&amp;lt;0.001). Antitumor effects persisted after therapy removal with exception of RAD001 alone or in combination with neratinib where a slow increase in tumour growth was observed. Conclusion The combination of RAD001, neratinib and endocrine agents may be effective in patients who have relapsed on endocrine therapy but retain a functional ER by combating ERBB pathway upregulation. Citation Format: Ricardo Ribas, Sunil Pancholi, Stephanie K. Guest, Aradhana Rani, Joanna Nikitorowicz-Buniak, Nikiana Simigdala, Allan Thornhill, Richard E. Cutler Jr, Alshad S. Lalani, Francesca Avogadri-Connors, Mitch Dowsett, Stephen R. Johnston, Lesley-Ann Martin. Co-blockade of mTORC1, ERBB and estrogen receptor signalling pathways in endocrine resistance breast cancer: Combating tumor plasticity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4157. doi:10.1158/1538-7445.AM2017-4157

Abstract 1078: Dinaciclib overcomes resistance to BKM120 in triple negative breast cancer patient-derived xenograft models

Abstract Purpose: Triple negative breast cancer (TNBC) is one of the most lethal subtypes of breast cancer with limited therapeutic options. Development of molecularly targeted agents for TNBC is an unmet clinical need. The phosphoinositide 3-kinase (PI3K) pathway, which is a major cell growth and survival pathway, is frequently activated in TNBC as a result of genetic aberrations such as loss of the negative regulator PTEN or gain of function mutations in PIK3CA, therefore an attractive therapeutic target. However, single agent PI3K inhibitors have shown limited anti-tumor activity in both preclinical models and in clinical trials. One of the most important mechanisms of resistance to PI3K inhibitor is amplification of Myc. Dinaciclib, a potent inhibitor of cyclin-dependent kinases (CDKs)1, 2, 5 and 9, has shown to be particularly effective in Myc dependent tumors in preclinical studies and in TNBC. We therefore hypothesized that dinaciclib could overcome tumor cell resistance to PI3K inhibitors and improve the therapeutic efficacy in TNBC. In this study, we evaluated the anti-tumor and molecular effect of dinaciclib and BKM120 (Pan-PI3K inhibitor), either alone or in combination, in a panel of patient derived xenograft (PDX) models of TNBC. Methods: Four TNBC PDXs models were selected for the study for in-vivo and ex-vivo response to vehicle, BKM120, dinaciclib, or the combination of BKM120 and dinaciclib. Tumor volume changes over time in each group were documented to calculate the percentage of tumor growth inhibition by either agent alone or in combination. Tumor tissues harvested post treatment were examined by immunohistochemistry for cleaved PARP to determine the extent of apoptosis and phospho-Histone H3 for G2 to M phase cell cycle progression. Western blot analysis and reverse protein phase array (RPPA) were also performed to determine treatment effect on PI3K downstream targets and cell cycle molecules. Results: The combination of BKM120 and dinaciclib induced significantly greater growth inhibitory effect on tumor growth than either single agent alone in TNBC PDX models. This is accompanied by an enhanced apoptotic induction and reduced cell cycle progression. In addition, the combination of dinaciclib and BKM120 significantly reduced the level of cyclin B and the key anti-apoptotic protein survivin as well as significant downregulation of pAKT, and pS6. Conclusions: These data suggest that dinaciclib and BKM120 combination is an effective approach in treating TNBC. Additional mechanistic investigation for the efficacy of this combination is underway. Citation Format: Sandeep Rajput, Fang Guo, Li Shun, Cynthia Ma. Dinaciclib overcomes resistance to BKM120 in triple negative breast cancer patient-derived xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1078. doi:10.1158/1538-7445.AM2017-1078

Abstract 4033: Combined MEK and BCL-2/XL inhibition as a potential drug combination for the treatment of high-grade serous ovarian cancer

Abstract High-grade serous ovarian cancer (HGSOC) accounts for 70-80% of ovarian cancer deaths. Despite an initial response to platinum-based chemotherapy, treatment resistance eventually occurs in most patients. The overall aim of our studies is to identify synergistic drug combinations for the treatment of HGSOC and biomarkers that predict sensitivity for future translation in clinical trials. In particular, this study focuses on the vulnerabilities of patient-derived ovarian cancer cells to combined inhibition of the MEK pathway and the anti-apoptotic proteins BCL-2 and BCL-XL. The Ras/MAPK pathway is activated in a subset of HGSOC by gene copy alterations. However, MEK inhibitors have not been evaluated in HGSOC. In this study, we used a collection of 14 primary samples derived from ascites cells of platinum-resistant HGSOC patients (Liu JF et al. 2016). We investigated the sensitivity of these tumor cells to the MEK inhibitor cobimetinib (GDC-0973). Treatment with GDC-0973 had no effects on cell viability in vitro in any of the patient-derived models. To investigate the response to MEK inhibition, we performed Reverse Phase Protein Array of the tumor cells treated with 0.3μM of GDC-0973 in vitro. We found that MEK inhibition by GDC-0973 reduced cell cycle progression markers and upregulated the pro-apoptotic protein BIM. Since more than half of the models express high levels of anti-apoptotic BCL-2 family proteins which neutralize BIM, we examined the effects of antagonism of BCL-2 pro-survival proteins in combination with GDC-0973. Treatment with a combination of GDC-0973 and the BCL-2/XL antagonist, navitoclax (ABT-263) significantly reduced cell number and increased cell death in 10 out of 14 patient-derived models. Protein levels of BIM following treatment with GDC-0973 correlated with sensitivity to the drug combination (R2=0.8 p&amp;lt;0.0001). Interestingly, the BIM levels after MEK inhibition correlate with BIM protein levels before the treatment, suggesting that baseline BIM levels could be used as biomarker of sensitivity. Levels of the pro-survival protein MCL-1 also played a critical role in patient-derived cells that were resistant to the drug combination. Indeed, treatment with a specific MCL-1 inhibitor, A-1210477, in combination with GDC-0973 and ABT-263 led to dramatic tumor cell killing in vitro compared to dual combination of GDC-0973 and ABT-263. Finally, we tested the combination of GDC-0973 and ABT-263 in vivo and preliminary results indicate that the drug combination is well tolerated and is able to significantly reduce tumor growth in patient-derived xenograft models (n=4). Our studies provide significant evidence that combined inhibition of MEK and BCL-2/XL may be an effective drug combination for treatment of HGSOC and that the pro-apoptotic protein BIM may serve as a predictive biomarker to stratify patients that can benefit from these targeted therapies Citation Format: Claudia Iavarone, Ioannis Zervantonakis, Laura M. Selfors, Sangeetha Palakurthi, Joyce F. Liu, Ursula A. Matulonis, Ronny I. Drapkin, Gordon B. Mills, Joel D. Leverson, Deepak Sampath, Joan S. Brugge. Combined MEK and BCL-2/XL inhibition as a potential drug combination for the treatment of high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4033. doi:10.1158/1538-7445.AM2017-4033

Coordination of Myeloid Differentiation with Reduced Cell Cycle Progression by PU.1 Induction of MicroRNAs Targeting Cell Cycle Regulators and Lipid Anabolism.

During macrophage development, myeloid progenitor cells undergo terminal differentiation coordinated with reduced cell cycle progression. Differentiation of macrophages from myeloid progenitors is accompanied by increased expression of the E26 transformation-specific transcription factor PU.1. Reduced PU.1 expression leads to increased proliferation and impaired differentiation of myeloid progenitor cells. It is not understood how PU.1 coordinates macrophage differentiation with reduced cell cycle progression. In this study, we utilized cultured PU.1-inducible myeloid cells to perform genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) analysis coupled with gene expression analysis to determine targets of PU.1 that may be involved in regulating cell cycle progression. We found that genes encoding cell cycle regulators and enzymes involved in lipid anabolism were directly and inducibly bound by PU.1 although their steady-state mRNA transcript levels were reduced. Inhibition of lipid anabolism was sufficient to reduce cell cycle progression in these cells. Induction of PU.1 reduced expression of E2f1, an important activator of genes involved in cell cycle and lipid anabolism, indirectly through microRNA 223. Next-generation sequencing identified microRNAs validated as targeting cell cycle and lipid anabolism for downregulation. These results suggest that PU.1 coordinates cell cycle progression with differentiation through induction of microRNAs targeting cell cycle regulators and lipid anabolism.