BET-bromodomain inhibition (BETi) has shown pre-clinical promise for MYC-amplified medulloblastoma. However, the mechanisms for its action, and ultimately for resistance, have not been fully defined. Here, using a combination of expression profiling, genome-scale CRISPR/Cas9-mediated loss of function and ORF/cDNA driven rescue screens, and cell-based models of spontaneous resistance, we identify bHLH/homeobox transcription factors and cell-cycle regulators as key genes mediating BETi's response and resistance. Cells that acquire drug tolerance exhibit a more neuronally differentiated cell-state and expression of lineage-specific bHLH/homeobox transcription factors. However, they do not terminally differentiate, maintain expression of CCND2, and continue to cycle through S-phase. Moreover, CDK4/CDK6 inhibition delays acquisition of resistance. Therefore, our data provide insights about the mechanisms underlying BETi effects and the appearance of resistance and support the therapeutic use of combined cell-cycle inhibitors with BETi in MYC-amplified medulloblastoma.

AIMS: Drug resistance is becoming a major clinical challenge to the success of breast cancer treatment. Compelling evidence has shown the association between the deregulated long non-coding RNAs (lncRNAs) and drug resistance in various malignancies. However, the effects of long intergenic noncoding RNA 00511 (LINC00511), a newly identified oncogenic lncRNA, on the drug resistance of breast cancer cells remain unknown.
MAIN METHODS: RT-qPCR was performed to detect the expressions of LINC00511, miR-29c, and cyclin dependent kinase 6 (CDK6) in breast cancer tissues and cells. Pearson correlation analysis was used to analyze the correlation between miR-29c, CDK6 and LINC00511 expression in breast cancer tissues. The interactions between LINC00511, CDK6 and miR-29c were explored by luciferase reporter assay, RT-qPCR and western blot. MTT assay and flow cytometry analysis were applied to evaluate paclitaxel cytotoxicity.
KEY FINDINGS: LINC00511 and CDK6 were upregulated while miR-29c was downregulated in breast cancer tissues and cells. miR-29c was negatively correlated with LINC00511 and CDK6 expression while LINC00511 was positively correlated with CDK6 expression in breast cancer tissues. LINC0051 directly interacted with miR-29c to suppress its expression. LINC00511 knockdown enhanced paclitaxel cytotoxicity in breast cancer cells by upregulating miR-29c. CDK6 was identified as a target of miR-29c. CDK6 knockdown attenuated the effects of miR-29c inhibition on paclitaxel cytotoxicity in breast cancer cells. LINC00511 positively regulated CDK6 expression in breast cancer cells.
SIGNIFICANCE: LINC00511 knockdown enhanced paclitaxel cytotoxicity in breast cancer cells via regulating miR-29c/CDK6 axis.

Adrenocortical cancer (ACC) is an aggressive malignancy with no available effective treatments; therefore, exploring the molecular mechanisms involved in the initiation and progression of ACC is quite important. Here, we found that the long noncoding RNA urothelial carcinoma-associated 1 (UCA1) was highly expressed in ACC tissues and closely associated with the TNM stage and metastasis of ACC patients. Overexpression of UCA1 significantly promoted the proliferation and suppressed the apoptosis of ACC cells. Mechanism study showed that UCA1 acted as sponge of miR-298 and decreased the expression abundance of miR-298 in ACC cells. Further investigation identified that miR-298 bound the 3'-UTR of the cyclin-dependent kinase 6 (CDK6) and inhibited the expression of CDK6. Consistently, ectopic expressed UCA1 suppressed miR-298 and up-regulated the expression of CDK6, which promoted the cell cycle progression of ACC cells. Taken together, our results identified the potential oncogenic function of UCA1 in ACC by regulating the miR-298-CDK6 axis.

Berberine has shown anticancer properties and has potential for a chemopreventive and/or chemotherapeutic agent for breast cancer. Berberine showed cytotoxicity to breast cancer cells, with an increase in the levels of p21/cip1 and p27/kip1, cyclin-dependent kinase inhibitors (CDKI), but mechanisms involved in up-regulating these molecules are largely unknown. Herein, we studied the key regulatory mechanisms involved in berberine-mediated up-regulation of p21/cip1 and p27/kip1. Berberine treatment for 24 and 48 h decreased the number of cells by 44-84% (P < 0.0001) and 38-78% (P < 0.0001), and increased cell death by 12-17% (P < 0.005) and 38-78% (P < 0.0001) in MCF-7 and MDA-MB-231 cells, respectively. Cells were arrested in G1 phase by berberine which was accompanied with up-regulation of mRNA and protein level of both p21/cip1 and p27/kip1. Berberine decreased the expression of protein levels of cyclin D1, cyclin E, CDK2, CDK4, and CDK6 to cause G1 phase arrest. Berberine caused nuclear localization of p21/cip1 in both the cell lines. Our data for the first time showed that the post-translational stability of both the proteins was strongly increased by berberine as examined by cycloheximide chase assay. Inhibition of Akt was associated with berberine-mediated up-regulation of p21/cip1 and also led to a decrease in cell viability accompanied with significant G1 phase cell cycle arrest. Our study revealed that berberine not only up-regulates mRNA and protein levels of p21/cip1 and p27/kip1 but also increases their nuclear localization and post-translational protein stability. Further, Akt inhibition was found to mediate berberine-mediated up-regulation of p21/cip1 but not the p27/kip1.

Long-chain fatty acids are the most abundant fatty acids and are essential for various physiological processes. Translocation of long-chain fatty acids across cell membrane is dependent on transport proteins. Solute carrier family 27 member 6 (SLC27A6) is a transport protein which mediates long-chain fatty acid uptake. The bioinformatic analysis revealed that the expression of SLC27A6 in non-tumoral breast tissue was higher than that in tumoral breast cancer in clinic samples. When SLC27A6 expression in non-tumorigenic cell H184B5F5/M10 was repressed, the fatty acids uptake capacity and cell proliferation was inhibited, and cell cycle was delayed. The protein expression of cell cycle regulators including cell division protein kinase 4 (CDK4), CDK6, and cyclin D1 was significantly decreased in SLC27A6-silenced H184B5F5/M10. By contrast, relatively low SLC27A6 expression in tumorigenic breast cancer cell Hs578T when compared to H184B5F5/M10. Repressing SLC27A6 expression did not affect these phenotypes in Hs578T. The interaction network of SLC27A6 was further investigated via STRING database. The function of these SLC27A6-associated proteins mainly involved in lipid biosynthesis, fatty acid metabolic process, and fatty acid transport. In conclusion, this study reveals inverse correlation between SLC27A6 expression and tumoral tissues and provides a new insight into SLC27A6-mediated cell growth and cell cycle regulation in non-tumorigenic breast cells.

BACKGROUND: Epithelial-mesenchymal transition (EMT) may be one of the reasons for the failure in some clinical trials regarding histone deacetylase inhibitors (HDACIs)-treated solid tumors. We investigated the effects of a pan-HDACI trichostatin A (TSA) on the proliferation and EMT of nasopharyngeal carcinoma (NPC) cells.
METHODS: Poorly-differentiated NPC cell line CNE2 and undifferentiated C666-1 were treated with various concentrations of TSA, the cell viability was assessed by CCK-8 assay, the morphology was photographed, and the mRNA level of HDACs was assessed by semiquantitative PCR. After determination the cell cycle distributions, cells were subjected to western blotting analysis of cell cycle and EMT-associated genes expression. And the changes in migration ability were assessed by transwell migration assay and scratch wound healing assay. Finally, histone deacetylases activator ITSA-1 was used to assess the reverse of TSA-induced changes in NPC cells.
RESULTS: TSA inhibited the proliferation of CNE2 and C666-1 cells in a concentration-dependent manner and arrested the cell cycle at G1 phases. TSA reduced PCNA, cyclin D1, cyclin E1, CDK2, p16 and p21 expressions and stimulated CDK6 levels. TSA stimulation for 48 h could effectively induce the EMT in CNE2 and C666-1 cells, which showed an increase of spindle-like cells and promoted expression of Vimentin and Snail1 expression in a concentration-dependent manner. Surprisingly, this short period of TSA treatment that induced EMT also impeded the migration ability of CNE2 and C666-1 cells. Interestingly, ITSA-1 rescued TSA-impeded CNE2 and C666-1 cells' proliferation, migration and HDACs expression, also re-induced the cells to turn into epithelial cell phenotypes.
CONCLUSIONS: These results indicate that short-term stimulation of TSA effectively inhibits cell proliferation and induce EMT-like changes in NPC cells but not increase its invasion ability.

The dysregulation of Fbxo4-cyclin D1 axis occurs at high frequency in esophageal squamous cell carcinoma (ESCC), where it promotes ESCC development and progression. However, defining a therapeutic vulnerability that results from this dysregulation has remained elusive. Here we demonstrate that Rb and mTORC1 contribute to Gln-addiction upon the dysregulation of the Fbxo4-cyclin D1 axis, which leads to the reprogramming of cellular metabolism. This reprogramming is characterized by reduced energy production and increased sensitivity of ESCC cells to combined treatment with CB-839 (glutaminase 1 inhibitor) plus metformin/phenformin. Of additional importance, this combined treatment has potent efficacy in ESCC cells with acquired resistance to CDK4/6 inhibitors in vitro and in xenograft tumors. Our findings reveal a molecular basis for cancer therapy through targeting glutaminolysis and mitochondrial respiration in ESCC with dysregulated Fbxo4-cyclin D1 axis as well as cancers resistant to CDK4/6 inhibitors.

Cellular senescence is the state of irreversible cell cycle arrest that provides a blockade during oncogenic transformation and tumor development. Avenanthramide A (AVN A) is an active ingredient exclusively extracted from oats, which possesses antioxidant, anti-inflammatory, and anticancer activities. However, the underlying mechanism(s) of AVN A in the prevention of cancer progression remains unclear. In the current study, we revealed that AVN A notably attenuated tumor formation in an azoxymethane/dextran sulfate sodium (AOM/DSS) mouse model. AVN A treatment triggered cellular senescence in human colon cancer cells, evidenced by enlarging cellular size, upregulating β-galactosidase activity, γ-H2AX positive staining, and G1 phase arrest. Moreover, AVN A treatment significantly increased the expression of miR-129-3p, which markedly repressed the E3 ubiquitin ligase Pirh2 and two other targets, IGF2BP3 and CDK6. The Pirh2 silencing by miR-129-3p led to a significant increase in protein levels of p53 and its downstream target p21, which subsequently induced cell senescence. Taken together, our data indicate that miR-129-3p/Pirh2/p53 is a critical signaling pathway in AVN A induced cellular senescence and AVN A could be a potential chemopreventive strategy for cancer treatment.

Several studies by our group and others have determined that expression levels of Bcl-2 and/or Bcl-xL, pro-survival molecules which are associated with chemoresistance, are elevated in patients with muscle invasive bladder cancer (MI-BC). The goal of this study was to determine whether combining Obatoclax, a BH3 mimetic which inhibits pro-survival Bcl-2 family members, can improve responses to cisplatin chemotherapy, the standard of care treatment for MI-BC. Three MI-BC cell lines (T24, TCCSuP, 5637) were treated with Obatoclax alone or in combination with cisplatin and/or pre-miR-34a, a molecule which we have previously shown to inhibit MI-BC cell proliferation via decreasing Cdk6 expression. Proliferation, clonogenic, and apoptosis assays confirmed that Obatoclax can decrease cell proliferation and promote apoptosis in a dose-dependent manner. Combination treatment experiments identified Obatoclax + cisplatin as the most effective treatment. Immunoprecipitation and Western analyses indicate that, in addition to being able to inhibit Bcl-2 and Bcl-xL, Obatoclax can also decrease cyclin D1 and Cdk4/6 expression levels. This has not previously been reported. The combined data demonstrate that Obatoclax can inhibit cell proliferation, promote apoptosis, and significantly enhance the effectiveness of cisplatin in MI-BC cells via mechanisms that likely involve the inhibition of both pro-survival molecules and cell cycle regulators.

AIMS: Cyclin dependent kinase 6 (CDK6) plays a crucial role in malignant tumor whereas less is reported in cervical cancer development. The aim of this study was to evaluate the effects of CDK6 3' untranslated region (3'UTR) polymorphisms on cervical cancer susceptibility among Uyghur females.
METHODS: The genotypes of the six CDK6 variants (rs8179, rs42032, rs42033, rs42034, rs42035, and rs42038) were identified among 306 cervical cancer cases and 310 healthy controls with the Agena MassARRAY platform. The associations of the candidate single nucleotide polymorphisms (SNPs) with the cervical cancer risk were evaluated under genetic models using conditional logistic regression analysis. Bioinformatics analysis was performed for SNP function prediction with the online databases. The expression differences between tumor tissues and normal cervix samples were also examined by Real-time PCR.
RESULTS: CDK6 rs8179 and rs42033 were correlated to the decreased risk of cervical cancer in Uyghurs under the allele model (rs8179 and rs42033: OR = 0.60, 95% CI: 0.37-0.99, p = 0.043) and log-additive model (rs8179 and rs42033: OR = 0.62, 95% CI: 0.38-1.00, p = 0.047). Rs8179, rs42032, and rs42033 were associated with susceptibility to high-grade cervical cancer in different genetic models as well (p < 0.05). Dataset-based analysis also uncovered the potential effects of these significant SNPs. In addition, aberrant expression of CDK6 were detected in cervical tumors.
CONCLUSIONS: Our results suggested the relationships between CDK6 3'UTR polymorphisms and cervical cancer pathogenesis, and the involvement of CDK6 in cervical cancer development among Uyghur females.

MicroRNAs (miRNAs or miRs) contribute to the development of various malignant neoplasms, including glioblastoma multiforme (GBM). The present study aimed to explore the pathogenesis of GBM and to identify latent therapeutic agents for patients with GBM, based on an in silico analysis. Gene chips that provide miRNA expression profiling in GBM were obtained from the Gene Expression Omnibus (GEO) database. Differentially expressed miRNAs (DEMs) were also determined via the RobustRankAggreg algorithm. The target genes of DEMs were predicted and then intersected with GBM‑associated genes that were collected from the Gene Expression Profiling Interactive Analysis. Gene Oncology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of the overlapping genes were then performed. Simultaneously, a connectivity map (CMap) analysis was performed to screen for potential therapeutic agents for GBM. A total of 10 DEMs (hsa‑miR‑196a, hsa‑miR‑10b, hsa‑miR‑196b, hsa‑miR‑18b, hsa‑miR‑542‑3p, hsa‑miR‑129‑3p, hsa‑miR‑1224‑5p, hsa‑miR‑876‑3p and hsa‑miR‑770‑5p) were obtained from three GEO gene chips (GSE25631, GSE42657 and GSE61710). Then, 1,720 target genes of the 10 miRNAs and 4,185 differently expressed genes in GBM were collected. By intersecting the aforementioned gene clusters, the present study identified 390 overlapping genes. GO and KEGG analyses of the 390 genes demonstrated that these genes were involved in certain cancer‑associated biological functions and pathways. Eight genes [(GTPase NRas (NRAS), calcium/calmodulin‑dependent protein kinase type II subunit Gamma (CAMK2G), platelet‑derived growth factor receptor alpha (PDGFRA), calmodulin 3 (CALM3), cyclin‑dependent kinase 6 (CDK6), calcium/calmodulin‑dependent protein kinase type II subunit beta (CAMK2B), retinoblastoma‑associated protein (RB1) and protein kinase C beta type (PRKCB)] that were centralized in the glioma pathway were selected for CMap analysis. Three chemicals (W‑13, gefitinib and exemestane) were identified as putative therapeutic agents for GBM. In summary, the present study identified three miRNA‑based chemicals for use as a therapy for GBM. However, more experimental data are needed to verify the therapeutic properties of these latent drugs in GBM.

Mushrooms comprise an unlimited source of active compounds that have beneficial health effects without known negative side effects and can potentially be used as important therapeutic products against cancer, which is the leading cause of death worldwide. In this study we investigated the cytotoxic, antiproliferative, apoptotic, and anti-invasion effects of Macrolepiota procera, which is valued as an edible and medicinal mushroom, on A549 lung cancer cells. The cytotoxic effect of the M. procera extract was determined by using the XTT method. Total RNA was isolated from cells with TRI Reagent to determine the apoptotic effect of the extract, after which complementary DNA was synthesized. Expression profiles of the target genes were determined by quantitative reverse-transcriptase polymerase chain reaction, and protein changes were determined by using Western blotting. We used the TUNEL assay to evaluate the apoptotic effects of the M. procera extract. Effects of M. procera on cell invasion were investigated by using a Matrigel chamber assay. The half-maximal inhibitory concentration of the M. procera extract was determined to be 2 mg/mL against A549 lung cancer cells at 72 hours. According to our results, expression of Cyclin Dl, CDK4, CDK6, Bcl-2, Akt, and NOXA genes significantly decreased and that of Bax, Caspase-3, Caspase-9, PTEN, PUMA, p21, and p53 increased in cells from the dose group compared with their expression in control cells. According to the results of the TUNEL assay, 28 ± 3.6% of cells were apoptotic in the dose group. The M. procera extract also reduced invasion in A549 cancer cells. The results suggest that M. procera has an antiproliferative effect in a dose- and time-dependent manner.

Cervical cancer remains one of the main factors leading to tumor-related death worldwide. Many strategies of cancer treatment such as chemotherapy are developed and used nowadays. However, for the cancer chemotherapy resistance, reduction of the limitation of cancer chemotherapy efficacy is one of the aims of several oncology teams. Moreover, the cyclin-dependent kinase 4/6-cyclin D-retinoblastoma protein-E2F pathway is an important mechanism for cell cycle control and its dysregulation is one of the key factors for cancers development including cervical cancer. Ribociclib is one of the selective CDK4/6 inhibitors and is a new therapeutic approach showing promise as a good strategy of therapy in many human cancers. However, there are not the studies regarding the investigation of effects of Ribociclib in cervical cancer yet. In the present study, by western blotting and immunofluorescence assay, we found respectively that CDK4, CDK6 and cyclin D1 are highly expressed and are mostly localized in the nucleus with some localized in the cytoplasm of cervical cancer cell lines. Moreover, Ribociclib induced cell cycle arrest in G0-G1 phase and cell apoptosis, and inhibited C33A cell proliferation in dose - dependent manner following by decreased expression of certain related genes such as CDK4, CDK6, E2F1, P-Rb, and increased Bax expression. In C33A xenografts, Ribociclib inhibited tumor growth associated with decreased expressions of CDK4, CDK6, cyclin D1, Rb and Ki-67, and also significantly increased tumor cell apoptosis. However, we didn't find side effect of Ribociclib concerning heart, liver and kidney perturbation and any Ribociclib anti-tumor effects on HeLa in vitro and in vivo which may be due to Hela cell infection by HPV. Based on our findings, the Rb-E2F pathway can be considered as an important factor in human cervical cancer pathogenesis and as a mechanism of Ribociclib, a potential strategy of treatment for the improvement of new therapeutic measures for the treatment of HPV-negative cervical cancer which application for HPV-positive cervical cancer is desired in further study.

Our previous study found that two novel cancer-related genes, PRR11 and SKA2, constituted a classic gene pair that was regulated by p53 and NF-Y in lung cancer. However, their role and regulatory mechanism in breast cancer remain elusive. In this study, we found that the expression levels of PRR11 and SKA2 were upregulated and have a negative prognotic value in breast cancer. Loss-of-function experiments showed that RNAi-mediated knockdown of PRR11 and/or SKA2 inhibited proliferation, migration, and invasion of breast cancer cells. Mechanistic experiments revealed that knockdown of PRR11 and/or SKA2 caused dysregulation of several downstream genes, including CDK6, TPM3, and USP12, etc. Luciferase reporter assays demonstrated that wild type p53 significantly repressed the PRR11-SKA2 bidirectional promoter activity, but not NF-Y. Interestingly, NF-Y was only essential for and correlated with the expression of PRR11, but not SKA2. Consistently, adriamycin-induced (ADR) activation of endogenous p53 also caused significant repression of the PRR11 and SKA2 gene pair expression. Notably, breast cancer patients with lower expression levels of either PRR11 or SKA2, along with wild type p53, exhibited better disease-free survival compared to others with p53 mutations and/or higher expression levels of either PRR11 or SKA2. Collectively, our study indicates that the PRR11 and SKA2 transcription unit might be an oncogenic contributor and might serve as a novel diagnostic and therapeutic target in breast cancer. [BMB Reports 2019; 52(2): 157-162].

Nerve growth factor receptor (NGFR), a member of kinase protein, is emerging as an important target for Glioblastoma (GBM) treatment. Overexpression of NGFR is observed in many metastatic cancers including GBM, promoting tumor migration and invasion. Hydrazones have been reported to effectively interact with receptor tyrosine kinases (RTKs). We report herein the synthesis of 23 arylhydrazones of active methylene compounds (AHAMCs) compounds and their anti-proliferative activity against GBM cell lines, LN229 and U87. Compound R234, 2-(2-(2,4-dioxopentan-3-ylidene)hydrazineyl)benzonitrile, was identified as the most active anti-neoplastic compound, with the IC

Esophageal adenocarcinoma (EAC) is a poor-prognosis cancer type with rapidly rising incidence. Understanding of the genetic events driving EAC development is limited, and there are few molecular biomarkers for prognostication or therapeutics. Using a cohort of 551 genomically characterized EACs with matched RNA sequencing data, we discovered 77 EAC driver genes and 21 noncoding driver elements. We identified a mean of 4.4 driver events per tumor, which were derived more commonly from mutations than copy number alterations, and compared the prevelence of these mutations to the exome-wide mutational excess calculated using non-synonymous to synonymous mutation ratios (dN/dS). We observed mutual exclusivity or co-occurrence of events within and between several dysregulated EAC pathways, a result suggestive of strong functional relationships. Indicators of poor prognosis (SMAD4 and GATA4) were verified in independent cohorts with significant predictive value. Over 50% of EACs contained sensitizing events for CDK4 and CDK6 inhibitors, which were highly correlated with clinically relevant sensitivity in a panel of EAC cell lines and organoids.

Tumor suppressor SMARCA4 (BRG1), a key SWI/SNF chromatin remodeling gene, is frequently inactivated in cancers and is not directly druggable. We recently uncovered that SMARCA4 loss in an ovarian cancer subtype causes cyclin D1 deficiency leading to susceptibility to CDK4/6 inhibition. Here, we show that this vulnerability is conserved in non-small cell lung cancer (NSCLC), where SMARCA4 loss also results in reduced cyclin D1 expression and selective sensitivity to CDK4/6 inhibitors. In addition, SMARCA2, another SWI/SNF subunit lost in a subset of NSCLCs, also regulates cyclin D1 and drug response when SMARCA4 is absent. Mechanistically, SMARCA4/2 loss reduces cyclin D1 expression by a combination of restricting CCND1 chromatin accessibility and suppressing c-Jun, a transcription activator of CCND1. Furthermore, SMARCA4 loss is synthetic lethal with CDK4/6 inhibition both in vitro and in vivo, suggesting that FDA-approved CDK4/6 inhibitors could be effective to treat this significant subgroup of NSCLCs.

Gliomas are the most common primary brain malignant tumors in humans. Glioblastoma multiforme(GBM) is the most malignant intracranial tumor with a relatively poor prognosis. There promote us to find effective anti-cancer therapies to reduce cancer mortality. By using bioinformatic analysis, we found SSFA2 as a gene with elevated expression in the glioma tissues. We detected the expression of SSFA2 in glioma tissues and in the glioma cell lines, as well as in normal brain tissues. SSFA2 expression was higher in glioma tissues, especially in glioblastoma multiforme than normal brain tissues. Subsequently, we found that down-regulate SSFA2 in glioma cell lines can regulate the cell cycle to reduce the proliferation ability and induce the early apoptosis rate in shSSFA2 cells relative to control cells. Moreover, we found that down-regulate SSFA2 in glioma cell line U87(shSSFA2-U87) inhibited the growth effectiveness compared to the control cell line U87. These result reveals us that SSFA2 may act as oncogene to promote the progression of glioma. For further research specific mechanisms of SSFA2 in gliomas, we used the gene chip to detect the downstream gene in U87. We found that 30 genes also may be as target gene of SSFA2, and we testify the protein expression by western-blot. The result reveal that IL1A, IL1B and CDK6 as target gene of SSFA2 to regulate the progression of glioma. These finding suggest that SSFA2 could be a new therapeutic target for gliomas.

Pancreatic ductal adenocarcinoma (PDAC), like many KRAS-driven tumors, preferentially loses CDKN2A that encodes an endogenous CDK4/6 inhibitor to bypass the RB-mediated cell cycle suppression. Analysis of a panel of patient-derived cell lines and matched xenografts indicated that many pancreatic cancers have intrinsic resistance to CDK4/6 inhibition that is not due to any established mechanism or published biomarker. Rather, there is a KRAS-dependent rapid adaptive response that leads to the upregulation of cyclin proteins, which participate in functional complexes to mediate resistance. In vivo, the degree of response is associated with the suppression of a gene expression signature that is strongly prognostic in pancreatic cancer. Resistance is associated with an adaptive gene expression signature that is common to multiple kinase inhibitors, but is attenuated with MTOR inhibitors. Combination treatment with MTOR and CDK4/6 inhibitors had potent activity across a large number of patient-derived models of PDAC underscoring the potential clinical efficacy.

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The t(8;21) translocation is one of the most frequent chromosome abnormalities associated with acute myeloid leukaemia (AML). This abberation deregulates numerous molecular pathways including the ERK signalling pathway among others. Therefore, the aim of the present study was to investigate the gene expression patterns following siRNA‑mediated suppression of RUNX1‑RUNX1T1 and MAPK1 in Kasumi‑1 and SKNO‑1 cells and to determine the differentially expressed genes in enriched biological pathways. BeadChip microarray and gene ontology analysis revealed that RUNX1‑RUNX1T1 and MAPK1 suppression reduced the proliferation rate of the t(8;21) cells with deregulated expression of several classical positive regulator genes that are otherwise known to enhance cell proliferation. RUNX1‑RUNX1T1 suppression exerted an anti‑apoptotic effect through the overexpression of BCL2, BIRC3 and CFLAR genes, while MAPK1 suppression induced apopotosis in t(8;21) cells by the apoptotic mitochondrial changes stimulated by the activity of upregulated TP53 and TNFSF10, and downregulated JUN gene. RUNX1‑RUNX1T1 suppression supported myeloid differentiation by the differential expression of CEBPA, CEBPE, ID2, JMJD6, IKZF1, CBFB, KIT and CDK6, while MAPK1 depletion inhibited the differentiation of t(8;21) cells by elevated expression of ADA and downregulation of JUN. RUNX1‑RUNX1T1 and MAPK1 depletion induced cell cycle arrest at the G0/G1 phase. Accumulation of cells in the G1 phase was largely the result of downregulated expression of TBRG4, CCNE2, FOXO4, CDK6, ING4, IL8, MAD2L1 and CCNG2 in the case of RUNX1‑RUNX1T1 depletion and increased expression of RASSF1, FBXO6, DADD45A and P53 in the case of MAPK1 depletion. Taken together, the current results demonstrate that MAPK1 promotes myeloid cell proliferation and differentiation simultaneously by cell cycle progression while suppresing apoptosis.

OBJECTIVE: Esophageal cancer is one of the deadliest cancers worldwide occurring at upper gastrointestinal tract. This study aimed to explore the possible role of long non-coding RNA X Inactive Specific Transcript (XIST) in the development of esophageal cancer.
MATERIAL AND METHODS: The lncRNA XIST expressions both in esophageal cancer tissues and in cells were investigated. The TE-1 and SKGT-4 cells were transfected with LV-sh-XIST and LV-scramble for the further detection of the effects of XIST expression on cell biological processes, including proliferation, apoptosis and the expression of apoptosis-related proteins, migration, invasion and the expression of epithelial-mesenchymal transition markers. Additionally, the regulatory relationships between lncRNA XIST and miR-494, between miR-494 and CDK6, between miR-494/CDK6 and JAK2/STAT3 pathway were investigated.
RESULTS: LncRNA XIST was overespressed in esophageal cancer tissues and cells. Suppression of XIST significantly inhibited the proliferation, migration and invasion, but induced apoptosis of two kinds of cells, TE-1 and SKGT-4. Moreover, miR-494 was down-regulated in esophageal cancer tissues and cells. XIST could sponge miR-494 and inhibition of miR-494 reversed the effects of XIST suppression on the malignant behaviors of TE-1 cells. Also, CDK6 was a target of miR-494 and CDK6 knockdown reversed the effects of miR-494 inhibition on the malignant behaviors of TE-1 cells. Besides, the expression of p-JAK2 and p-STAT3 was increased after miR-494 inhibition, which was reversed after inhibition of miR-494 and CDK6 at the same time.
CONCLUSIONS: The data presented in this study revealed that XIST abnormal expression may play an oncogenic role in the development of esophageal cancer via miR-494/CDK6 axis through JAK2/STAT3 signal pathway. This study may provide theoretical basis for the molecular mechanism investigation of esophageal cancer.

We aimed to confirm the role of miR-1296-5p in gastric cancer and to identify its target genes. The expression of miR-1296-5p was measured in gastric cancer tissues and cell lines. The function of miR-1296-5p was examined by the overexpression and inhibition of its expression in typical gastric cell lines as well as SGC-7901 and MGC-803 cells. The targets of miR-1296-5p were identified by a luciferase activity assay. We found that miR-1296-5p was down-regulated in gastric cancer tissue and cell lines, and low expression levels of miR-1296-5p were associated with advanced clinical stage. Moreover, miR-1296-5p inhibited cell proliferation, migration, and invasion in SGC-7901 and MGC-803 cells. Then, we identified CDK6 and EGFR as novel targets of miR-1296-5p by a luciferase activity assay. Furthermore, the overexpression of miR-1296-5p suppressed the expression of CDK6 and EGFR. Our results indicated a tumor-suppressive role of miR-1296-5p through the translational repression of oncogenic CDK6 and EGFR in gastric cancer.

Aberrant expression of programmed death ligand-1 (PD-L1) in tumor cells promotes cancer progression by suppressing cancer immunity. The retinoblastoma protein RB is a tumor suppressor known to regulate the cell cycle, DNA damage response, and differentiation. Here, we demonstrate that RB interacts with nuclear factor κB (NF-κB) protein p65 and that their interaction is primarily dependent on CDK4/6-mediated serine-249/threonine-252 (S249/T252) phosphorylation of RB. RNA-seq analysis shows a subset of NF-κB pathway genes including PD-L1 are selectively upregulated by RB knockdown or CDK4/6 inhibitor. S249/T252-phosphorylated RB inversely correlates with PD-L1 expression in patient samples. Expression of a RB-derived S249/T252 phosphorylation-mimetic peptide suppresses radiotherapy-induced upregulation of PD-L1 and augments therapeutic efficacy of radiation in vivo. Our findings reveal a previously unrecognized tumor suppressor function of hyperphosphorylated RB in suppressing NF-κB activity and PD-L1 expression and suggest that the RB-NF-κB axis can be exploited to overcome cancer immune evasion triggered by conventional or targeted therapies.

BRD4 assembles transcriptional machinery at gene super-enhancer regions and governs the expression of genes that are critical for cancer progression. However, it remains unclear whether BRD4-mediated gene transcription is required for tumor cells to develop drug resistance. Our data show that prolonged treatment of luminal breast cancer cells with AKT inhibitors induces FOXO3a dephosphorylation, nuclear translocation, and disrupts its association with SirT6, eventually leading to FOXO3a acetylation as well as BRD4 recognition. Acetylated FOXO3a recognizes the BD2 domain of BRD4, recruits the BRD4/RNAPII complex to the CDK6 gene promoter, and induces its transcription. Pharmacological inhibition of either BRD4/FOXO3a association or CDK6 significantly overcomes the resistance of luminal breast cancer cells to AKT inhibitors in vitro and in vivo. Our study reports the involvement of BRD4/FOXO3a/CDK6 axis in AKTi resistance and provides potential therapeutic strategies for treating resistant breast cancer.

The upregulation of long non‑coding RNA (lncRNA) human ovarian cancer‑specific transcript 2 (HOST2) has been identified in breast cancer. The present study aimed to investigate whether lncRNA HOST2 regulated the proliferation of triple negative breast cancer (TNBC) cells, and the underlying molecular mechanism. In total, 30 patients with primary TNBC, who were treated at Wuhai People's Hospital (Wuhai, China), were recruited for the present study. Reverse transcription‑quantitative polymerase chain reaction analysis was used for the examination of gene expression levels. A Cell Counting kit‑8 (CCK‑8) assay was used for the detection of cell proliferation. Phases of the cell cycle were evaluated by flow cytometry. Western blot analysis was performed to detect protein expression levels. A dual luciferase activity assay was performed to examine the interaction between microRNA (miRNA) and the 3' untranslated region (UTR) of target mRNA. The results revealed increased expression levels of HOST2 in tumor tissues from patients with TNBC. A positive correlation was identified between the expression of HOST2 and cyclin‑dependent kinase 6 (CDK6) in tumor tissues. The silencing of HOST2 induced cell proliferation inhibition and cell cycle redistribution in MDA‑MB‑231 and MDA‑MB‑468 TNBC cells. In these two cell lines, HOST2 silencing caused a decrease in the phosphorylation of RB1 and CDK6, which was observed at the mRNA and protein levels. However, the silencing of CDK6 did not alter the expression of HOST2. It was hypothesized and confirmed that let‑7b, a previously reported target miRNA of HOST2, was able to directly bind to the 3'UTR of CDK6 and repress its expression. The expression of let‑7b was negatively correlated with the expression of HOST2 and CDK6 in tumor tissues. Overall, the data suggested that lncRNA HOST2 acts as an oncogene in TNBC via the upregulation of CDK6.

CDKN2A is one of the most studied tumor suppressor genes. It encodes the p16-INK4a protein that plays a critical role in the cell cycle progression, differentiation, senescence, and apoptosis. Mutations in CDKN2A or dysregulation of its functional activity are frequently associated with various types of human cancer. As a cyclin-dependent kinase inhibitor, p16-INK4a forms a complex with cyclin-dependent kinases 4/6 (CDK4/6) thereby competing with cyclin D. It is believed that the helix-turn-helix structures in the content of tandem ankyrin repeats in p16-INK4a are required for the protein interaction with CDK4. Until recently, the mechanisms considered to be involved in the regulation of p16-INK4a functions and cancer development have been mutations in DNA, homozygous or heterozygous gene loss, and methylation of CDKN2A promoter region. In this review, we discuss recent findings on the regulation of p16-INK4a by covalent modifications at both transcriptional and post-translational levels.

BACKGROUND: Following up on previous work demonstrating the involvement of the transcription factor forkhead box M1 (FOXM1) in the biology and outcome of a high-risk subset of newly diagnosed multiple myeloma (nMM), this study evaluated whether FOXM1 gene expression may be further upregulated upon tumor recurrence in patients with relapsed multiple myeloma (rMM). Also assessed was the hypothesis that increased levels of FOXM1 diminish the sensitivity of myeloma cells to commonly used myeloma drugs, such as the proteasome inhibitor bortezomib (Bz) and the DNA intercalator doxorubicin (Dox).
METHODS: FOXM1 message was evaluated in 88 paired myeloma samples from patients with nMM and rMM, using gene expression microarrays as measurement tool. Sources of differential gene expression were identified and outlier analyses were performed using statistical methods. Two independent human myeloma cell lines (HMCLs) containing normal levels of FOXM1 (FOXM1
RESULTS: Upregulation of FOXM1 occurred in 61 of 88 (69%) patients with rMM, including 4 patients that exhibited > 20-fold elevated expression peaks. Increased FOXM1 levels in FOXM1
CONCLUSIONS: These findings enhanced our understanding of the emerging FOXM1 genetic network in myeloma and provided preclinical support for the therapeutic targeting of the FOXM1-NEK2 and CDK4/6-Rb-E2F pathways using small-drug CDK and NEK2 inhibitors. Clinical research is warranted to assess whether this approach may overcome drug resistance in FOXM1

BACKGROUND/AIMS: The purpose of this study was to probe the clinico-pathological significance and the underlying mechanism of miR-30d-5p expression in non-small cell lung cancer (NSCLC).
METHODS: We initially examined the level of miR-30d-5p expression in NSCLC and non-cancer tissues using RT-qPCR. Then, a series of validation analyses including a meta-analysis of data from microarray chips in Gene Expression Omnibus (GEO), data mining of the cancer genome atlas (TCGA) and an integrated meta-analysis incorporating GEO microarray chips, TCGA data, in-house RT-qPCR and literature studies were performed to examine the clinico-pathological value of miR-30d-5p expression in NSCLC. In vitro experiments were further conducted to investigate the impact of miR-30d-5p on NSCLC cell growth. The molecular mechanism by which miR-30d-5p regulates the pathogenesis of NSCLC was probed through a bioinformatics analysis of its target genes. Moreover, dual luciferase reporter assay was conducted to verify the targeting regulatory relationship between miR-30d-5p and CCNE2.
RESULTS: Based on results from RT-qPCR, GEO meta-analysis, TCGA data mining and the integrated meta-analysis incorporating GEO microarray chips, TCGA data, in-house RT-qPCR and literature studies, miR-30d-5p expression was decreased in NSCLC tissues, and patients with NSCLC who presented with lower miR-30d-5p expression tended to display an advanced clinical progression. Significant pathways including the Mucin type O-glycan biosynthesis pathway, cell cycle pathway and cysteine and methionine metabolism pathway (all P< 0.05) revealed potential roles of the target genes of miR-30d-5p in the oncogenesis of NSCLC. Results from in vitro experiments indicated that miR-30d-5p could attenuate proliferation and viability of NSCLC cells. Among the 12 identified hub genes, nine genes including E2F3, CCNE2, SKP2, CDK6, TFDP1, LDHA, GOT2, DNMT3B and ST6GALNAC1 were validated by Pearson's correlation test and the human protein atlas (HPA) database as targets of miR-30d-5p with higher probability. Specifically, dual luciferase reporter assay confirmed that CCNE2 was directly targeted by miR-30d-5p.
CONCLUSION: In summary, miR-30d-5p expression is decreased in NSCLC, and it might play the role as tumor suppressor in NSCLC by regulating target genes.

Dermatopontin (DPT), a noncollagenous extracellular matrix component, has been illustrated to regulate cellular proliferation and invasiveness in several types of neoplasms. Nevertheless, the biological functions of DPT in cell proliferation, especially papillary thyroid cancer (PTC) cell proliferation, remain unknown, as do the mechanisms underlying its effects. In this study, we detected low DPT expression in PTC, which was related to higher T classifications. Ectopic DPT expression impeded cell proliferation both in vitro and in vivo. Furthermore, we illustrated that DPT down-regulated MYC, which in turn targeted CDK4, CDK6 and p21, through the ERK pathway. These results suggest that DPT regulates CDK4, CDK6 and p21, through MEK-ERK-MYC signaling to repress PTC proliferation.