Fas Knockout Research Articles

In vivo analysis of CRISPR-edited germinal center murine B cells.

The germinal center (GC) reaction is crucial for somatic hypermutation, affinity maturation, and the selection of high-affinity B cells, all of which are hallmarks of the humoral immune response. Understanding the distinct roles of various B cell genes is essential for elucidating the selection mechanisms within the GC reaction. Traditionally, studying B cell gene function in the GC reaction involved generating knock-out mice, a highly time-consuming method that necessitates complex vectors. The advent of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology has simplified the creation of knock-out mice. However, even with CRISPR, the generation of knock-out mice still faces challenges, including being time-consuming, costly, having low knock-out efficiency, and raising ethical concerns regarding animal use. To address these challenges, we developed an alternative method to traditional knock-out mouse generation. Our approach entails the ex vivo CRISPR editing of B cells from transgenic donor mice with different B cell receptor affinities followed by their adoptive transfer into recipient mice. We present a cost-effective, rapid, versatile, and adaptable CRISPR-Cas9 method for in vivo loss-of-function studies of individual murine B cell genes within the context of the GC reaction. This method provides a valuable tool for investigating the complex roles of different B cell genes in the GC selection process. As proof of concept, we validated our approach by examining the role of the pro-apoptotic gene Fas in the GC selection process. We adoptively transferred a mix of Fas knock-out (FasKO) low-affinity B cells, Fas wild-type (FasWT) low-affinity B cells, and FasWT high-affinity B cells into recipient mice. From our results, FasKO low-affinity B cells were still outcompeted by the FasWT high-affinity B cells for selection in the GC. An important observation was the accumulation of FasKO low-affinity GC B cells when compared to the FasWT low-affinity B cells, which suggested a role of Fas in the GC selection process.

FAS receptor regulates NOTCH activity through ERK-JAG1 axis activation and controls oral cancer stemness ability and pulmonary metastasis

Pulmonary metastasis occurring via the colonization of circulating cancer stem cells is a major cause of oral squamous cell carcinoma (OSCC)-related death. Thus, understanding the mechanism of OSCC pulmonary metastasis may provide a new opportunity for OSCC treatment. FAS, a well-known apoptosis-inducing death receptor, has multiple nonapoptotic, protumorigenic functions. Previously, we found that SAS OSCC cells with FAS receptor knockout did not affect orthotopic tumor growth or cervical lymph node metastasis. However, FAS knockout cells could not colonize in distant organs to form metastases upon intravenous injection, which hinted at the cancer stemness function of the FAS receptor. Immunohistochemistry staining indicated that the FAS receptor serves as a poor prognosis marker in OSCC patients. FAS knockout inhibited in vitro cancer spheroid formation, migration and invasion, and prevented mesenchymal transition in OSCC cells and inhibited OSCC pulmonary metastasis in vivo. To determine the regulatory mechanism by which the FAS receptor exerts its oncogenic function, we utilized cDNA microarrays and phosphoprotein arrays to discover key candidate genes and signaling pathway regulators. JAG1 expression and NOTCH pathway activation were controlled by the FAS receptor through ERK phosphorylation. Both JAG1 and NOTCH1 silencing decreased in vitro cancer spheroid formation. In OSCC cells, FAS ligand or JAG1 protein treatment increased NOTCH pathway activity, which could be abolished by FAS receptor knockout. In FAS knockout cells, restoring the NOTCH1 intracellular domain stimulated cancer spheroid formation. Both JAG1 and NOTCH1 silencing decreased in vivo OSCC growth. In conclusion, we found a novel FAS-ERK-JAG1-NOTCH1 axis that may contribute to OSCC stemness and pulmonary metastasis.

FAS receptor regulates HNSCC cancer stemness ability and pulmonary metastasis through Notch pathway

Up to 80% of head and neck squamous cell carcinoma (HNSCC) distance metastasis is occurring in lung through seeding of circulating cancer stem cells. It is crucial to understand the genes that are involved in HNSCC pulmonary colonization which may provide an important therapy niche in treating HNSCC patients successfully. Here, we found the FAS receptor knockout SAS cells could diminished the in vitro stemness ability and completely abolished the HNSCC pulmonary colonization in the immunodeficiency NSG mice. Even pretreated with single dose of FAS neutralize antibody could also significantly reduce the lung colonies forming numbers in vivo. FAS knockout could also prevent the in vitro cell migration and invasion abilities in the Boyden chamber assay. Using the reporter assays of common oncogenic signaling pathways, our results showed that Notch, HIF1A, RhoA, and STAT1 were suppressed in the FAS knockout cells. Moreover, the microarray analysis revealed that FAS knockout could also decrease stemness gene expressions, such as XIST, WDR66, HMGA2, and the ligand of Notch, Jag1 expressions. These genes and pathways reversal and complementation assays were performed and confirmed that indeed Notch pathway is one of the major downstream pathways of FAS‐mediated HNSCC stemness signaling to promote lung cancer metastasis. Taken together, our study found the novel FAS‐XIST axis might be mediated through Jag1/Notch pathway that may be contributing to HNSCC patient's lung metastasis.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract LB-341: CD95/FAS promotes tumorigenesis

Abstract FAS/CD95 has been known as a typical death receptor that enhances cell apoptosis mainly in the immune system. Recent studies indicate that CD95 expression is rarely absent or down-regulated in solid tumors and CD95L, the cognate ligand that binds cell membrane CD95, is constantly shown to be elevated in patient serum as well as tumor microenviroment, raising the question whether CD95 acturally enhances tumorigenesis and through what mechanisms. By stably knocking down endogeneous CD95 expression level using lentivirus based shRNA in six different tumor cell lines that represent liver (HepG2), colon (HCT116), renal (CAKI-1), breast (MCF7), ovary (SKOV3ip1 and HeyA8), in all cases the CD95 knockdown tumor cell lines show a clear reduction in proliferation rate comparing with the corresponding vector control cells. Injection CD95 knockdown SKOV3ip1 cells i.p. into nude mice according to method of well established xenograft ovary cancer animal model again shows a reduced tumor load and this is also confirmed by using a early passage human ovary cancer cell line MONTY-1. Human neutralizing anti-CD95L mAb (NOK-1) rather than mouse anti-CD95 mAb (MFL3) injection into these xenografted nude mice significantly reduced the tumor load, indicating that it's the CD95L secreted by tumor cells rather than the mice microenviroment that stimulates the tumor proliferation. Knocking down FasL expression from the six tumor cell lines mentioned above reduced cell proliferation even more dramatically than knocking down Fas and some cell lines, like HepG2, CAKI-1, MCF7 and HeyA8, even stop growing with reduced FasL expression. The reduced tumor formation by losing FAS/CD95 was also confirmed in vivo in two animal models: DEN induced liver cancer model by using FAS/CD95 liver specific knockout mice and Ade-Cre induced ovary cancer mouse model by generating Fas knockout mice from a mice stain with background of mutated k-ras and deleted Pten expression (LSK-K-ras(G12D/+)Pten(loxP/loxP)). The tumorigenic activity of CD95 is further identified as mediated through the CD95L-CD95-JNK-c-Jun-Fos and EGR1 pathway. These results demonstrate that CD95 plays a major growth promoting role during tumorigenesis and suggest that efforts to inhibit its activity rather than to enhance its activation should be considered during cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-341.

Chemokine receptor expression on BAL lymphocytes isolated from lung transplant recipients

group received a 28-day course of ZVAD-fmk (1 mg/kg/day) via subcutaneous mini-osmotic pumps, starting at the time of transplantation. For the apoptosis time course, allografts were harvested on post-op days 1, 3, 4, 7, 14, 21 and 28. Fas knockout and ZVAD-treated allografts were harvested on day 28. Histological, morphometric and in-situ TUNEL analyses were performed in a blinded fashion. Results: From days 1-7, allograft airway epithelial cells appear to be undergoing apoptosis. On day 28, the mean airway obliteration in both the Fas knockout group (65.63% /20.90 (mean /S.D.), p 0.004) and the ZVAD group (70.83% /14.71, p 0.011) were significantly reduced compared to controls (97.92% /4.07 and 98.98% /0.82, respectively). The airway epithelium was not preserved in Fas knockout allografts or controls. Conclusion: While apoptosis of airway epithelial cells does occur in the setting of allograft airway transplantation, inhibition of apoptosis, through broad-spectrum caspase inhibition or Fas pathway blockade, only partially prevents airway obliteration.