Numerous Human Tumors Research Articles

ZNF460-mediated upregulation of APCDD1L-DT promotes cholangiocarcinoma development by inhibiting the ubiquitin-mediated degradation of DVL2.

Cholangiocarcinoma (CCA), known for its aggressive nature, poses a formidable challenge in the current medical landscape, particularly in targeted therapies. Against this backdrop, long non-coding RNAs (lncRNAs) have captured the attention of researchers. These unique RNAs are believed to play pivotal roles in various cancers, offering promising avenues for the development of more effective treatment strategies. Previous studies have substantiated the aberrant expression of the APCDD1L-DT in numerous human tumors, demonstrating its positive regulatory roles in disease progression. Nevertheless, the biological functions of APCDD1L-DT in CCA are still not fully understood. This study marks the inaugural documentation of APCDD1L-DT exhibiting aberrant expression in CCA specimen, establishing a close correlation with the TNM staging of tumor patients. Furthermore, suppressing APCDD1L-DT expression hinders both the viability and motility of tumor cells. Mechanistically, the abnormal activation of the transcription factor ZNF460 positively regulated APCDD1L-DT expression in CCA. This activation, in turn, propels the abnormal activation of the Wnt pathway, fostering tumor development by impeding the ubiquitin-mediated degradation of DVL2. Broadly speaking, this study provides auspicious perspectives for comprehending CCA and furnishes support for addressing this daunting malignancy.

Doublecortin-likekinase3(DCLK3)isassociatedwithbadclinicaloutcomeofpatientswithgastriccancerandregulatestheferroptosisandmitochondriafunctioninvitroandinvivo.

Doublecortin-like kinase 3 (DCLK3), a member of tubulin superfamily, has been proved to be closely associated with the pathogenesis of numerous human tumors. However, the expression pattern and regulatory mechanisms of DCLK3 in gastric cancer (GC) remain unknown. DCLK3 expression in GC cells was assessed by RT-qPCR and western blotting. The correlation between DCLK3 levels and the overall survival of GC patients was assessed via TCGA, ACLBI, and Kaplan-Meier plotter databases. Additionally, key proteins (TCF4) involved in the regulation of DCLK3 on GC progression were screened by ACLBI database. Cell proliferation, ferroptotic cell death, and oxidative stress markers were measured by EdU staining, immunofluorescence, ELISA, and western blotting assays. DCLK3 was upregulated in GC, and high DCLK3 expression was significantly associated with poor survival of GC patients. Here, DCLK3 knockdown reduced GC cell proliferation, induced ferroptotic cell death, and exacerbated oxidative stress level. Logistic regression analysis showed that TCF4 was an independent prognostic indicator of GC. Mechanistically, DCLK3 promoted TCF4 expression and subsequently upregulated the expression of TCF4 downstream target genes (c-Myc and Cyclin D1). Furthermore, DCLK3 overexpression enhanced GC cell proliferation, but mitigating ferroptotic cell death and oxidative stress. The regulatory mechanism may involve the upregulation of TCF4, c-Myc, and cyclin D1. Our research suggests that DCLK3 modulates the levels of iron and reactive oxygen and may involve regulation of TCF4 pathway, thereby promoting the GC cell growth, indicating that DCLK3 may use as a prognostic marker and therapeutic target for GC patients.

N6-Methyladenosine Modification of CIRCKRT17 Initiated by METTL3 Promotes Osimertinib Resistance of Lung Adenocarcinoma by EIF4A3 to Enhance YAP1 Stability.

Circular RNAs (circRNAs) play a key role in regulating the drug resistance of numerous human tumors. However, whether circKRT17 involves in the osimertinib resistance of lung adenocarcinoma (LUAD) remains undetermined. Relative mRNA/circRNA and protein levels were detected by qRT-PCR and western blotting. Localization of circKRT17 and YAP1 was determined by FISH and immunofluorescence staining. Cell growth and apoptosis were evaluated using colony formation, EdU assays, and flow cytometry. The N6-methyladenosine (m6A) modification was analyzed by MeRIP. The interplay between EIF4A3 and circKRT17 or YAP1 was verified by RNA pull-down or/and RIP assays. Subcutaneous tumor growth was monitored in nude mice, and Ki-67 and TUNEL staining were carried out to evaluate cell proliferation and apoptosis, respectively. CircKRT17 and METTL3 were elevated in osimertinib-insensitive LUAD tissues and cells. Knockdown of circKRT 17 and METTL3 increased the sensitivity of LUAD cells to osimertinib. Knockdown of METTL3 decreased the expression of circKRT17 by inhibiting m6A modification. CircKRT17 promoted the stability and nuclear transportation of YAP1 by recruiting EIF4A3 in LUAD cells. Overexpression of YAP1 abolished the impacts of circKRT17 knockdown on the osimertinib sensitivity of LUAD cells. CircKRT17 knockdown increased the repressive effects of osimertinib on tumor growth in vivo by inhibiting YAP1 signaling. METTL3 initiated the m6A modification of circKRT17, thus promoting osimertinib resistance of LUAD by enhancing YAP1 stability through EIF4A4 recruitment.

A pan-tumor-siRNA aptamer chimera to block nonsense-mediated mRNA decay inflames and suppresses tumor progression

Immune-checkpoint blockade (ICB) therapy has changed the clinical outcome of many types of aggressive tumors, but there still remain many cancer patients that do not respond to these treatments. There is an unmet need to develop a feasible clinical therapeutic platform to increase the rate of response to ICB. Here we use a previously described clinically tested aptamer (AS1411) conjugated with SMG1 RNAi (AS1411-SMG1 aptamer-linked siRNA chimeras [AsiCs]) to inhibit the nonsense-mediated RNA decay pathway inducing tumor inflammation and improving response to ICB. The aptamer AS1411 shows binding to numerous mouse and human tumor cell lines tested. AS1411 induces tumor cytotoxicity in long incubation times, which allows for the use of the aptamer as a carrier to target the RNAi inhibition to the tumor. The AS1411-SMG1 AsiCs induce a strong antitumor response in local and systemic treatment in different types of tumors. Finally, AS1411-SMG1 AsiCs are well tolerated with no detected side effects.

Abstract 3567: Comprehensive preclinical studies of BT-001: An oncolytic vaccinia virus armed with Treg-depleting @CTLA4 and GM-CSF

Abstract Background Immune checkpoint blockade (ICB) is a clinically proven concept to treat cancer. Still, a majority of cancer patients including those with poorly immune infiltrated “cold” tumors are resistant to currently available ICB therapies. Oncolytic viruses are natural immunomodulators able to turn “cold” tumors “hot” and, for some of them, to deliver therapeutic payloads intratumorally. Payloads of choice are those with tolerability and/or pharmacokinetic/biodistribution issues, such as @CTLA4 antibodies and GM-CSF respectively. Here, Transgene and BioInvent present a preclinical characterization of a highly efficacious and potentially safe strategy to target CTLA-4 and GM-CSF using vectorization in an oncolytic vaccinia virus. Methods A novel human IgG1 CTLA-4 antibody (4-E03) was identified using function-first screening for mAbs and targets associated with checkpoint blocking and superior Treg depleting activity. A tumor-selective oncolytic Vaccinia vector was then engineered to encode this novel αCTLA-4 as full-length antibody (i.e. distinct transgenes for light and heavy chains) and GM-CSF (VVGM-αhCTLA4, BT-001). Viruses encoding matching mouse surrogate payloads were additionally generated, enabling proof-of-concept studies in syngeneic immune competent mouse tumor models. Results Our studies demonstrate that two chains cloning approach allowed to express @CTLA4 at a high level in numerous infected human tumor cell lines without affecting replication, oncolytic activity or genetic stability of the virus. Intratumoral (i.t.) administration of VVGM-αCTLA4 achieved tumor-restricted CTLA-4 receptor saturation at least for 10 days, Treg-depletion antigen cross-presentation, stronger systemic expansion of tumor-specific CD8+ T cells and antitumor immunity compared with systemic αCTLA-4 antibody or unarmed virus therapies. Remarkably, in a clinically relevant mouse model resistant to systemic immune checkpoint blockade, i.t. VVGM-αCTLA4 synergized with αPD-1 to reject “cold” tumors. Conclusion Our findings demonstrate in vivo proof-of-concept for tumor delivery by Vaccinia virus of GM-CSF and strongly Treg-depleting, immune checkpoint blocking, vectorized αCTLA-4. This vector-based delivery is a highly effective and safe strategy to target CTLA-4 which overcomes current limitations of approved αCTLA-4 regimens. A clinical trial evaluating i.t. VVGM-αhCTLA4 (BT-001) alone and in combination with αPD-1 in metastatic or advanced solid tumors has been initiated. Citation Format: Jean-Baptiste Marchand, Monika Semmrich, Christelle Ziller, Matilda Rehn, Laetitia Fend, Petra Holmkvist, Nathalie Silvestre, Carolin Svensson, Kleinpeter Patricia, Jules Deforges, Fred Junghus, Linda Mårtensson, Johann Foloppe, Ingrid Teige, Björn Frendéus, Eric Quéméneur. Comprehensive preclinical studies of BT-001: An oncolytic vaccinia virus armed with Treg-depleting @CTLA4 and GM-CSF [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3567.

The Anti-Tumoral Potential of Phosphonate Analog of Sulforaphane in Zebrafish Xenograft Model.

Isothiocyanates (ITCs) show strong activity against numerous human tumors. Five structurally diverse ITCs were tested in vivo using the zebrafish embryos 6 and 48 h post-fertilization (hpf). The survival rate, hatching time, and gross morphological changes were assessed 24, 48, and 72 h after treatment with all compounds in various doses (1–10 µM). As a result, we selected a phosphonate analog of sulforaphane (P-ITC; 1–3 µM) as a non-toxic treatment for zebrafish embryos, both 6 and 48 hpf. Furthermore, the in vivo anti-cancerogenic studies with selected 3 µM P-ITC were performed using a set of cell lines derived from the brain (U87), cervical (HeLa), and breast (MDA-MB-231) tumors. For the experiment, cells were labeled using red fluorescence dye Dil (1,1′-Dioctadecyl-3,3,3′,3′-Tetramethylindocarbocyanine, 10 μg/mL) and injected into the hindbrain ventricle, yolk sac region and Cuvier duct of zebrafish embryos. The tumor size measurement after 48 h of treatment demonstrated the significant inhibition of cancer cell growth in all tested cases by P-ITC compared to the non-treated controls. Our studies provided evidence for P-ITC anti-cancerogenic properties with versatile activity against different cancer types. Additionally, P-ITC demonstrated the safety of use in the living organism at various stages of embryogenesis.

Abstract 1716: WTX-330, a conditionally activated IL-12 INDUKINE, releases IL-12 selectively in the tumor microenvironment to activate anti-tumor immune responses and induce regressions in mouse tumor models

Abstract Systemic therapy with proinflammatory immune modulators is a promising approach to treat cancer by activating innate and adaptive anti-tumor immunity. However, dose-limiting toxicities such as inflammation, cytokine release syndrome, and tissue damage rendered the strategy impractical for cytokines such as the cytotoxic T and NK cell activating interleukin 12 (IL-12). WTX-330 is a novel systemically delivered therapy with targeted intra-tumoral activation to deliver wild-type IL-12 in the tumor microenvironment identified using the Predator™ discovery platform. WTX-330 is a member of a class of inducible polypeptides (INDUKINE™ therapies) designed to be an inactive IL-12 pro-drug with a half-life extension (HLE) domain to support infrequent systemic administration. The INDUKINE™ polypeptide is kept inactive in the periphery via high affinity antibody blockade tethered to IL-12 via a tumor protease-sensitive linker. Cleavage of the same linker also removes the HLE domain producing a PK profile similar to wild-type IL-12. WTX-330 was selected as a lead molecule due to its beneficial in vitro profile. The mouse MC38 syngeneic tumor model was used to demonstrate strong anti-tumor activity of lead mouse INDUKINE™ polypeptides. Intraperitoneal (i.p.) administration of mouse WTX-330 led to complete regression of all MC38 tumors at dose levels which only moderately induced systemic IFNγ and were well tolerated by the mice. Similar efficacy was observed in CT26, B16F10, and EMT-6 syngeneic models. Importantly, wild-type IL-12, while active in mouse tumor models, was not tolerated by the mice after i.p. injection at equimolar amounts compared to the IL-12 INDUKINE™ polypeptide. Mouse WTX-330 strongly activated NK and CD8+ cell responses and induced APC and effector cell markers in MC38 tumors supporting a mechanism of action as described for wild-type IL-12. In non-human primates, WTX-330 was well tolerated at exposure levels which vastly exceeded the levels needed for efficacy in mice. In addition, plasma levels of free IL-12 after dosing with WTX-330 were very low compared to tolerated levels after treatment with wild-type IL-12. Ex vivo incubation of WTX-330 with numerous primary human tumors led to the rapid release of IL-12, while WTX-330 proved to be stable in human serum and normal tissues. Pharmacological and translational data obtained so far clearly support continued preclinical development with the goal of moving this innovative and differentiated engineered IL-12 therapy into human clinical testing. Citation Format: Philipp Steiner, Heather Brodkin, Dan Hicklin, Nesreen Ismail, Kristin Morris, Christopher Nirschl, Andres Salmeron, Cindy Seidel-Dugan, Zoe Steuert, Jenna Sullivan, William Winston. WTX-330, a conditionally activated IL-12 INDUKINE, releases IL-12 selectively in the tumor microenvironment to activate anti-tumor immune responses and induce regressions in mouse tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1716.

New Insights into YES-Associated Protein Signaling Pathways in Hematological Malignancies: Diagnostic and Therapeutic Challenges.

Simple SummaryYES-associated protein (YAP) is a co-transcriptional activator that binds to transcriptional factors to increase the rate of transcription of a set of genes, and it can intervene in the onset and progression of different tumors. Most of the data in the literature refer to the effects of the YAP system in solid neoplasms. In this review, we analyze the possibility that YAP can also intervene in hematological neoplasms such as lymphomas, multiple myeloma, and acute and chronic leukemias, modifying the phenomena of cell proliferation and cell death. The possibilities of pharmacological intervention related to the YAP system in an attempt to use its modulation therapeutically are also discussed.The Hippo/YES-associated protein (YAP) signaling pathway is a cell survival and proliferation-control system with its main activity that of regulating cell growth and organ volume. YAP operates as a transcriptional coactivator in regulating the onset, progression, and treatment response in numerous human tumors. Moreover, there is evidence suggesting the involvement of YAP in the control of the hematopoietic system, in physiological conditions rather than in hematological diseases. Nevertheless, several reports have proposed that the effects of YAP in tumor cells are cell-dependent and cell-type-determined, even if YAP usually interrelates with extracellular signaling to stimulate the onset and progression of tumors. In the present review, we report the most recent findings in the literature on the relationship between the YAP system and hematological neoplasms. Moreover, we evaluate the possible therapeutic use of the modulation of the YAP system in the treatment of malignancies. Given the effects of the YAP system in immunosurveillance, tumorigenesis, and chemoresistance, further studies on interactions between the YAP system and hematological malignancies will offer very relevant information for the targeting of these diseases employing YAP modifiers alone or in combination with chemotherapy drugs.

Platelet‑derived growth factor‑BB mediates pancreatic cancer malignancy via regulation of the Hippo/Yes‑associated protein signaling pathway.

Platelet-derived growth factor (PDGF) is a potent mitogen and chemoattractant that serves a role in the development of several types of solid cancer, and abnormal PDGF activity has been reported in numerous human tumors. Tumor-derived PDGF ligands are considered to act in either a paracrine or autocrine manner, serving roles in the phosphorylation of receptors on tumor and stromal cells in the tumor microenvironment. Despite the well-established association between PDGF and tumor progression, the precise mechanisms of autocrine PDGF signaling in pancreatic tumor cells remain elusive. Therefore, the present study aimed to analyze the influence of PDGF-BB in pancreatic cancer. Pancreatic adenocarcinoma BxPC-3 cells were cultured and treated with recombinant human PDGF-BB in vitro. Cell proliferation was tested using an MTT assay. Cell apoptosis was measured using flow cytometry. Tumor cell migration and invasion were examined via wound-healing and Transwell assays, respectively. The expression and subcellular localization of Yes-associated protein (YAP) was determined using western blotting and immunofluorescence. The transcriptional activity of target genes was tested using a luciferase assay and reverse transcription-quantitative PCR. The present study revealed that PDGF-BB significantly promoted cell proliferation in pancreatic adenocarcinoma BxPC-3 cells and enhanced the aggressiveness of this cell line, as demonstrated by Transwell and wound-healing assays. Anoikis resistance is an important mechanism by which metastatic cells avoid apoptosis when detaching from adjacent cells or the extracellular matrix. PDGF-BB treatment inhibited anoikis under anchorage-independent conditions. Mechanistic experiments revealed that PDGF-BB promoted the upregulation and activation of the transcriptional coactivator YAP, an effector of the Hippo signaling pathway. RhoA or protein phosphatase-1 (PP-1) inhibition partially abolished the accumulation and activation of YAP, suggesting PDGF-BB-mediated YAP dephosphorylation and transactivation via the RhoA/PP-1 cascade. Pharmacologic inhibition of the PDGF receptor directly downregulated YAP activity and the expression levels of downstream genes. Furthermore, verteporfin, a small molecular inhibitor of the Hippo/YAP signaling pathway, partially reversed the effects of PDGF-BB on cell proliferation, anoikis resistance and cell migration. In conclusion, the present study revealed that the Hippo/YAP signaling pathway may be involved in the tumor-promoting activity of PDGF-BB in pancreatic cancer.

MicroRNA-3666 Suppresses Cell Growth in Head and Neck Squamous Cell Carcinoma Through Inhibition of PFKFB3-Mediated Warburg Effect.

PurposeMicroRNA-3666 (miR-3666) is aberrantly expressed and plays critical roles in numerous human tumors. However, the expression pattern, biological role, and mechanisms of action of miR-3666 in head and neck squamous cell carcinoma (HNSCC) remain unknown. Therefore, we attempted to determine the expression status and function of miR-3666 in HNSCC and to explore the underlying mechanisms in detail.MethodsIn this study, quantitative real-time polymerase chain reaction was carried out to measure the expression of miR-3666 HNSCC tissues. A series of experiments, including a Cell Counting Kit-8 assay, colony formation assay, BrdU incorporation and apoptosis analysis, were applied to test whether miR-3666 affects the growth of HNSCC cells. Glucose uptake and lactate production measurements and extracellular acidification and oxygen consumption rate assays were conducted to determine the effect of miR-3666 on glycolysis.ResultsWe found that miR-3666 showed a decreased expression in HNSCC tissues. Further functional studies demonstrated that miR-3666 inhibited the growth of HNSCC cells by suppressing cell proliferation and promoting apoptosis. Bioinformatics analysis and luciferase reporter assays identified phosphofructokinase-2/fructose-2,6-bisphosphatase 3 (PFKFB3), a key enzyme regulating glycolysis, as a direct target of miR-3666. Through inhibition of PFKFB3, miR-3666 decreased glycolysis in HNSCC cells by reducing the production of F2,6BP. Importantly, glycolysis suppression caused by miR-3666 was found to be required for its inhibitory effect on HNSCC cell growth.ConclusionOur data suggest that miR-3666 functions as a tumor suppressor by decreasing the rate of glycolysis through inhibition of PFKFB3 activity, and this miRNA may present a potential candidate for HNSCC therapy.

Linalool inhibits the growth of human T cell acute lymphoblastic leukemia cells with involvement of the MAPK signaling pathway.

Linalool can inhibit the malignant proliferation of numerous human malignant solid tumors, including hepatocellular carcinoma, breast cancer, small cell carcinoma and malignant melanoma. However, the role of linalool in T cell acute lymphoblastic leukaemia (T-ALL) remains unclear. In the present study, human T-ALL cell lines (Jurkat, H9, Molt-4 and Raji cells) and peripheral blood mononuclear cells (PBMCs) from healthy donors were treated with various concentrations of linalool (3.75, 7.50, 15.00, 30.00, 60.00 and 120.00 µM, respectively). A CCK-8 assay was used to analyse cell viability and it demonstrated that linalool inhibited the growth of T-ALL cells in a dose-dependent manner, but did not significantly affect normal PBMCs. Flow cytometry was used to detect the cell cycle and apoptosis and demonstrated that linalool reduced the percentage of T-ALL cells at the G0/G1 phase, and induced the apoptosis of T-ALL cells. RNA sequencing was conducted on an Illumina HiSeq X Series 2500 before and after treatment with linalool followed by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis. It was demonstrated that the mitogen-activated protein kinase (MAPK) pathway was involved in the effect of linalool on T-ALL cells. Real-time quantitative PCR and western blotting were performed to verify the mRNA and protein levels, respectively of the genes in the signaling pathway identified. In addition, it was found that linalool significantly inhibited phosphorylated (p)-ERK1/2 protein expression and enhanced p-JNK protein expression of T-ALL cells. In conclusion, the present study revealed that linalool inhibits T-ALL cell survival with involvement of the MAPK signaling pathway. JNK activation and ERK inhibition may play a functional role in apoptosis induction of T-ALL cells. Linalool may be developed as a novel anti T-ALL agent.

Overexpression of microRNA-130a predicts adverse prognosis of primary gastrointestinal diffuse large B-cell lymphoma.

Primary gastrointestinal diffuse large B-cell lymphoma (PGI-DLBCL) is a highly heterogeneous type of non-Hodgkin lymphoma. A number of studies have demonstrated that microRNA-130a (miR-130a) serves a role in the tumorigenesis and prognosis of numerous human tumors. However, to the best of our knowledge, the prognostic significance of miR-130a in PGI-DLBCL remains unknown. The present study explored the association between miR-130a and the clinical outcomes of PGI-DLBCL. Relative miR-130a expression was assessed by reverse transcription-quantitative PCR. Immunohistochemistry was used to detect expression levels of BCL-2, c-MYC, neprilysin, B-cell lymphoma 6 protein, PWWP domain-containing DNA repair factor 3A and proliferation marker protein Ki-67. A receiver operating characteristic curve was constructed to analyze the specificity and sensitivity of microRNA levels in the diagnosis of PGI-DLBCL. Survival curves were constructed using the Kaplan-Meier method. In the present study, miR-130a expression was notably higher in patients with PGI-DLBCL compared with in the controls (P<0.0001). miR-130a overexpression was closely associated with a high International Prognostic Index score (3–5) and drug resistance (P=0.017 and P=0.044, respectively). No significant difference in other clinical features was observed. Patients with increased expression levels of miR-130a had lower overall survival [hazard ratio (HR), 2.998; 95% CI, 1.347-6.673; P=0.007] and progression-free survival (HR, 3.325; 95% CI, 1.488-7.429; P=0.003) compared with patients who had lower expression levels of miR-130a. Furthermore, multivariate Cox regression analysis suggested that miR-130a was a negative prognostic parameter in PGI-DLBCL. Therefore, upregulation of miR-130a could become a potential prognostic marker for PGI-DLBCL. Additionally, further study of these results may have important guiding significance for the prognosis of patients with PGI-DLBCL in the clinical setting.

Ginsenoside CK induces apoptosis and suppresses proliferation and invasion of human osteosarcoma cells through the PI3K/mTOR/p70S6K1 pathway

Osteosarcoma is one of the most malignant bone tumors, and its major threats are aggressive invasion and early tumor metastasis, which result in a poor prognosis and high mortality. Accumulating evidence indicates that ginsenoside compound K (CK) has a significant antitumor effect, particularly on the inhibition of proliferation and invasion of numerous human tumors. In the present study, it was revealed that CK inhibited the viability and proliferation of osteosarcoma cells. Moreover, it was demonstrated that CK induced apoptosis and inhibited the migration and invasion of osteosarcoma cells via apoptotic staining, Annexin V/PI staining, and Transwell invasion assays. Furthermore, at the molecular level, the present results confirmed that apoptosis and invasion-related proteins were regulated by CK, which was possibly related to the blockade of the PI3K/mTOR/p70S6K1 signaling pathway. In summary, the present findings indicated that CK inhibited viability and proliferation, induced apoptosis, and inhibited the migration and invasion of osteosarcoma cells through the PI3K/mTOR/p70S6K1 signaling pathway.

ELK1-activated GPC3-AS1/GPC3 axis promotes the proliferation and migration of cervical cancer cells.

Long non-coding RNA (lncRNA) is reported to be involved in multiple biological processes in numerous human tumors. Furthermore, an increasing number of studies have confirmed the involvement of lncRNA in the initiation and development of human cancers, including cervical cancer (CC). The present study explored the potential role of lncRNA glypican 3 antisense transcript 1 (GPC3-AS1) with respect to regulating CC cell proliferation and migration. A quantitative reverse transcriptase-polymerase chain reaction confirmed that GPC3-AS1 was up-regulated in CC cells compared to normal CRL-2614 cells. Loss-of-function assays demonstrated the negative effect of GPC3-AS1 depletion on CC cell proliferation and migration. GPC3-AS1 positively regulated its nearby gene glypican 3 (GPC3). Significant up-regulation of GPC3 was also observed in CC cells, consistently with GPC3-AS1. In addition, GPC3-AS1 and GPC3 synergistically promoted the proliferative and migratory abilities of CC cells. Mechanistic investigation showed that ELK1 acted as the transcription activator of GPC3-AS1 and GPC3, thus contributing to their up-regulation in CC cells. Rescue assays indicated that the ELK1-induced GPC3-AS1/GPC3 axis promoted cell proliferation and migration in CC. The results of the present study have revealed a novel molecular pathway that can regulate CC cell proliferation and migration, thus providing a new basis for investigating the molecular mechanism associated with CC progression.

Overexpression of SMYD3 in Ovarian Cancer is Associated with Ovarian Cancer Proliferation and Apoptosis via Methylating H3K4 and H4K20.

Background: Epigenetic regulation has been verified as a key mechanism in tumorigenesis. SET and MYND domain-containing protein 3 (SMYD3), a histone methyltransferase, is a promising epigenetic therapeutic target and is overexpressed in numerous human tumors. SMYD3 can promote oncogenic progression by methylating lysines to integrate cytoplasmic kinase signaling cascades or by methylating histone lysines to regulate specific gene transcription. However, the exact role of SMYD3 in the progression of ovarian cancer is still unknown.Methods: Immunohistochemistry was employed to test SMYD3 expression in ovarian cancer tissues from clinical patients. CCK-8 assay, Real-time cell analysis (RTCA), colony formation assay, cell cycle and apoptosis tested by Flow cytometer were employed to test the effects of SMYD3 on cell proliferation and apoptosis in ovarian cancer cell lines. A PCR array was used to identify the downstream targets of SMYD3. And, PCR and Western blot were used to verify their expression. The binding of SMYD3 on the promoter of target genes were tested by ChIP assays. We also use nude mice subcutaneous tumor model and patient-derived xenograft (PDX) model to investigate the tumor promotive function of SMYD3 in vivo.Results: SMYD3 expression was higher in ovarian cancer tissues and cell lines than in normal ovarian epithelial tissue and human ovarian surface epithelial cells (HOSEpiC). After silencing SMYD3, the proliferation of ovarian cancer cells was significantly inhibited in vitro. In addition, the SMYD3-specific small-molecule inhibitor BCI-121 suppressed ovarian cancer cell proliferation. Downregulation of SMYD3 led to S phase arrest and increased the cell apoptosis rate. Furthermore, a PCR array revealed that SMYD3 knockdown caused the upregulation of the cyclin-dependent kinase (CDK) inhibitors CDKN2A (p16INK4), CDKN2B (p15INK4B), CDKN3 and CDC25A, which may be responsible for the S phase arrest. In addition, the upregulation of CD40LG and downregulation of BIRC3 may explain the increased cell apoptosis rate after silencing SMYD3. We also discovered that SMYD3 bound on the promoter of CDKN2A and down-regulated its expression by triple-methylating H4K20. In addition, SMYD3 bound on the promoter of BIRC3 and up-regulated its expression by triple-methylating H3K4. Finally, knocking down SMYD3 could inhibit ovarian cancer growth in nude mice subcutaneous tumor model and PDX model.Conclusion: Our results demonstrated that SMYD3 was overexpressed in ovarian cancer and contributes to the regulation of tumor proliferation and apoptosis via SMYD3-H4K20me3-CDKN2A pathway and SMYD3-H3K4me3-BIRC3 pathway. Thus, SMYD3 is a promising epigenetic therapeutic target for ovarian cancer.

MiR-25 promotes invasion of human non-small cell lung cancer via CDH1

ABSTRACT MicroRNA-25 (miR-25) has been reported to be overexpressed in numerous human tumors and plays a key role in tumor promotor. However, there are few reports about miR-25 expression and function in non-small cell lung cancer (NSCLC). In this study, we investigated the biological role of miR-25 in NSCLC and its underlying molecular mechanisms. We found that the upregulation of miR-25 was correlated with lymph node metastasis and TNM stage in 113 NSCLC patients. Moreover, the up-regulation of miR-25 was associated with poor survival of NSCLC patients and might be used as an independent prognostic factor. Moreover, forced expression of miR-25 enhanced H358 and Calu-1 cell migration and invasion, but not apoptosis and proliferation in vitro. Elevation of invasion and metastasis by miR-25 directly and significantly correlated with inactivation of CDH1 expression. Therefore, patients with up-regulated miR-25 are prone to lymph node metastasis and thus have a poor prognosis.

Induction of Oxidative DNA Damage in Bovine Herpesvirus 1 Infected Bovine Kidney Cells (MDBK Cells) and Human Tumor Cells (A549 Cells and U2OS Cells).

Bovine herpesvirus 1 (BoHV-1) is an important pathogen of cattle that causes lesions in mucosal surfaces, genital tracts and nervous systems. As a novel oncolytic virus, BoHV-1 infects and kills numerous human tumor cells. However, the mechanisms underlying the virus-induced cell damages are not fully understood. In this study, we demonstrated that virus infection of MDBK cells induced high levels of DNA damage, because the percentage of comet tail DNA (tailDNA%) determined by comet assay, a direct indicator of DNA damage, and the levels of 8-hydroxyguanine (8-oxoG) production, an oxidative DNA damage marker, consistently increased following the virus infection. The expression of 8-oxoguanine DNA glycosylase (OGG-1), an enzyme responsible for the excision of 8-oxoG, was significantly decreased due to the virus infection, which corroborated with the finding that BoHV-1 infection stimulated 8-oxoG production. Furthermore, the virus replication in human tumor cells such as in A549 cells and U2OS cells also induced DNA damage. Chemical inhibition of reactive oxidative species (ROS) production by either ROS scavenger N-Acetyl-l-cysteine or NOX inhibitor diphenylene iodonium (DPI) significantly decreased the levels of tailDNA%, suggesting the involvement of ROS in the virus induced DNA lesions. Collectively, these results indicated that BoHV-1 infection of these cells elicits oxidative DNA damages, providing a perspective in understanding the mechanisms by which the virus induces cell death in both native host cells and human tumor cells.

The Metabolism of Separase Inhibitor Sepin-1 in Human, Mouse, and Rat Liver Microsomes.

Separase, a known oncogene, is widely overexpressed in numerous human tumors of breast, bone, brain, blood, and prostate. Separase is an emerging target for cancer therapy, and separase enzymatic inhibitors such as sepin-1 are currently being developed to treat separase-overexpressed tumors. Drug metabolism plays a critical role in the efficacy and safety of drug development, as well as possible drug–drug interactions. In this study, we investigated the in vitro metabolism of sepin-1 in human, mouse, and rat liver microsomes (RLM) using metabolomic approaches. In human liver microsomes (HLM), we identified seven metabolites including one cysteine–sepin-1 adduct and one glutathione–sepin-1 adduct. All the sepin-1 metabolites in HLM were also found in both mouse and RLM. Using recombinant CYP450 isoenzymes, we demonstrated that multiple enzymes contributed to the metabolism of sepin-1, including CYP2D6 and CYP3A4 as the major metabolizing enzymes. Inhibitory effects of sepin-1 on seven major CYP450s were also evaluated using the corresponding substrates recommended by the US Food and Drug Administration. Our studies indicated that sepin-1 moderately inhibits CYP1A2, CYP2C19, and CYP3A4 with IC50 < 10 μM but weakly inhibits CYP2B6, CYP2C8/9, and CYP2D6 with IC50 > 10 μM. This information can be used to optimize the structures of sepin-1 for more suitable pharmacological properties and to predict the possible sepin-1 interactions with other chemotherapeutic drugs.

KEAP1 has a sweet spot: A new connection between intracellular glycosylation and redox stress signaling in cancer cells

ABSTRACTThe KEAP1/NRF2 pathway is a master regulator of the redox stress response and is dysregulated in numerous human tumors. We discovered that NRF2 signaling is controlled by the site-specific glycosylation of KEAP1, revealing a potentially broad link among nutrient sensing, proteostasis and stress resistance in both normal and cancer cells.

HMGB1-mediated autophagy confers resistance to gemcitabine in hormone-independent prostate cancer cells.

As a main treatment of prostate cancer, castration therapy has been widely applied in the clinic. However, the therapeutic strategy for hormone-independent prostate cancer (HIPC) was not satisfied. Gemcitabine is an important chemotherapeutic agent that has been approved for the treatment of numerous human solid tumors, including HIPC, whereas the gemcitabine resistance has become a serious problem in clinical chemotherapy. In the present study, the mechanisms of resistance to gemcitabine were investigated in HIPC cell lines. The results demonstrated that the autophagy markers were induced significantly in HIPC cells subsequent to gemcitabine treatment. Meanwhile, administration of gemcitabine to HIPC cells increased the expression of high mobility group box1 (HMGB1). Furthermore, the gemcitabine-induced autophagy response was attenuated in stable HIPC cells harboring HMGB1 shRNA. Notably, the HIPC cells stably transfected with HMGB1 shRNA or treated with autophagy inhibitors were more sensitive to gemcitabine compared with the control group. These data suggested that inhibition of HMGB1 increased the sensitivity to gemcitabine by decreasing autophagy response in HIPC cells. Overall, the present findings demonstrate a new mechanism for the resistance to gemcitabine in HIPC cell lines.