Novel transduction of nutrient stress to Notch pathway by RasGRP3 promotes malignant aggressiveness in human esophageal squamous cell carcinoma

Abstract

In the process of enlarging of tumors, the dissolving tissue structures and remodeling endothelial cells for restoring gas exchange and nutritional support, further facilitate tumor cell invasion and metastasis. Activation of Ras plays a critical role in the development of esophageal squamous cell carcinoma (ESCC), but the underlying mechanisms remain poorly understood. We therefore investigated whether Ras guanyl-releasing protein 3 (RasGRP3), a Ras activator, could promote metastasis by inducing vascular regeneration and further epithelial-mesenchymal transition under nutrient stress (NS). In the present study, we explored that the accumulation of RasGRP3 regulated vascular endothelial growth factor-A production, co-stimulated Notch pathway with high expression of Notch intracellular domain (NICD) and Hes1. Moreover, ESCC cells under NS increased the expression of vimentin, Snail, Slug and MMP9 proteins; while inhibition of Notch activation by DAPT (a γ-secretase inhibitor) or RasGRP3-targeted RNA interference prevented from the effect. In conclusion, these findings provide a new insight into the upregulation of RasGRP3 involved in Notch pathway activation in the development of ESCC, especially under nutrient deprivation.

SREBP1 silencing inhibits the proliferation and motility of human esophageal squamous carcinoma cells via the Wnt/β‑catenin signaling pathway

“…A previous study reported that SREBP1 can suppress VEGF expression through indirect interaction with the proximal tandem Sp1 sites in human smooth muscle cells (54). VEGF-C, as a lymphangiogenic switch, is a specific factor involved in the invasiveness and metastasis of ESCC (7,11,39,55). Furthermore, according to the significant relationship between SREBP1 expression levels and lymphatic metastasis shown in the present study, SREBP1 was identified as a promoting factor regulating VEGF-C formation and secretion.…”

“…Tissue samples were collected during surgical resection after informed written consent had been provided by patients with ESCC (Table I). The patients were diagnosed by two pathologists in accordance with the World Health Organization classification without chemotherapy or radiotherapy before resection, as described previously (39). The 77 paired ESCC tumor and adjacent normal tissue samples that were 2 cm away from the tumors' edge, were collected between February 2018 and July 2019 at the Affiliated Hospital of Jiangsu University (Zhenjiang, China).…”

“…The 77 paired ESCC tumor and adjacent normal tissue samples that were 2 cm away from the tumors' edge, were collected between February 2018 and July 2019 at the Affiliated Hospital of Jiangsu University (Zhenjiang, China). The IHC staining procedure used to determine SREBP1 expression levels was performed as previously described (7,39). The sections were incubated with antibody against SREBP1 (1:100; catalog no.…”

“…ESCCs were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS; Biological Industries) and 1% penicillin-streptomycin (Gibco; Thermo Fisher Scientific, Inc.) at 37˚C in a thermostatic incubator containing 5% CO 2 . Immortalized human normal esophageal epithelial cells, Het-1A (BeNa Culture Collection), were maintained in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS and containing 1% penicillin-streptomycin, as described previously (39). Wnt/β-catenin inhibitor ICG-001 was obtained from Selleck Chemicals and dissolved in dimethyl sulfoxide as previously described (7,40).…”

“…Immunofluorescence was used to detect the location and expression of target proteins (E-cadherin, Vimentin) as described previously ( 7 , 39 ). Cells were grown on glass coverslips and fixed in 4% paraformaldehyde at 4°C for 30 min, rinsed three times with PBS, permeabilized with 0.5% Triton X-100 for 10 min and blocked at room temperature for 30 min using PBS containing 5% BSA.…”