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BACKGROUND: The low efficiency of clustered, regularly interspaced, palindromic repeats-associated Cas (CRISPR/Cas) system editing genes in vivo limits the application. A components of the extracellular matrix (ECM), the extra domain A positive fibronectin (EDA+FN), may be a target for CRISPR/Cas system for the pro-oncogenic effects. The exclusion of EDA exon would alter the microenvironment and inhibit tumor progression, even the frequency of gene editing is still limited. RESULTS: The pro-oncogenic effects were confirmed by the exclusion of EDA exon from the fibronectin gene, as illustrated by the down-regulated proliferation, migration and invasion of CNE-2Z or SW480 cells (P<0.05). Furthermore, although the efficacy of EDA exon knockout through CRISPR/Cas system was shown to be low in vivo, the EDA+FN protein levels decrease obviously, inhibiting the tumor growth rate significantly (P<0.05), which was accompanied by a decrease in Ki-67 expression and microvessel numbers, and increased E-cadherin or decreased Vimentin expression (P<0.05). METHODS AND MATERIALS: Human nasopharyngeal carcinoma cell line CNE-2Z, and the colorectal carcinoma cell line SW480 were transfected with CRISPR/Cas9 plasmids targeting EDA exon. The effects of the exclusion of EDA on the cell proliferation, motility and epithelial-mesenchymal transition (EMT) were investigated, and the western blot and real-time PCR were performed to analyze the underlying mechanisms. Furthermore, CRISPR/Cas9 plasmids were injected into xenograft tumors to knockout EDA exon in vivo, and tumor growth, cell proliferation, EMT rate, or vascularization were investigated using western blot, PCR and immunohistochemistry. CONCLUSION: CRISPR/Cas system targeting ECM components was shown to be an effective method for the inhibition of tumor progression, as these paracrine or autocrine molecules are necessary for various tumor cells. This may represent a novel strategy for overcoming the drug evasion or resistance, in addition, circumventing the low efficiency of CRISPR/Cas system in vivo.
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Cancer cell-derived immunoglobulin G (cancer-IgG) has been implicated in the pathogenesis and progression of various types of cancer. However, its role in salivary adenoid cystic carcinoma (SACC) remains unclear. The present study aimed to investigate the effects of cancer-IgG on metastasis and prognosis in 96 patients with SACC. Immunohistochemical staining showed that cancer-IgG expression was present in all 96 individual SACC tissues. Additionally, high cancer-IgG expression was significantly correlated with metastasis, nerve invasion and recurrence in SACC (P<0.05). Moreover, cancer-IgG expression was significantly correlated with the survival duration of patients with SACC (P<0.05). Proliferation, cell motility and invasion all decreased significantly following knockdown of cancer-IgG in SACC cells (P<0.05) through population-doubling time, wound healing and transwell invasion assays. Additionally, cancer-IgG-knockdown in SACC cells induced the increased expression of E-cadherin and matrix metalloproteinase 9, and promoted the epithelial-mesenchymal transition, but decreased the expression of F-actin filaments. Taken together, these results showed that the high expression of cancer-IgG was strongly associated with metastasis, recurrence and invasion in SACC, suggesting that cancer-IgG expression could serve as a useful biomarker to predict the prognosis of the disease.
RESUMEN
OBJECTIVE: Cancer-IgG is a newly-discovered molecule, mainly derived from epithelial carcinoma cells and is significantly correlated with differentiation, metastasis, local invasion, and poor prognosis of many cancers. In our previous study we detected IgG expression in oral epithelial carcinoma, including salivary adenoid cystic carcinoma (SACC), using an IgG-specific commercial antibody. Here, we explored the correlation between cancer-IgG and clinicopathological features of SACC. DESIGN: A total of 68 human SACC tissue specimens and 2 siRNAs were used to analyze the correlation between cancer-IgG and extra domain A (EDA+)-containing fibronectin using the cancer-IgG-specific monoclonal antibody, RP215. RESULTS: We found an unexpected correlation between cancer-IgG and EDA+ fibronectin, both of which showed aberrant expression in SACC tissue samples. Both were highly expressed in SACC with nerve invasion. In our previous study, EDA+ fibronectin overexpression in SACC cells decreased N-cadherin expression. In the present study, we used SACC-83 cells, wherein EDA+ fibronectin is overexpressed and cancer-IgG is knocked down. EDA+ fibronectin expression was reduced with cancer-IgG knockdown, while cancer-IgG expression did not affect EDA+ fibronectin overexpression. Furthermore, knockdown of non-B cell-derived IgG in SACC cells decreased cellular motility (P<0.05) as well as increased E-cadherin and alpha-smooth muscle actin levels. CONCLUSION: The results suggest that cancer IgG potentially regulates EDA+ fibronectin expression, thereby suggesting possible new therapeutic approaches for SACC.