Abstract
uPAR is a globular protein, tethered to the cell membrane by a GPI-anchor involved in several cancer-related properties and its overexpression commonly correlates with poor prognosis and metastasis. We investigated the consequences of uPAR irreversible loss in human melanoma and colon cancer cell lines, knocking out its expression by CRISPR/Cas9. We analyzed through flow cytometry, western blotting and qPCR, the modulation of the most known cancer stem cells-associated genes and the EGFR while we observed the proliferation rate exploiting 2D and 3D cellular models. We also generated uPAR “rescue” expression cell lines as well as we promoted the expression of only its 3’UTR to demonstrate the involvement of uPAR mRNA in tumor progression. Knocking out PLAUR, uPAR-encoding gene, we observed an inhibited growth ratio unexpectedly coupled with a significant percentage of cells acquiring a stem-like phenotype. In vivo experiments demonstrated that uPAR loss completely abrogates tumorigenesis despite the gained stem-like profile. Nonetheless, we proved that the reintroduction of the 3’UTR of PLAUR gene was sufficient to restore the wild-type status validating the hypothesis that such a region may act as a “molecular sponge”. In particular miR146a, by binding PLAUR 3’ UTR region might be responsible for uPAR-dependent inhibition of EGFR expression.
Highlights
The urokinase plasminogen activator receptor is a membrane receptor characterized by three globular domains, involved in several typical cancer features such as survival, invasion and migration, angiogenesis and intra-tumor recruitment of inflammatory cells [1,2,3,4]
I.e. low proliferation rate, oxidative phosphorylation (OXPHOS) predominant metabolism paired with mitochondrial biogenesis deregulation and high mitochondrial membrane potential, are all typical features of cancer stem cells and led us to investigate if uPAR KO cells may have gained a stem-like profile [102,103,104]
Taken together, such data support the idea that the partial stem status induced by loss of uPAR in cancer cells may confer to such cells the characteristics required to give origin to the minimal residual disease [111]
Summary
The urokinase plasminogen activator (uPA) receptor (uPAR) is a membrane receptor characterized by three globular domains, involved in several typical cancer features such as survival, invasion and migration, angiogenesis and intra-tumor recruitment of inflammatory cells [1,2,3,4]. In one of our previous studies, we decided to exploit the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/ Cas technique to establish two melanoma and one colon carcinoma cell lines with a complete uPAR KO [18], to better understand its role in tumor progression, examining the typical cancer hallmarks. This wide-spreading technology, based on a naturally-occurring system that protects bacteria from phages infections [19], is useful being uPAR commonly modulated by many extracellular factors such as hypoxia, cytokines and transcription factors such as NF-kB and TCF/ LEF [20] and by cell-cell contact [21]. In the present study we investigated all these aspects in the obtained uPAR KO clones, demonstrating in vitro and in vivo tumor growth inhibition coupled with unexpected features, sometimes unrelated to what previously reported by using short-term silencing methods, such as appearance of stemness markers and loss of EGFR
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