RGD-p21Ras-scFv expressed prokaryotically on a pilot scale inhibits ras-driven colorectal cancer growth by blocking p21Ras-GTP.

BACKGROUND: Ras gene mutation and/or overexpression are drivers in the progression of cancers, including colorectal cancer. Blocking the Ras signaling has become a significant strategy for cancer therapy. Previously, we constructed a recombinant scFv, RGD-p21Ras-scFv by linking RGD membrane-penetrating peptide gene with the anti-p21Ras scFv gene. Here, we expressed prokaryotically RGD-p21Ras-scFv on a pilot scale, then investigated the anti-tumor effect and the mechanism of blocking Ras signaling. METHODS: The E. coli bacteria which could highly express RGD-p21Ras-scFv was screened and grown in 100 L fermentation tank to produce RGD-p21Ras-scFv on optimized induced expression conditions. The scFv was purified from E. coli bacteria using His Ni-NTA column. ELISA was adopted to test the immunoreactivity of RGD-p21Ras-scFv against p21Ras proteins, and the IC50 of RGD-p21Ras-scFv was analyzed by CCK-8. Immunofluorescence colocalization and pull-down assays were used to determine the localization and binding between RGD-p21Ras-scFv and p21Ras. The interaction forces between RGD-p21Ras-scFv and p21Ras after binding were analyzed by molecular docking, and the stability after binding was determined by molecular dynamics simulations. p21Ras-GTP interaction was detected by Ras pull-down. Changes in the MEK-ERK /PI3K-AKT signaling paths downstream of Ras were detected by WB assays. The anti-tumor activity of RGD-p21Ras-scFv was investigated by nude mouse xenograft models. RESULTS: The technique of RGD-p21Ras-scFv expression on a pilot scale was established. The wet weight of the harvested bacteria was 31.064 g/L, and 31.6 mg RGD-p21Ras-scFv was obtained from 1 L of bacterial medium. The purity of the recombinant antibody was above 85%, we found that the prepared on a pilot scale RGD-p21Ras-scFv could penetrate the cell membrane of colon cancer cells and bind to p21Ras, then led to reduce of p21Ras-GTP (active p21Ras). The phosphorylation of downstream effectors MEK-ERK /PI3K-AKT was downregulated. In vivo antitumor activity assays showed that the RGD-p21Ras-scFv inhibited the proliferation of colorectal cancer cell lines. CONCLUSION: RGD-p21Ras-scFv prokaryotic expressed on pilot-scale could inhibited Ras-driven colorectal cancer growth by partially blocking p21Ras-GTP and might be able to be a hidden therapeutic antibody for treating RAS-driven tumors.

MCM6 is a Poor Prognostic Biomarker and Promotes Progression in Breast Cancer.

BACKGROUND: Breast cancer is the commonest global malignancy and the primary cause of carcinoma death. MCM6 is vital to carcinogenesis, but the pathogenesis of MCM6 remains unclear. METHODS: MCM6 expression in patients with breast cancer was examined through The Cancer Genome Atlas (TCGA) database, immunohistochemistry, Quantitative Real-Time PCR (qRT‒PCR) and Western blotting. The prognostic factors were assessed by the Kaplan‒Meier method and Cox regression. On the basis of the key factors selected by multivariable Cox regression analysis, a nomogram risk prediction model was adopted for clinical risk assessment. The TCGA database was utilized to determine how MCM6 is correlated with chemotherapy sensitivity, immune checkpoint-related genes (ICGs), tumor-infiltrating immune cells, along with tumor mutation burden (TMB) and methylation. The impact of MCM6 on carcinoma cells was investigated in terms of proliferation, cell cycle as well as migrating and invasive behavior through CCK assays, flow cytometry, wound healing assays, Transwell assays and xenotransplantation experiments. RESULTS: MCM6 expression was upregulated, which is closely associated with the size of the tumor (p = 0.001) and lymph node metastasis (p = 0.012) in patients with breast cancer. Multivariate analysis revealed MCM6 to be an independent risk factor for prognosis in patients with breast carcinoma. The nomograph prediction model included MCM6, age, ER, M and N stage, which displayed good discrimination with a C index of 0.817 and good calibration. Overexpression of MCM6 correlated with chemotherapy sensitivity, immune checkpoint-related genes (ICGs), tumor-infiltrating immune cells, tumor mutation burden (TMB), and methylation. Silencing MCM6 significantly inhibited proliferation, prolonged the G1 phase of the cell cycle, and restrained the proliferation, migration and invasive behavior of cancerous cells and inhibited tumor growth in vivo. CONCLUSIONS: Our research shows that MCM6 is highly expressed in breast cancer and can be used as an independent prognostic factor, which is expected to become a new target for the treatment of breast cancer in the future.