Knockdown Induced Cell Cycle Arrest Research Articles

<italic>UHRF1</italic> knockdown induces cell cycle arrest and apoptosis in breast cancer cells through the ZBTB16/ANXA7/Cyclin B1 axis

<italic>UHRF1</italic> knockdown induces cell cycle arrest and apoptosis in breast cancer cells through the ZBTB16/ANXA7/Cyclin B1 axis

ZNF146 regulates cell cycle progression via TFDP1 and DEPDC1B in ovarian cancer cells.

Aberration in cell cycle progression is one of the essential mechanisms underlying tumorigenesis, making regulators of cell cycle reasonable anti-cancer therapeutic targets. Here, we dissected the regulatory mechanism involving the novel axis ZNF146/TFDP1/DEPDC1B in the cell cycle in ovarian cancer. Ovarian cancer (OC) is the third most common kind of gynecological tumor, in addition to being the most lethal. Transcription factor Dp-1 (TFDP1) functions as a binding partner for E2F transcription factors, and its target genes include those involved in DNA synthesis, cell cycle, and apoptosis. However, the regulatory role of TFDP1 in OC remains incompletely understood. This study aimed to investigate the role and mechanism of TFDP1 in OC. TFDP1 was highly expressed in the ovarian epithelial tissues of OC patients, and the expression of TFDP1 in OC cells was higher than that in normal ovarian epithelial cells. Silencing of TFDP1 inhibited the biological activity of OC cells and hindered cell cycle entry. Zinc finger protein 146 (ZNF146) knockdown induced cell cycle arrest at the G0/G1 phase and tumor growth by blocking TFDP1 transcription, which was overturned by ectopic expression of TFDP1. TFDP1 stimulated DEP domain-containing protein 1B (DEPDC1B) expression through transcriptional activation. DEPDC1B increased the proportion of OC cells in the G2/M phase and potentiated tumor malignant progression in nude mice inhibited by sh-ZNF146. Taken together, these findings demonstrate that ZNF146 participates in TFDP1/DEPDC1B activation and plays a vital role in the cell cycle in OC.

AKR1C3 silencing inhibits autophagy-dependent glycolysis in thyroid cancer cells by inactivating ERK signaling.

Thyroid cancer is a highly differentiated and poorly malignant tumor. Interfering with glycolysis has become an effective means of controlling cancer progression and autophagy is negatively correlated with glycolysis. Aldo-keto reductase family 1 member C3 (AKR1C3) has been demonstrated to be highly expressed in thyroid cancer tissue and the higher AKR1C3 expression predicted the worse prognosis. We aimed to explore whether AKR1C3 could affect thyroid cancer progression by regulating autophagy-dependent glycolysis. AKR1C3 expression in thyroid cancer cells was detected by western blot. Then, AKR1C3 was knocked down by transfection with short hairpin RNA specific to AKR1C3 in the absence or presence of 3-methyladenine (3-MA) or PMA treatment. Cell cycle and apoptosis was detected by flow cytometry. Immunofluorescence staining was used to analyze LC3B expression. Extracellular acidification, glucose uptake and lactic acid secretion were detected. To evaluate the tumorigenicity of AKR1C3 insufficiency on thyroid cancer in vivo, TPC-1 cells with AKR1C3 knockdown were injected subcutaneously into nude mice. Then, cyclinD1 and Ki67 expression in tumorous tissues was measured by immunohistochemical analysis. Apoptosis was assessed by terminal-deoxynucleoitidyl transferase mediated nick end labeling staining. Additionally, the expression of proteins related to cell cycle, apoptosis, glycolysis, autophagy, and extracellular signal-regulated kinase (ERK) signaling in cells and tumor tissues was assessed by western blot. Highly expressed AKR1C3 was observed in thyroid cancer cells. AKR1C3 knockdown induced cell cycle arrest and apoptosis of TPC-1 cells. Besides, autophagy was activated and glycolysis was inhibited following AKR1C3 silencing, and 3-MA treatment restored the impacts of AKR1C3 silencing on glycolysis. The further experiments revealed that AKR1C3 insufficiency inhibited ERK signaling and PMA application reversed AKR1C3 silencing-induced autophagy in TPC-1 cells. The in vivo results suggested that AKR1C3 knockdown inhibited the development of subcutaneous TPC-1 tumors in nude mice and inactivated the ERK signaling. Collectively, AKR1C3 silencing inhibited autophagy-dependent glycolysis in thyroid cancer by inactivating ERK signaling.

The long non-coding RNA PVT1 promotes tumorigenesis of cutaneous squamous cell carcinoma via interaction with 4EBP1

The long non-coding RNA (lncRNA) plasmacytoma variant translocation 1 (PVT1) plays an oncogenic role in multiple cancers due to its high expression. However, the expression and associated regulatory mechanisms of PVT1 in cutaneous squamous cell carcinoma (cSCC) remain unclear. Our results revealed that PVT1 was highly upregulated in cSCC tissues and cSCC cell lines. To determine the functional role of PVT1 in cSCC, we constructed a stable knockdown cell model of PVT1 in the A431 and COLO16 cell lines using a lentiviral approach. Xenograft tumor experiments of nude mice in vivo, and colony formation, CCK-8, and EdU assays in vitro demonstrated that knockdown of PVT1 could widely suppress cell proliferation in vivo and in vitro. In addition, PVT1 knockdown induced cell cycle arrest and promoted apoptosis, as detected by flow cytometry analysis. Wound healing and transwell assays revealed that PVT1 knockdown significantly inhibited the migration and invasion of CSCC cell lines. To gain insight into the tumorigenic mechanism and explore the potential target molecules of PVT1, we employed label-free quantitative proteomic analysis. The GO, KEGG enrichment, and protein–protein interaction (PPI) networks suggested that 4E-binding protein 1 (4EBP1) is the possible downstream target effector of PVT1, which was validated by western blot analysis. PVT1 silencing markedly decreased 4EBP1 protein expression levels and directly bound 4EBP1 in the cytoplasm of cSCC cells. 4EBP1 overexpression counteracted the effects of PVT1 knockdown on tumorigenesis in cSCC cells, including cell proliferation, apoptosis, migration, and invasion. Our findings provide strong evidence that PVT1 is an oncogene which plays a role in tumorigenesis of cSCC, that PVT1 may interact with 4EBP1 in the cytoplasm as an underlying mechanism in cSCC carcinogenesis, and that PVT1 combined with 4EBP1 may serve as a potential new therapeutic target for cSCC.

NUCKS1 promotes the progression of colorectal cancer via activating PI3K/AKT/mTOR signaling pathway.

Nuclear ubiquitous casein and cyclin-dependent kinase substrate 1 (NUCKS1) is highly expressed in a variety of malignant tumors and functions as an oncogene; however, its role in colorectal cancer (CRC) remains unclear. We aimed to explore the function and regulatory mechanisms of NUCKS1 and potential therapeutic agents targeting NUCKS1 in CRC. We knocked down and overexpressed NUCKS1 in CRC cells and explored its effects in vitro and in vivo. Flow cytometry, CCK-8, Western blotting, colony formation, immunohistochemistry, in vivo tumorigenic, and transmission electron microscopy analyses were performed to determine the effects of NUCKS1 on CRC cell function. LY294002 was used to examine the mechanism of NUCKS1 expression in CRC cells. Potential therapeutic agents for NUCKS1-high CRC patients were analyzed using the CTRP and PRISM datasets, and the function of selected agents was determined by CCK-8 and Western blotting. We revealed that NUCKS1 was highly expressed in CRC tissues and clinically correlated with poor prognosis in CRC patients. NUCKS1 knockdown induces cell cycle arrest, inhibits CRC cell proliferation, and promotes apoptosis and autophagy. These results were reversed when NUCKS1 was overexpressed. Mechanistically, NUCKS1 exerts a cancer-promoting function by activating the PI3K/AKT/mTOR signaling pathway. This was reversed when LY294002 was used to inhibit the PI3K/AKT pathway. Furthermore, we determined that mitoxantrone exhibited high drug sensitivity in NUCKS1-overexpressing CRC cells. This work demonstrated NUCKS1 plays a crucial role in CRC progression via the PI3K/AKT/mTOR signaling pathway. Additionally, mitoxantrone may be a potential therapeutic agent for CRC treatment. Therefore, NUCKS1 represents a promising anti-tumor therapeutic target.

Phosphofructokinase Platelet Overexpression Accelerated Colorectal Cancer Cell Growth and Motility.

Background: Glycolysis is a glucose metabolism pathway that generates the high-energy compound adenosine triphosphate, which supports cancer cell growth. Phosphofructokinase platelet (PFKP) plays a crucial role in glycolysis regulation and is involved in human cancer progression. However, the biological function of PFKP remains unclear in colorectal cancer (CRC). Methods: We analyzed the expression levels of PFKF in colon cancer cells and clinical samples using real-time PCR and western blot techniques. To determine the clinical significance of PFKP expression in colorectal cancer (CRC), we analyzed public databases. In addition, we conducted in vitro assays to investigate the effects of PFKP on cell growth, cell cycle, and motility. Results: An analysis by the Cancer Genome Atlas database revealed that PFKP was significantly overexpressed in CRC. We examined the levels of PFKP mRNA and protein, revealing that PFKP expression was significantly increased in CRC. The results of the univariate Cox regression analysis showed that high PFKP expression was linked to worse disease-specific survival (DSS) and overall survival (OS) [DSS: crude hazard ratio (CHR) = 1.84, 95% confidence interval (CI): 1.01-3.36, p = 0.047; OS: CHR=1.91, 95% CI: 1.06-3.43, p = 0.031]. Multivariate Cox regression analysis revealed that high PFKP expression was an independent prognostic biomarker for the DSS and OS of patients with CRC (DSS: adjusted HR = 2.07, 95% CI: 1.13-3.79, p = 0.018; AHR = 2.34, 95% CI: 1.29-4.25, p = 0.005). PFKP knockdown reduced the proliferation, colony formation, and invasion of CRC cells. In addition, the knockdown induced cell cycle arrest at the G0/G1 phase by impairing cell cycle-related protein expression. Conclusion: Overexpression of PFKP contributes to the growth and invasion of CRC by regulating cell cycle progression. PFKP expression can serve as a valuable molecular biomarker for cancer prognosis and a potential therapeutic target for treating CRC.

Engulfment and Cell Motility 1 (ELMO1) Regulates Tumor Cell Behavior and Predicts Prognosis in Colorectal Cancer.

Engulfment and cell motility 1 (ELMO1) plays a crucial role in the process of migration, chemotaxis, and metastasis of tumor cells. ELMO1 has been implicated in the pathogenesis of various cancers. However, the distinct function of ELMO1 in colorectal cancer (CRC) is unclear. We determined whether ELMO1 affects the oncogenic behavior of CRC cells and investigated its prognostic value in CRC patients. We investigated the impact of ELMO1 on tumor cell behavior using small interference RNA (siRNA) in CRC cell lines, including SW480 and DLD1. The expression of ELMO1 was investigated by reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA) in cancer tissues and sera obtained from CRC patients. ELMO1 knockdown suppressed tumor cell proliferation in SW480 and DLD1 cells. ELMO1 knockdown-induced apoptosis through up-regulation of caspase-3, -7, and PARP activities and down-regulation of the anti-apoptotic Mcl-1 protein. ELMO1 knockdown-induced cell-cycle arrest by decreasing cyclin D1, cyclin-dependent kinase 2, 4 and 6, and the 25C cell division cycle (CDC25C). ELMO1 knockdown suppressed tumor cell invasion and migration. The expression of E-cadherin was increased, while that of Vimentin and Claudin 1 decreased following ELMO1 knockdown. The phosphorylation levels of PDK1, Akt, and GSK-3β and were down-regulated after ELMO1 knockdown. The expression of ELMO1 was found up-regulated in cancer tissues and sera taken from CRC patients. ELMO1 expression was significantly associated with tumor stage, lymph node metastasis, distant metastases, and poor survival. ELMO1 mediates tumor progression by increasing tumor cell motility and inhibiting apoptosis in human CRC.

NUF2 Expression Promotes Lung Adenocarcinoma Progression and Is Associated With Poor Prognosis.

Aberrant expression of the gene encoding the Ndc80 kinetochore complex component (NUF2) reportedly contributes to the progression of several human cancers. However, the functional roles of NUF2 and their underlying mechanisms in lung adenocarcinoma (LUAD) are largely unknown. The current study aimed to investigate the role of NUF2 in LUAD tumorigenesis. Here, TCGA, ONCOMINE, the Human Protein Atlas, UALCAN, and the results of our cohort were used to analyze the expression of NUF2 in LUAD. A Kaplan–Meier analysis and univariate and multivariate Cox regression analyses were performed to estimate the prognostic values of NUF2 expression in the Cancer Genome Atlas cohort. We studied the effects of NUF2 expression on proliferation, migration, invasion, and tumor growth using LUAD cell lines. Gene set enrichment analysis (GSEA) was used to analyze the pathways and biological function enrichment of NUF2 in LUAD. The ssGSEA database was used to analyze the relationship between NUF2 expression and immune cell infiltration in LUAD. Results revealed elevated expression of NUF2 in LUAD specimens. Patients overexpressing NUF2 had poor prognoses relative to those with low NUF2 expression. Knockdown of NUF2 suppressed the proliferation, migration, invasion, epithelial-mesenchymal transition, and colony formation of LUAD cells. Moreover, NUF2 knockdown induced cell cycle arrest at the G0/G1 phase. Gene Ontology and GSEA analyses suggested that NUF2 may be involved in immunity, proliferation, and apoptosis-related pathways. NUF2 overexpression was positively correlated with differential immune cell infiltration. In conclusion, NUF2 expression was associated with the clinical phenotype of LUAD and hence has potential implications in LUAD treatment.

Metformin inhibits gastric cancer cell proliferation by regulation of a novel Loc100506691-CHAC1 axis

Long noncoding RNAs (lncRNAs) are a group of nonprotein coding transcripts that play a critical role in cancer progression. However, the role of lncRNA in metformin-induced inhibition of cell growth and its biological function in gastric cancer remain largely unknown. In this study, we identified an oncogenic lncRNA, Loc100506691, the expression of which was decreased in gastric cancer cells with metformin treatment. Moreover, Loc100506691 was significantly overexpressed in gastric cancer compared with adjacent normal tissues (p < 0.001), and high Loc100506691 expression was significantly correlated with poor survival of patients with gastric cancer. Additionally, Loc100506691 knockdown could significantly suppress gastric cancer cell growth in vitro, and ectopic Loc100506691 expression accelerated tumor growth in an in vivo mouse model. Analysis of the cell cycle revealed that Loc100506691 knockdown induced cell cycle arrest at the G2/M phase by impairing cell entry from the G2/M to G1 phase. Loc100506691 negatively regulated CHAC1 expression by modulating miR-26a-5p/miR-330-5p expression, and CHAC1 knockdown markedly attenuated Loc100506691 knockdown-induced gastric cancer cell growth and motility suppression. We concluded that anti-proliferative effects of metformin in gastric cancer may be partially caused by suppression of the Loc100506691-miR-26a-5p/miR-330-5p-CHAC1 axis.

Comprehensive analysis of differentially expressed genes reveals the promotive effects of UBE2T on colorectal cancer cell proliferation.

Colorectal cancer (CRC) is one of the most common malignancies worldwide. Via analysis using The Cancer Genome Atlas database, the present study identified 1,835 genes that were differentially expressed in CRC, including 811 upregulated and 1,024 downregulated genes. Enrichment analyses using the Database for Annotation, Visualization and Integrated Discovery tool revealed that these differentially expressed genes were associated with the regulation of CRC progression by modulating multiple pathways, such as ‘Cell Cycle, Mitotic’, ‘DNA Replication’, ‘Mitotic M-M/G1 phases’ and ‘ATM pathway’. To identify the key genes in CRC, protein-protein interaction (PPI) network analysis was performed and the hub modules in upregulated and downregulated PPI networks were identified. Ubiquitin-conjugating enzyme E2 T (UBE2T), a member of the E2 family, was identified to be a key regulator in CRC. To the best of our knowledge, the present study was the first to demonstrate that UBE2T expression was upregulated in CRC samples compared with normal tissues. Kaplan-Meier analysis revealed that higher expression levels of UBE2T were associated with worse prognosis compared with lower UBE2T expression levels in CRC. Additionally, the present study demonstrated that knockdown of UBE2T inhibited CRC cell proliferation. Flow cytometry assays revealed that UBE2T knockdown induced cell cycle arrest at G1 phase and apoptosis in vitro. These results suggested that UBE2T may be a novel potential biomarker for CRC.

LCAT3, a novel m6A-regulated long non-coding RNA, plays an oncogenic role in lung cancer via binding with FUBP1 to activate c-MYC

BackgroundLong non-coding RNAs (lncRNAs) are important epigenetic regulators, which play critical roles in diverse physiological and pathological processes. However, the regulatory mechanism of lncRNAs in lung carcinogenesis remains elusive. Here, we characterized a novel oncogenic lncRNA, designated as Lung Cancer Associated Transcript 3 (LCAT3).MethodsWe predicted and validated LCAT3 by analyzing RNA-sequencing (RNA-seq) data of lung cancer tissues from TCGA. Methylated RNA immunoprecipitation was performed to assess m6A modification on LCAT3. The LCAT3-FUBP1-MYC axis was assessed by dual-luciferase reporter, RNA immunoprecipitation and Chromatin immunoprecipitation assays. Signaling pathways altered by LCAT3 knockdown were identified using RNA-seq. Furthermore, the mechanism of LCAT3 was investigated using loss-of-function and gain-of-function assays in vivo and in vitro.ResultsLCAT3 was found to be up-regulated in lung adenocarcinomas (LUAD), and its over-expression was associated with the poor prognosis of LUAD patients. LCAT3 upregulation is attributable to N6-methyladenosine (m6A) modification mediated by methyltransferase like 3 (METTL3), leading to LCAT3 stabilization. Biologically, loss-of-function assays revealed that LCAT3 knockdown significantly suppressed lung cancer cell proliferation, migration and invasion in vitro, and inhibited tumor growth and metastasis in vivo. LCAT3 knockdown induced cell cycle arrest at the G1 phase. Mechanistically, LCAT3 recruited Far Upstream Element Binding Protein 1 (FUBP1) to the MYC far-upstream element (FUSE) sequence, thereby activating MYC transcription to promote proliferation, survival, invasion and metastasis of lung cancer cells.ConclusionsTaken together, we identified and characterized LCAT3 as a novel oncogenic lncRNA in the lung, and validated the LCAT3-FUBP1-MYC axis as a potential therapeutic target for LUAD.

Hsa_circ_0074371 Regulates Proliferation, Apoptosis, Migration, and Invasion via the miR-582-3p/LRP6 Axis in Trophoblast Cells.

Fetal growth restriction (FGR) ranks second among causes of perinatal death. Circular RNA hsa_circ_0074371 (hsa_circ_0074371) is reported to be downregulated in FGR placentae. However, the role and regulatory mechanism of hsa_circ_0074371 in FGR pathogenesis are indistinct. Expression of hsa_circ_0074371, microRNA (miR)-582-3p, and low-density lipoprotein receptor-related protein 6 (LRP6) mRNA in FGR placentae and trophoblast cells (HTR-8/SVneo) was detected by quantitative real-time polymerase chain reaction (qRT-PCR). The relationship between hsa_circ_0074371 or LRP6 and miR-582-3p was verified by dual-luciferase reporter and/or RNA immunoprecipitation (RIP), RNA pull-down assays. The proliferation, cell cycle progression, apoptosis, migration, and invasion of HTR-8/SVneo cells were evaluated by Cell Counting Kit-8 (CCK-8), flow cytometry, wound healing, or transwell assays. Caspase3 activity was analyzed with a commercial kit. The protein levels of c-myc, cyclinD1, matrix metalloproteinase (MMP) 2, MMP9, and LRP6 were examined by western blotting. We observed that hsa_circ_0074371 and LRP6 were downregulated while miR-582-3p was upregulated in FGR placentae and HTR-8/SVneo cells. Hsa_circ_0074371 modulated LRP6 expression via sponging miR-582-3p. Hsa_circ_0074371 knockdown induced cell cycle arrest, apoptosis, and inhibited cell proliferation, migration, and invasion in HTR-8/SVneo cells. MiR-582-3p inhibitor reversed hsa_circ_0074371 silencing me on proliferation, migration, and invasion of HTR-8/SVneo cells. LRP6 overexpression overturned the inhibitory effect of miR-582-3p mimic on proliferation, migration, and invasion of HTR-8/SVneo cells. In conclusion, hsa_circ_0074371 downregulation inhibited proliferation, migration, and invasion of trophoblast cells via sponging miR-582-3p and decreasing LRP6 expression, providing a new mechanism related to FGR pathogenesis.

KIAA0101 knockdown inhibits cell proliferation and induces cell cycle arrest and cell apoptosis in chronic lymphocytic leukemia cells.

BackgroundChronic lymphocytic leukemia (CLL) is a heterogeneous disease with intense cytogenetic aberrations. Importantly, our recent report indicated that thyroid hormone receptor interactor 13 (TRIP13) is a potential new therapeutic target in CLL. In this study, we predicted 20 TRIP13-related genes and found that KIAA0101 is a novel gene that regulates cell proliferation and the cell cycle of CLL cells.MethodsCD19+ B cells were isolated from the peripheral blood of 26 CLL patients and 6 healthy donors through magnetic cell sorting. Cell proliferation was assessed by the CCK-8 assay. The mRNA and protein levels of genes were examined through RT-qPCR and western blot assays, respectively. Cell cycle and cell apoptosis were measured through Annexin V-based flow cytometry and the caspase 3/7 activity assay. Potential targets of KIAA0101 were identified through microarray analysis. 20 TRIP13 related genes was predicted by Ingenuity Pathway Analysis (IPA). KIAA0101-regulated functions and molecular pathways were predicted through IPA.ResultsKIAA0101 knockdown had the strongest inhibitory effect on CLL cell proliferation among the 20 TRIP13-related genes. KIAA0101 was highly expressed in CD19+ B cells of CLL patients. KIAA0101 knockdown induced cell cycle arrest and cell apoptosis, and inhibited FOXO1, MYD88, and TLR4 expression in CLL cells.ConclusionsTaken together, we demonstrated that KIAA0101 plays a critical role in cell proliferation and the cell cycle of human CLL cells. KIAA0101 knockdown induced cell apoptosis, and reduced FOXO1, MYD88, and TLR4 expression, and may therefore be used as a therapeutic target of CLL.

Semaphorin 4D is a potential biomarker in pediatric leukemia and promotes leukemogenesis by activating PI3K/AKT and ERK signaling pathways.

Semaphorin 4D (Sema4D) is highly expressed in a variety of tumors and is associated with high invasion, poor prognosis and poor therapeutic response. However, the expression and role of Sema4D in leukemia remains unclear. The present study investigated the expression of Sema4D in pediatric leukemia and its effects in leukemia cells. The results demonstrated that Sema4D protein was highly expressed in peripheral blood mononuclear cells of patients with pediatric leukemia, and high levels of soluble Sema4D were also observed in the plasma of these patients. Sema4D knockdown induced cell cycle arrest in G0/G1 phase, inhibited proliferation and promoted apoptosis in BALL-1 cells, while Sema4D overexpression exhibited the opposite effect. In Jurkat cells, Sema4D knockdown inhibited proliferation and promoted apoptosis, while Sema4D overexpression decreased the abundance of the cells in the G0/G1 phase of the cell cycle and promoted proliferation. Sema4D overexpression also increased the migratory capacity of Jurkat cells and the invasive capacity of BALL-1 cells. The phosphorylation level of PI3K was decreased in both Sema4D knocked-down Jurkat and BALL-1 cells, and the phosphorylation level of ERK was decreased in Sema4D knocked-down BALL-1 cells. The phosphorylation levels of PI3K, ERK and AKT were elevated in patients with pediatric leukemia, and were correlated to the increased Sema4D expression. Sema4D overexpression was associated with a shorter overall survival in patients with acute myeloid leukemia. Overall, the results of the present study indicated that Sema4D serves an important role in leukemia development by activating PI3K/AKT and ERK signaling, and it may be used as a potential target for the diagnosis and treatment of leukemia.

BORA regulates cell proliferation and migration in bladder cancer

BackgroundBladder cancer is having a gradually increasing incidence in China. Except for the traditional chemotherapy drugs, there are no emerging new drugs for almost 30 years in bladder cancer. New potential therapeutic targets and biomarkers are urgently needed.MethodsBORA is the activator of kinase Aurora A and plays an important role in cell cycle progression. To investigate the function of BORA in BCa, we established BORA knockdown and overexpression cell models for in vitro studies, xenograft and pulmonary metastasis mouse models for in vivo studies.ResultsOur results indicated that BORA was upregulated in human bladder cancer (BCa) compared to the normal bladder and paracancerous tissues at transcriptional and translational levels. We found that BORA was positively related to BCa cell proliferation. Furthermore, BORA knockdown induced cell cycle arrest in G2/M phase while BORA overexpression decreased the proportion of cells in G2/M, associated with PLK1–CDC25C–CDK1 alteration. Interestingly, we observed that knockdown of BORA inhibited BCa cell migration and invasion, accompanied with alterations of epithelial–mesenchymal transition (EMT) pathway related proteins. In vivo studies confirmed the inhibition effect of BORA knockdown on BCa cell growth and migration.ConclusionsOur study indicates that BORA regulates BCa cell cycle and growth, meanwhile influences cell motility by EMT, and could be a novel biomarker and potential therapeutic target in BCa.

Down-regulation of lncRNA BLACAT1 inhibits ovarian cancer progression by suppressing the Wnt/β-catenin signaling pathway via regulating miR-519d-3p.

Ovarian cancer has the highest mortality in gynecologic malignancies. LncRNA BLACAT1 serves crucial functions in various cancers, but its role in ovarian cancer has not been investigated. In this article, our team explored the role and the potential regulatory mechanism of BLACAT1 in ovarian cancer. Quantitative RT-PCR showed that BLACAT1 was aberrantly up-regulated in ovarian cancer tissues compared with normal tissues. In vitro, BLACAT1 knockdown induced cell cycle arrest and inhibited the proliferation, migration and invasion of ovarian cancer cells using flow cytometry, MTT and EdU assays, wound healing assay and transwell assay, respectively. Luciferase assay verified the binding relationship between microRNA-519d-3p and lncRNA BLACAT1, and BLACAT1 negatively regulated the expression of miR-519d-3p. We also found that miR-519d-3p overexpression could inhibit ovarian cancer cells proliferation, migration and invasion. Further, Western blot demonstrated that the expression of RPS15A and nuclear β-catenin expression was markedly reduced by BLACAT1 knockdown, and cytoplasmic β-catenin level was not obviously affected. In vivo, BLACAT1 knockdown inhibited the tumor growth, and immunohistochemistry showed that ki67 expression was decreased by BLACAT1 suppression. Inhibition of BLACAT1 was sufficient to down-regulate the expression of RPS15A and nuclear β-catenin but did not cause an obvious change in cytoplasmic β-catenin expression. Taken together, BLACAT1 knockdown inhibited the progression of ovarian cancer by suppressing the Wnt/β-catenin signaling pathway via regulating miR-519d-3p. Our work provided a proper understanding of the critical roles of BLACAT1 in ovarian cancer.

Abstract P3-02-15: Integrin a2 promotes stemness and lung metastasis in triple negative breast cancer

Abstract Accumulating evidence demonstrates that cancer plasticity and stem cell properties (stemness) drive both tumorigenesis and metastasis, and how to eliminate metastasis remains one of the biggest challenges in the clinic. Here we report that the integrin receptor CD49b-encoding ITGA2 is an oncogenic target of miR-206 regulating cancer stemness and metastasis through pathways linked to lipid metabolism and cell cycle regulation in metastatic triple negative breast cancer (TNBC). ITGA2 knockdown suppressed self-renewal related mammosphere formation and pluripotency marker expression, inhibited cell cycling, compromised migration and invasion, and therefore decreased lung metastasis of TNBC. RNA sequencing analyses revealed that ITGA2 knockdown inhibits newly identified pathways such as lipid metabolism and protein kinase activity. ACLY-encoded ATP citrate lyase is one of the top targets in CD49b promoting lipid biosynthesis and self-renewal of breast cancer cells. Overexpression of CCND1 rescues the phenotype of ITGA2 knockdown-induced cell cycle arrest. High expression levels of the ITGA2/ACLY/CCND1 axis are associated with an unfavorable relapse-free survival of patients with high grade breast cancer, in both basal-like and other subtypes. This study identifies ITGA2 as a potential therapeutic target of TNBC stemness, metabolism, and metastasis. Citation Format: Valery Adorno-Cruz, Andrew Hoffmann, Xia Liu, Ruth Keri, Huiping Liu. Integrin a2 promotes stemness and lung metastasis in triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-02-15.

ITGA2 promotes expression of ACLY and CCND1 in enhancing breast cancer stemness and metastasis

Cancer metastasis is largely incurable and accounts for 90% of breast cancer deaths, especially for the aggressive basal-like or triple negative breast cancer (TNBC). Combining patient database analyses and functional studies, we examined the association of integrin family members with clinical outcomes as well as their connection with previously identified microRNA regulators of metastasis, such as miR-206 that inhibits stemness and metastasis of TNBC. Here we report that the integrin receptor CD49b-encoding ITGA2, a direct target of miR-206, promotes breast cancer stemness and metastasis. ITGA2 knockdown suppressed self-renewal related mammosphere formation and pluripotency marker expression, inhibited cell cycling, compromised migration and invasion, and therefore decreased lung metastasis of breast cancer. ITGA2 overexpression reversed miR-206-caused cell cycle arrest in G1. RNA sequencing analyses revealed that ITGA2 knockdown inhibits genes related to cell cycle regulation and lipid metabolism, including CCND1 and ACLY as representative targets, respectively. Knockdown of CCND1 or ACLY inhibits mammosphere formation of breast cancer cells. Overexpression of CCND1 rescues the phenotype of ITGA2 knockdown-induced cell cycle arrest. ACLY-encoded ATP citrate lyase is essential to maintain cellular acetyl-CoA levels. CCND1 knockdown further mimics ITGA2 knockdown in abolishing lung colonization of breast cancer cells. We identified that the low levels of miR-206 as well as high expression levels of ITGA2, ACLY and CCND1 are associated with an unfavorable relapse-free survival of the patients with estrogen receptor-negative or high grade breast cancer, especially basal-like or TNBC, possibly serving as potential biomarkers of cancer stemness and therapeutic targets of breast cancer metastasis.

Deficiency of PTEN leads to aberrant chromosome segregation through downregulation of MAD2

Proper spindle formation and accurate chromosome segregation are essential for ensuring mitotic fidelity. Phosphatase and tensin homolog (PTEN) is a multifunctional protein, which is able to maintain the stability of the genome and chromosomes. The present study described an essential role of PTEN in regulating chromosome segregation to prevent gross genomic instability via regulation of mitotic arrest deficient 2 (MAD2). PTEN knockdown induced cell cycle arrest and abnormal chromosome segregation, which manifested as the formation of anaphase bridges, lagging chromosomes and premature chromatid separation. In addition, MAD2 was identified as a potential target of PTEN. Furthermore, the present study revealed that PTEN knockdown resulted in MAD2 degradation via the ubiquitin-proteasomal pathway, while restoration of MAD2 expression partially ameliorated the mitotic defects induced by PTEN loss. The results from the present study proposed a novel mechanism by which PTEN maintains chromosome stability.

Knockdown of REG Iα Enhances the Sensitivity to 5-Fluorouracil of Colorectal Cancer Cells via Cyclin D1/CDK4 Pathway and BAX/BCL-2 Pathways.

Objective: The reverse of chemoresistance and the improvement of sensitivity to chemotherapeutic agents of colorectal cancer cells have great clinical significance and the mechanism underlying the drug resistance is still unclear. REG Iα was reported to be upregulated in colorectal cancer tissues, but the roles of chemoresistance are still unclear. Materials and Methods: The expression of REG Iα in colorectal cancer cell lines was assessed by quantitative real-time polymerase chain reaction (Q-PCR). The expression of REG Iα in HCT116 and LOVO cells was knockdown by siRNA. The cell viability and IC50 (half maximal inhibitory concentration) values were analyzed by the CCK8 assay. The proportion of apoptosis and cell cycles were analyzed by flow cytometry. The migration potency of HCT116 and LOVO cells was analyzed by cell migration assay. The protein level of Cyclin D1, CDK4 (cyclin-dependent kinase 4), Bax and Bcl-2 were analyzed by western blot. Results: Knockdown of REG Iα enhances the sensitivity to 5-Fu of colorectal cancer cells. REG Iα knockdown promoted the cell apoptosis of HCT116 and LOVO under the 5-Fu treatment. The cell migration and cycle of colorectal cancer cells was also inhibited by REG Iα knockdown. We also found that REG Iα knockdown induced cell cycle arrest and cell apoptosis by Cyclin D1/CDK4 pathway and BAX/BCL-2 pathways. Conclusions: Knockdown of REG Iα enhances the sensitivity to 5-Fu of colorectal cancer cells via cyclin D1/CDK4 pathway and BAX/BCL-2 pathways.