Functional CDKN2A assay identifies frequent deleterious alleles misclassified as variants of uncertain significance

Pathogenic germline CDKN2A variants are associated with an increased risk of pancreatic ductal adenocarcinoma (PDAC). CDKN2A variants of uncertain significance (VUSs) are reported in up to 4.3% of patients with PDAC and result in significant uncertainty for patients and their family members as an unknown fraction are functionally deleterious, and therefore, likely pathogenic. Functional characterization of CDKN2A VUSs is needed to reclassify variants and inform clinical management. 29 germline CDKN2A VUSs previously reported in patients with PDAC or in ClinVar were evaluated using a validated in vitro cell proliferation assay. 12 of the 29 CDKN2A VUSs were functionally deleterious (11 VUSs) or potentially functionally deleterious (1 VUS) and were reclassified as likely pathogenic variants. Thus, over 40% of CDKN2A VUSs identified in patients with PDAC are functionally deleterious and likely pathogenic. When incorporating VUSs found to be functionally deleterious, and reclassified as likely pathogenic, the prevalence of pathogenic/likely pathogenic CDKN2A in patients with PDAC reported in the published literature is increased to up to 4.1% of patients, depending on family history. Therefore, CDKN2A VUSs may play a significant, unappreciated role in risk of pancreatic cancer. These findings have significant implications for the counselling and care of patients and their relatives.

Enhancement of the Anti-Angiogenic Effects of Delphinidin When Encapsulated within Small Extracellular Vesicles

(1) Background: The anthocyanin delphinidin exhibits anti-angiogenic properties both in in vitro and in vivo angiogenesis models. However, in vivo delphinidin is poorly absorbed, thus its modest bioavailability and stability reduce its anti-angiogenic effects. The present work takes advantage of small extracellular vesicle (sEV) properties to enhance both the stability and efficacy of delphinidin. When encapsulated in sEVs, delphinidin inhibits the different stages of angiogenesis on human aortic endothelial cells (HAoECs). (2) Methods: sEVs from immature dendritic cells were produced and loaded with delphinidin. A method based on UHPLC-HRMS was implemented to assess delphinidin metabolites within sEVs. Proliferation assay, nitric oxide (NO) production and Matrigel assay were evaluated in HAoECs. (3) Results: Delphinidine, 3-O-β-rutinoside and Peonidin-3-galactoside were found both in delphinidin and delphinidin-loaded sEVs. sEV-loaded delphinidin increased the potency of free delphinidin 2-fold for endothelial proliferation, 10-fold for endothelial NO production and 100-fold for capillary-like formation. Thus, sEV-loaded delphinidin exerts effects on the different steps of angiogenesis. (4) Conclusions: sEVs may be considered as a promising approach to deliver delphinidin to target angiogenesis-related diseases, including cancer and pathologies associated with excess vascularization.

Anti-Myeloma Effects of Panobinostat in Combination with Duvelisib

Introduction: Panobinostat (Secura Bio) is a non-selective histone deacetylase inhibitor (HDACi) that has shown anti-tumor activity in preclinical studies in both solid and hematological malignancies. Duvelisib (Secura Bio) is an FDA-approved oral drug and a potent small molecule inhibitor of the delta (δ) and gamma (γ) isoforms of phosphoinositide-3 kinase (PI3K) with potential immunomodulating and antineoplastic activities. There is no data regarding in combination of an HDACi with duvelisib for multiple myeloma (MM) treatment. We evaluated the anti-myeloma effects of the panobinostat in combination with duvelisib. Methods: The human MM cell lines U266, RPMI 8226 and MM1s were obtained from ATCC. Primary MM tumor cells were isolated from MM patients' bone marrow (BM) aspirates and mononuclear cells (MCs) were isolated. The cells were seeded at 10 5 cells/well in 96-well plates and incubated for 24 h in the presence of vehicle, panobinostat or duvelisib alone, or the two drugs together for 48 h. Cell viability was quantified using the MTS cell proliferation assay. For the apoptosis assay, U266, RPMI 8226, MM1s, or primary MM tumor cells were cultured at 1 x 10 6 cells per well for 48 hours at 37°C in 5% CO2 with or without the addition of panobinostat or duvelisib. Next, the cells were washed twice with PBS, re-suspended in binding buffer, and stained with FITC-conjugated annexin V and fluorescent dye propidium iodide (PrI) following annexin V assay standard protocol (BioVision, USA). For each drug treatment, 1 x 10 5 gated events were recorded. Results and Discussion: The MTS cell proliferation assay showed that panobinostat alone inhibited cell proliferation of the MM cell lines U266 and MM1s but not RPMI8226. Panobinostat alone induced concentration-dependent inhibition of BMMCs. Panobinostat (20nM) in combination with duvelisib markedly increased inhibition of primary MM cell proliferation. Panobinostat alone induced MM cell apoptosis in all three MM cell lines. Panobinostat in combination with duvelisib enhanced MM cell apoptosis in MM1s but not U266 and RPMI 8226 cells. We further examined apoptosis of primary MM cell treated with panobinostat alone and in combination with duvelisib. The results showed panobinostat alone induced primary MM cell apoptosis and in combination with duvelisib markedly increased the induction of apoptosis. This study illustrates that the combination of panobinostat and duvelisib shows potent anti-MM effects in vitro and we are currently evaluating the anti-MM effects of this combination in vivo using our human MM xenograft models. *The study was partially funded by Secura Bio, Inc Disclosures No relevant conflicts of interest to declare.

SPTBN2, a New Biomarker of Lung Adenocarcinoma

ObjectivesThe roles played by β-III-spectrin, also known as spectrin beta, non-erythrocytic 2 (SPTBN2), in the occurrence and development of lung adenocarcinoma (LUAD) have not been previously examined. Our study aimed to reveal the relationship between the SPTBN2 expression and LUAD.Materials and MethodsTwenty pairs of LUAD tissues and adjacent tissues were collected from patients diagnosed and treated at the Thoracic Surgery Department of The First Affiliated Hospital of Zhengzhou University from July 2019 to September 2020. RNA sequencing (RNA-seq) analysis determined that the expression of SPTBN2 was higher in LUAD samples than in adjacent normal tissues. The expression levels of SPTBN2 were examined in various databases, including the Cancer Cell Line Encyclopedia (CCLE), Gene Expression Omnibus (GEO), and Human Protein Atlas (HPA). The Search Tool for the Retrieval of Interacting Genes (STRING) online website was used to examine protein–protein interactions involving SPTBN2, and the results were visualized by Cytoscape software. The Molecular Complex Detection (MCODE) plug-in for Cytoscape software was used to identify functional modules of the obtained protein–protein interaction (PPI) network. Gene enrichment analysis was performed, and survival analysis was conducted using the Kaplan–Meier plotter. The online prediction website TargetScan was used to predict SPTBN2-targeted miRNA sequences by searching for SPTBN2 sequences. Finally, we verified the expression of SPTBN2 in the obtained tissue samples using real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). The human lung cancer cell lines A549 and H1299 were selected for the transfection of small interfering RNA (siRNA) targeting SPTBN2 (si-SPTBN2), and the knockdown efficiency was evaluated by RT-qPCR. The cellular proliferation, migration, and invasion capacities of A549 and H1299 cells were determined using the cell counting kit-8 (CCK-8) proliferation assay; the wound-healing assay and the Transwell migration assay; and the Matrigel invasion assay, respectively.ResultsThe expression of SPTBN2 in non–small cell lung cancer (NSCLC) ranked 13th among cancer cell lines based on the CCLE database. At the mRNA and protein levels, the expression levels of SPTBN2 were higher in LUAD tissues than in normal lung tissues. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that proteins related to SPTBN2 were enriched in apoptotic and phagosomal pathways. Kaplan–Meier survival analysis revealed that SPTBN2 expression was significantly related to the prognosis of patients with LUAD. The TargetScan database verified that miR-16 was a negative regulator of SPTBN2 mRNA expression. The results of the CCK-8 cell proliferation assay revealed that SPTBN2 knockdown significantly inhibited the cell proliferation abilities of A549 and H1299 cells. The wound-healing assay indicated that SPTBN2 knockdown resulted in reduced migration after 48 h compared with the control group. The Transwell migration and invasion test revealed that the migration and invasion abilities were greatly decreased by SPTBN2 knockdown compared with control conditions.ConclusionWe uncovered a novel gene, SPTBN2, that was significantly upregulated in LUAD tissues relative to normal tissue expression. SPTBN2 is highly expressed in LUAD, positively correlated with poor prognosis, and can promote the proliferation, migration, and invasion of LUAD cells.

Circular RNA circNF1 siRNA Silencing Inhibits Glioblastoma Cell Proliferation by Promoting the Maturation of miR-340

It has been reported that circNF1, a type of circular RNA (circRNA), promotes gastric cancer. This study aimed to analyze the role of circNF1 in glioblastoma (GBM). The expression of circNF1, mature miR-340, and miR-340 precursor in paired GBM and non-cancer tissues from GBM patients (n = 50) was analyzed by RT-qPCR. GBM cells were transfected with circNF1 siRNA, followed by the analysis of the expression of mature miR-340 and miR-340 precursor, to study the effects of circNF1 knockdown on the maturation of miR-340. The CCK-8 assay was carried out to explore the role of circNF1 and miR-340 in the proliferation of GBM cells. circNF1 expression was found to be upregulated in GBM and was correlated with patient survival. In glioma tissue, circNF1 was inversely correlated with mature miR-340, but not with the miR-340 precursor. In GBM cells, circNF1 siRNA silencing resulted in the upregulation of mature miR-340, but not the miR-340 precursor. The cell proliferation assay showed that circNF1 siRNA silencing and miR-340 overexpression decreased the proliferation of GBM cells. In addition, the miR-340a inhibitor suppressed the role of circNF1 siRNA silencing in cell proliferation. Therefore, circNF1 siRNA silencing may inhibit GBM cell proliferation by promoting the maturation of miR-340.

Chemotherapy of HER2- and MDM2-Enriched Breast Cancer Subtypes Induces Homologous Recombination DNA Repair and Chemoresistance

Analyzing the TCGA breast cancer database, we discovered that patients with the HER2 cancer subtype and overexpression of MDM2 exhibited decreased post-treatment survival. Inhibition of MDM2 expression in the SKBR3 cell line (HER2 subtype) diminished the survival of cancer cells treated with doxorubicin, etoposide, and camptothecin. Moreover, we demonstrated that inhibition of MDM2 expression diminished DNA repair by homologous recombination (HR) and sensitized SKBR3 cells to a PARP inhibitor, olaparib. In H1299 (TP53−/−) cells treated with neocarzinostatin (NCS), overexpression of MDM2 WT or E3-dead MDM2 C478S variant stimulated the NCS-dependent phosphorylation of ATM, NBN, and BRCA1, proteins involved in HR DNA repair. However, overexpression of chaperone-dead MDM2 K454A variant diminished phosphorylation of these proteins as well as the HR DNA repair. Moreover, we demonstrated that, upon NCS treatment, MDM2 K454A interacted with NBN more efficiently than MDM2 WT and that MDM2 WT was degraded more efficiently than MDM2 K454A. Using a proliferation assay, we showed that overexpression of MDM2 WT, but not MDM2 K454A, led to acquisition of resistance to NCS. The presented results indicate that, following chemotherapy, MDM2 WT was released from MDM2-NBN complex and efficiently degraded, hence allowing extensive HR DNA repair leading to the acquisition of chemoresistance by cancer cells.

HPV16 E6-178G/E7-647G Promotes Proliferation and Inhibits Apoptosis in Cervical Cancer C33A Cells

Background：HPV16 is the main cause of cervical cancer. In our study, we aimed to investigate the role of HPV mutants HPV16 E6-178G/E7-647G in the proliferation and apoptosis of cervical cancer C33A cells. Methods：Plasmids encoding the HPV16 E7 prototype (E7-647A)-GV144, E7 mutant (E7-647G)-GV144, HPV16 E6/E7 prototype (E6-178T/E7-647A)-GV144, and E6/E7 mutant (E6-178G/E7-647G)-GV144 were stably transfected into cervical cancer C33A cells. Western blot analysis, CCK8 proliferation assay, cell cloning assay and flow cytometry were used to detect the effects of the different polymorphism sites in HPV16 on cell proliferation and apoptosis. Results：HPV16 mutations promoted the proliferation and inhibited the apoptosis of cervical cancer C33A cells, and the effect of the E6-178G/E7-647G co-mutation was significantly greater than that of the single E7-647G mutant (P<0.05). Conclusions：HPV16 E6-178G/E7-647G can thus promote the proliferation and inhibit the apoptosis of cervical cancer cells.