EpCAM associates with integrin and regulates cell adhesion in cancer cells

Yang, Jie; Isaji, Tomoya; Zhang, Guowei; Qi, Feng; Duan, Chengwei; Fukuda, Tomohiko; Gu, Jianguo

doi:10.1016/j.bbrc.2019.11.152

Cited by 20 publications

(15 citation statements)

References 26 publications

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“…Genetic deletion of tumor associated genes using clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR associated protein-9 nuclease (CRISPR/Cas9) has great potential in anti-cancer therapy 22 – 25 . CRISPR/Cas9 gene-editing relies on the guidance of a single-guide RNA (sgRNA) 26 , 27 to disrupt targeted genes 28 – 31 . However, target specificity (off-target effects) and gene-editing efficiency are still major challenges for applying CRISPR/Cas9 technology in biomedical and clinical applications 32 – 35 .…”

Section: Introductionmentioning

confidence: 99%

Dual-sgRNA CRISPR/Cas9 knockout of PD-L1 in human U87 glioblastoma tumor cells inhibits proliferation, invasion, and tumor-associated macrophage polarization

Fierro

DiPasquale

Perez

et al. 2022

Sci Rep

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Programmed death ligand 1 (PD-L1) plays a key role in glioblastoma multiforme (GBM) immunosuppression, vitality, proliferation, and migration, and is therefore a promising target for treating GBM. CRISPR/Cas9-mediated genomic editing can delete both cell surface and intracellular PD-L1. This systemic deliverable genomic PD-L1 deletion system can be used as an effective anti-GBM therapy by inhibiting tumor growth and migration, and overcoming immunosuppression. To target PD-L1 for CRISPR/Cas9 gene editing, we first identified two single guide RNA (sgRNA) sequences located on PD-L1 exon 3. The first sgRNA recognizes the forward strand of human PD-L1 near the beginning of exon 3 that allows editing by Cas9 at approximately base pair 82 (g82). The second sgRNA recognizes the forward strand of exon 3 that directs cutting at base pair 165 (g165). A homology-directed repair template (HDR) combined with the dual-sgRNAs was used to improve PD-L1 knockout specificity and efficiency. sgRNAs g82 and g165 were cloned into the multiplex CRISPR/Cas9 assembly system and co-transfected with the HDR template in human U87 GBM cells (g82/165 + HDR). T7E1 analysis suggests that the dual-sgRNA CRISPR/Cas9 strategy with a repair template was capable of editing the genomic level of PD-L1. This was further confirmed by examining PD-L1 protein levels by western blot and immunofluorescence assays. Western blot analysis showed that the dual-sgRNAs with the repair template caused a 64% reduction of PD-L1 protein levels in U87 cells, while immunostaining showed a significant reduction of intracellular PD-L1. PD-L1 deletion inhibited proliferation, growth, invasion and migration of U87 cells, indicating intracellular PD-L1 is necessary for tumor progression. Importantly, U87 cells treated with g82/165 + HDR polarized tumor-associated macrophages (TAM) toward an M1 phenotype, as indicated by an increase in TNF-α and a decrease in IL-4 secretions. This was further confirmed with flow cytometry that showed an increase in the M1 markers Ly6C + and CD80 +, and a decrease in the M2 marker CD206 + both in vitro and in vivo. Utilizing dual-sgRNAs and an HDR template with the CRISPR/Cas9 gene-editing system is a promising avenue for the treatment of GBM.

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Section: Introductionmentioning

confidence: 99%

Dual-sgRNA CRISPR/Cas9 knockout of PD-L1 in human U87 glioblastoma tumor cells inhibits proliferation, invasion, and tumor-associated macrophage polarization

Fierro

DiPasquale

Perez

et al. 2022

Sci Rep

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“…Similarly, Yang, et al [ 21 ] also stably transfected gRNA, Cas9 enzyme, and GFP expressing plasmids and sorted for green fluorescing cells when creating their EpCAM KO human cancer cell pool, but only targeted Exon 6. When only Exon 7 was targeted here, half of the KO clone's sequenced cDNAs had mutations that had the potential to produce a soluble portion of EpCAM, which appeared to be detected in cell supernatants ( Supplemental Fig.…”

Section: Discussionmentioning

confidence: 99%

CRISPR/Cas9 disruption of EpCAM Exon 2 results in cell-surface expression of a truncated protein targeted by an EpCAM specific T cell engager

Bagheri

Culp

DuBridge

et al. 2022

Biochemistry and Biophysics Reports

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CRISPR/Cas9 gene-editing technology allows researchers to study protein function by specifically introducing double-stranded breaks in the gene of interest then analyze its subsequent loss in sensitive biological assays. To help characterize one of a series of highly potent, conditionally active, T cell engaging bispecific molecules called COBRA™, the human EpCAM gene was disrupted in HT29 cells using CRISPR/Cas9 and guide RNA targeting its Exon 2. Although a commercially available antibody indicated loss of cell-surface expression, the EpCAM targeting bispecific COBRA was still able to lyse these cells in a T cell dependent cellular cytotoxicity assay. RT-PCR sequence analysis of these cells showed a major alternative transcript generated after CRISPR/Cas9, with Exon 1 and 3 spliced together in-frame, skipping Exon 2 completely, to express a truncated cell-surface receptor recognized by the EpCAM-COBRA. Researchers who use CRISPR/Cas9 must be cognizant of this potential to express alternative versions of their proteins and use sensitive orthogonal detection methods to ensure complete gene disruption.

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“…The interaction with claudin 7 (and with TEMs) is an important lead to pursue, as it could impact not only on their mutual stability [ 24 , 25 ], but also on subcellular localization and/or on PKC inhibition. EpCAM has also been detected as an interactor of integrins [ 18 , 56 , 57 , 58 ] ( Figure 2 D), but the physiological implications are so far unclear.…”

Section: Perspectives: From Here Now Where Do We Go?mentioning

confidence: 99%

EpCAM as Modulator of Tissue Plasticity

Fagotto

2020

Cells

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The Epithelial Cell Adhesion Molecule or EpCAM is a well-known marker highly expressed in carcinomas and showing a strong correlation with poor cancer prognosis. While its name relates to its proposed function as a cell adhesion molecule, EpCAM has been shown to have various signalling functions. In particular, it has been identified as an important positive regulator of cell adhesion and migration, playing an essential role in embryonic morphogenesis as well as intestinal homeostasis. This activity is not due to its putative adhesive function, but rather to its ability to repress myosin contractility by impinging on a PKC signalling cascade. This mechanism confers EpCAM the unique property of favouring tissue plasticity. I review here the currently available data, comment on possible connections with other properties of EpCAM, and discuss the potential significance in the context of cancer invasion.

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EpCAM associates with integrin and regulates cell adhesion in cancer cells

Cited by 20 publications

References 26 publications

Dual-sgRNA CRISPR/Cas9 knockout of PD-L1 in human U87 glioblastoma tumor cells inhibits proliferation, invasion, and tumor-associated macrophage polarization

Dual-sgRNA CRISPR/Cas9 knockout of PD-L1 in human U87 glioblastoma tumor cells inhibits proliferation, invasion, and tumor-associated macrophage polarization

CRISPR/Cas9 disruption of EpCAM Exon 2 results in cell-surface expression of a truncated protein targeted by an EpCAM specific T cell engager

EpCAM as Modulator of Tissue Plasticity

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