Snail Mucus Induces Cytotoxicity and Chemosensitivity of Triple-Negative Breast Cancer Cells via Activation of Fas Signaling Pathway

BackgroundTriple-negative breast cancers (TNBCs) patients showed poor survival outcomes due to chemoresistance, and the development of new therapeutic strategies is urgently needed. The development of cancer is mainly related to chronic inflammation. Therefore, the pharmacological reduction of inflammation by natural extract components may promote anti-cancer activity and increase chemosensitivity. Snail mucus has been reported to possess the ability against inflammation, a process closely related to tumorigenesis, suggesting a potential anti-cancer function. The direct cytotoxic activity of snail mucus in cancer cells was investigated in this study.Methods The effect of snail mucus on cell viability in breast cancer cells and normal epithelial cells were measured by MTT and IncuCyte Live-cell analysis. The active fractions of snail mucus were isolation by performing MPLC and the anti-cancer ingredients were identified by NMR spectrometer analysis.Results Snail mucus significantly decreased the proliferation and viability of TNBC cells with relatively lower cytotoxicity to normal breast epithelial cells and enhanced their response to chemotherapy. Mechanistically, snail mucus induces an extrinsic apoptotic pathway through activation of Fas signaling by suppressing nucleolin. Two possible peptide fractions have also been identified as the anti-cancer ingredients of the snail mucus.ConclusionsIn summary, snail mucus can induce programmed cell death via the extrinsic apoptotic pathway and might have therapeutic potential with chemo-sensitizing effect for TNBCs.

Bio-Molecular Characteristics of Whey Proteins with Relation to Inflammation

Whey proteins in bovine milk are a mixture of globular proteins manufactured from whey which is a byproduct of cheese industry. Whey protein is categorized to contain plethora of healthy components due to wide range of pH, promising nutritional profile with cost effective and diverse functionality. Reportedly there are three categories of whey protein, whey protein concentrate (WPC) (29–89%); whey protein isolate (WPI) 90% and whey protein hydrolysate (WPH) on the basis of proteins present in them. Whey proteins is composed of β-lactoglobulin (45–57%), immunoglobulins (10–15%) α-lactalbumin (15–25%), glicomacropeptide (10–15%), lactoperoxidase (<1%) and lactoferrin nearly (1%). Whey protein plays an important role and is validated to confer anti-inflammatory and immunostimulatory roles related to all metabolic syndromes. According to molecular point of view whey proteins decrease inflammatory cytokines (IL-1α, IL-1β, IL-10 and TNF- α); inhibits ACE and NF-κB expression; promotes Fas signaling and caspase-3 expression; elevates GLP-1, PYY, CCK, G1P and leptin; chelate and binds Fe+3, Mn+3 and Zn+2. In this chapter we will discuss significant biological role of whey proteins related to inflammatory health issues.

A coordinated function of lncRNA HOTTIP and miRNA-196b underpinning leukemogenesis by targeting Fas signaling

MicroRNAs (miRNAs) may modulate more than 60% of human coding genes and act as negative regulators, while long non-coding RNAs (lncRNAs) regulate gene expression on multiple levels by interacting with chromatin, functional proteins, and RNAs such as mRNAs and microRNAs. However, the crosstalk between lncRNA HOTTIP and miRNAs in leukemogenesis remains elusive. Using combined integrated analyses of global miRNA expression profiling and state-of-the-art genomic analyses of chromatin such as ChIRPseq., (genome wide HOTTIP binding analysis), ChIP-seq., and ATACseq., we found that miRNA genes are directly controlled by HOTTIP. Specifically, the HOX cluster miRNAs (miR-196a, miR-196b, miR-10a and miR-10b), located cis & trans, were most dramatically regulated and significantly decreased in HOTTIP knockout (KO) AML cells. HOTTIP bound to the miR-196b promoter, and HOTTIP deletion reduced chromatin accessibility and enrichment of active histone modifications at HOX cluster associated miRNAs in AML cells, while reactivation of HOTTIP restored miR gene expression and chromatin accessibility in the CTCF-boundary-attenuated AML cells. Inactivation of HOTTIP or miR-196b promotes apoptosis by altering the chromatin signature at the FAS promoter and increasing FAS expression. Transplantation of miR-196b knockdown MOLM13 cells in NSG mice increased overall survival compared to wild-type cells. Thus, HOTTIP remodels the chromatin architecture around miRNAs to promote their transcription, consequently repressing tumor suppressors and promoting leukemogenesis.

Elevation of MicroRNA-126 Levels in Intracranial Aneurysm and Bioinformatic Analysis of Potential Molecular Mechanisms

The study is to investigation of microRNA-126 levels in patients with intracranial aneurysm and bioinformatic analysis of the molecular mechanisms involved. A total of 166 patients with ICA who were hospitalized or examined in our hospital from September 2015 to December 2017 were used as the experimental group (ICA group). This group included 120 patients with unruptured intracranial aneurysm (UICA; UICA group) and 46 patients with ruptured intracranial aneurysm (RICA); RICA group). The UICA group was further subdivided into 42 surgical groups (S group) and 78 nonsurgical groups (NS group). Sixty-three normal people without intracranial aneurysms were selected as the control group. RT-PCR was used to quantitatively detect the relative expression of microRNA- 126 in peripheral blood mononuclear cells at the time of admission and immediately after surgery. The UCSC database was used to analyze the gene locus and homology of microRNA-126. The TargetScan database and CoMeTa database were used to predict the potential target genes of microRNA-126. The DAVID database was used to enrich the function of potential target genes of microRNA-126 (GO enrichment) and KEGG pathway enrichment for analysis. The expression level of microRNA-126 in peripheral blood was significantly higher in the ICA group than in the control group (P <0.01), significantly higher in the RICA group than in the UICA group (P <0.05). Expression was also higher in the NS group than in the S group but the difference was nonsignificant (P >0.05). A total of 15 potential target genes including ITGA6, CRK, PCDH7, and ADAM9 were identified through the target gene prediction software and GO analysis and KEGG pathway analysis showed that the function of the microRNA-126 target gene was mainly focused on protein binding and the FAS signaling pathway. In Conclusion the microRNA-126 is up-regulated in ICA patients and affects ICA by regulating multiple target genes in the FAS signaling pathway.

Blunted Fas signaling favors RIPK1-driven neutrophil necroptosis in critically ill COVID-19 patients

Critically ill COVID-19 patients are characterized by a severely dysregulated cytokine profile and elevated neutrophil counts, which are thought to contribute to disease severity. However, to date it remains unclear how neutrophils contribute to pathophysiology during COVID-19. Here, we assessed the impact of the dysregulated cytokine profile on the tightly regulated cell death program of neutrophils. We show that in a subpopulation of neutrophils, canonical apoptosis was skewed towards rapidly occurring necroptosis. This phenotype was characterized by abrogated caspase-8 activity and increased RIPK1 levels, favoring execution of necroptosis via the RIPK1-RIPK3-MLKL axis, as further confirmed in COVID-19 biopsies. Moreover, reduction of sFas-L levels in COVID-19 patients and hence decreased signaling to Fas directly increased RIPK1 levels and correlated with disease severity. Our results suggest an important role for Fas signaling in the regulation of cell death program ambiguity via the ripoptosome in neutrophils during COVID-19 and a potential therapeutic target to curb inflammation and thus influence disease severity and outcome.

90 Potentiation of T-cell mediated tumor killing via modulation of the fas/fasL pathway

BackgroundT-cell based immunotherapies such as CAR-T, bispecific mAb, transgenic T cells and checkpoint blockade have profound efficacy in multiple tumor types but share a common limitation – target antigen (Ag) escape.1 2 One approach to address this limitation has been therapy directed at a ‘parallel’ target (e.g. CD22 after CD19 loss), however, these lineage markers are frequently lost together.3 Here, we describe an alternate, broadly applicable, approach: potentiating fasL/fas-signaling to increase localized bystander killing of Ag-tumor cells and thereby prevent Ag escape.MethodsWe used a CRISPR/Cas9 library to screen for tumor expressed molecules that inhibit or facilitate T-cell killing. We then evaluated one candidate -fas- using murine transgenic T cells, murine and human CAR-T cells, bispecific mAb redirected PBMC, and tumoral RNAseq data from a large CAR-T clinical trial.ResultsGFP-specific (JEDI) CD8 T cells were co-cultured with on-target (GFP+) and bystander (mCherry+) lymphoma cells that had been transfected with a CRISPR/Cas9 library; this screen revealed several tumor-expressed candidate molecules inhibiting or facilitating T-cell killing. Notably, we observed a marked dependence on fas for on-target tumor killing and then, surprisingly, an exquisite dependence on fas for localized bystander tumor killing. (figure 1).Because bystander tumor killing appeared critically fas-dependent, we hypothesized that potentiating fas-signaling might increase bystander killing. An in vitro screen of small molecules that modulate fas-pathway revealed several candidates, including inhibitors of histone deacetylases (HDAC), inhibitors of apoptosis proteins (IAP) and Bcl-2 family members in murine and human systems (figure 2). To validate these candidates, we demonstrated that HDACi increased GFP-specific T cell killing of both on-target and bystander lymphoma cells, in a completely fas-dependent manner (figure 3). Similarly, using a bispecific antibody-based system, we demonstrated increased, fas-dependent, T cell killing of both on-target and bystander human lymphoma cells with inhibitors of IAP and bcl-2 family members (e.g. MCL1).Abstract 90 Figure 1See text for descriptionAbstract 90 Figure 2See text for descriptionAbstract 90 Figure 3See text for descriptionConclusionsT-cell mediated tumor killing can be potentiated with fas pathway modulators. This augmentation improves both fas-dependent Ag+ and Ag-tumor cell death. Further studies of modulating the fas pathway alongside T-cell based immunotherapies are needed as potential treatments to prevent antigen escape and improve patient outcomes.AcknowledgementsWe thank the flow cytometry core facility, microscopy core facility, and the CCMS animal facility at ISMMS.Ethics ApprovalThe studies were approved by The Mount Sinai Institutional Review Board.ReferencesZaretsky J, Garcia-Diaz A, Shin D, et al. Mutations Associated with Acquired Resistance to PD-1 Blockade in Melanoma. N Engl J Med 2016: 375(9); 819–20.Majzner R, Mackall C. Tumor antigen escape from CAR T-cell therapy. Cancer Discov 2018;8(10):1219–1226.Jacoby E, Nguyen S, Fountaine T, et al. CD19 CAR immune pressure induces B-precusor acute lymphoblastic leukaemia lineage switch exposing inherent leukaemic plasticity. Nat Commun 2016; 7:12320.