Exogenous Cripto-1 Suppresses Self-Renewal of Cancer Stem Cell Model

Abstract

Cripto-1 is a glycophosphatidylinositol (GPI) anchored signaling protein of epidermal growth factor (EGF)-Cripto-1-FRL1-Cryptic (CFC) family and plays a significant role in the early developmental stages and in the different types of cancer cells, epithelial to mesenchymal transition and tumor angiogenesis. Previously, we have developed cancer stem cells (miPS-LLCcm) from mouse iPSCs by culturing them in the presence of conditioned medium of Lewis Lung Carcinoma (LLC) cells for four weeks. Nodal and Cripto-1 were confirmed to be expressed in miPS-LLCcm cells by quantitative reverse transcription PCR (rt-qPCR) implying that Cr-1 was required in maintaining stemness. To investigate the biological effect of adding exogenous soluble CR-1 to the cancer stem cells, we have prepared a C-terminally truncated soluble form of recombinant human CR-1 protein (rhsfCR-1), in which the GPI anchored moiety was removed by substitution of a stop codon through site-directed mutagenesis. rhsfCR-1 effectively suppressed the proliferation and sphere forming ability of miPS-LLCcm cells in a dose-dependent manner in the range of 0 to 5 µg/mL, due to the suppression of Nodal-Cripto-1/ALK4/Smad2 signaling pathway. Frequency of sphere-forming cells was dropped from 1/40 to 1/69 by rhsfCR-1 at 1 µg/mL. Moreover, rhsfCR-1 in the range of 0 to 1 µg/mL also limited the differentiation of miPS-LLCcm cells into vascular endothelial cells probably due to the suppression of self-renewal, which should reduce the number of cells with stemness property. As demonstrated by a soluble form of exogenous Cripto-1 in this study, the efficient blockade would be an attractive way to study Cripto-1 dependent cancer stem cell properties for therapeutic application.

Exosomal Cripto-1 Serves as a Potential Biomarker for Perihilar Cholangiocarcinoma

“…CD24, located in lipid rafts through its GPI anchor, is demonstrated to be found in high levels in exosomes isolated from ovarian cancer cells, compared to other tumor biomarkers such as CA-125, EGFR and MUC18 ( 30 ). Since Cripto-1 has been reported as a cell surface GPI-linked glycoprotein ( 31 ), this study tested whether it can be found in exosomes. Cripto-1 was found in exosomes from the culture medium of PHCCA cell lines and the peripheral blood of mice implanted PHCCA cells.…”

A Novel Artificially Humanized Anti-Cripto-1 Antibody Suppressing Cancer Cell Growth

“…The phages with the affinity to recombinant soluble human CR-1 protein (rshCR-1) [ 28 ] were concentrated by bio-panning. After three rounds of bio-panning, phage clones exhibiting the affinity to rshCR-1 were isolated.…”

New Insights into Cancer Targeted Therapy: Nodal and Cripto-1 as Attractive Candidates

“…In 2017, the results obtained by Watanabe and colleagues were replicated in esophageal squamous cell carcinoma (ESCC) cell lines [67]. Mouse-induced CSCs exposed to soluble CR-1, which compete with endogenous CR-1 for Nodal signaling, display a reduction in the self-renewal ability, as well as a decrease in the ability to migrate and form spheroids in vitro [68]. In a recent study, it was demonstrated that the expression of CR-1 in human CSCs from colorectal cancers (CRC) spheroids originated from patients is not stationary.…”