CRISPR/Cas9 uPAR Gene Knockout Results in Tumor Growth Inhibition, EGFR Downregulation and Induction of Stemness Markers in Melanoma and Colon Carcinoma Cell Lines

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.

Enhanced Vasculogenic Capacity Induced by 5-Fluorouracil Chemoresistance in a Gastric Cancer Cell Line

“…In particular, 5FUr showed some very large cellular bodies with big nuclei which might be generated by the fusion of several cells in a sort of syncytial formation, while CISr demonstrated reduced dimensions. We monitored cell proliferation through CFSE labeling assay, showing that at 24 h only CISr have a lower proliferation ratio, but after 48 h we observed a decreased proliferation index (the average number of divisions that all responding cells have undergone since the initiation of the culture [ 16 ]) in all resistant cells ( Figure 2 b). As shown in Figure 2 c, such inhibited proliferation rate is consistent with an accumulation of 5FUr and TAXr in S phase while CISr encountered a delay in G0/G1 phase.…”

“…proliferation index (the average number of divisions that all responding cells have undergone since the initiation of the culture [16]) in all resistant cells (Figure 2b). As shown in Figure 2c, such inhibited proliferation rate is consistent with an accumulation of 5FUr and TAXr in S phase while CISr encountered a delay in G0/G1 phase.…”

“…We then evaluated several endothelial or VM-related markers through WB (Figure 3b) and we demonstrated an upregulation of VEGFR2 and Ephrin A2 in 5FUr only, while Galectin-3 was upregulated in both 5FUr and CISr. Laminin proliferation index (the average number of divisions that all responding cells have undergone since the initiation of the culture [16]) in all resistant cells (Figure 2b). As shown in Figure 2c, such inhibited proliferation rate is consistent with an accumulation of 5FUr and TAXr in S phase while CISr encountered a delay in G0/G1 phase.…”

Strategies to overcome the main challenges of the use of CRISPR/Cas9 as a replacement for cancer therapy

“… [ 65 ] Colon cancer uPAR CRL1619, CCL247 - Knockout Okayama-Berg vector Knockout of the uPAR gene Leads to tumor growth inhibition, EGFR downregulation, and an increase in stemness markers. [ 66 ] Prostate cancer Tceal1 Mouse: SP1 Human: PC3M, LNCaP, DU145, CWR22, RWPE - Knockdown lentivirus TCEAL1 deletion causes a different cell cycle profile than docetaxel alone, with more subG1 cell death and polyploidy. [ 67 ] Prostate cancer miRNA (miR)205, miR221, miR222, miR30c, miR224, miR4553, miR23b, miR505 LNCaP - Knockout Lentivirus Functional classification of prostate cancer-associated miRNAs through CRISPR/Cas9 mediated gene knockout [ 68 ] Prostate cancer BRAF CWR-R1 - Knockout lentiviral MAPK/AR co-targeting may help patients with active MAPK pathways, especially those with oncogenic BRAF mutations.…”

PLAUR as a Potential Biomarker Associated with Immune Infiltration in Bladder Urothelial Carcinoma

“…It has multifaceted roles in diseases, especially cancer. 8–11 PLAUR involves in cell–cell and cell–extracellular matrix interactions, and participates in various cancer-specific processes, such as cell differentiation, 12 proliferation, 13 metastasis 14 and angiogenesis, 15 etc. Rysenkova et al found that uPAR was an important regulator of EGFR and ERK1/2 signaling and implicated that uPAR was closely involved in neuroblastoma cell survival and differentiation.…”