Elevated ITGA5 facilitates hyperactivated mTORC1-mediated progression of laryngeal squamous cell carcinoma via upregulation of EFNB2

Abstract

Background: Laryngeal squamous cell carcinoma (LSCC) is one of the most common malignant tumors of the head and neck, and it has shown increasing incidence and mortality. The mechanistic target of rapamycin complex 1 (mTORC1) is frequently dysregulated in LSCC, but its underlying mechanisms remain unclear. Methods: Establishment of a novel LSCC cell line using primary LSCC tumor tissues with dysregulated mTORC1 activity and then stable knockdown of Raptor (an mTORC1 specific component) in this cell line. Transcriptomic sequencing, quantitative real-time PCR, western blot analysis, and immunofluorescence assays were used to identify the crucial downstream effector of mTORC1. A series of experiments were conducted to investigate the functions and underlying mechanisms of the mTORC1 target gene in LSCC progression. Clinical LSCC samples were used to evaluate the association of mTORC1 and its downstream targets with clinicopathological features and patient prognosis. Finally, the influence on cisplatin (CDDP) sensitivity upon depletion of the mTORC1 target gene was assessed using a cell culture system, a cell line-derived xenograft (CDX) model, and a patient-derived xenograft (PDX) model. Results:We successfully established a novel LSCC cell line with hyperactivated mTORC1 activity and then identified integrin subunit alpha 5 (ITGA5) as a novel functional downstream effector of mTORC1 in the progression of LSCC. Elevated ITGA5 promotes LSCC progression through augmentation of ephrin-B2 (EFNB2). Clinical data analysis indicated that the activation of the mTORC1-ITGA5-EFNB2 signaling pathway is associated with malignant progression and poor prognosis of LSCC patients. Inhibition of ITGA5 significantly sensitized LSCC cells to CDDP. Conclusions: Our findings highlight a novel molecular mechanism for the tumorigenesis driven by deregulated mTORC1 signaling in LSCC, suggesting that the ITGA5-EFNB2 axis may be a therapeutic target for the treatment of mTORC1-related LSCC.

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“…Western blot analysis has been described previously [ 24 ]. Briefly, cells and tissues were denatured and electrophoresed by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes (Millipore, Billerica, MA, USA).…”

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“…Immunoblotting was performed according to standard procedures [ 24 ]. Briefly, cell lysates were prepared using NuPAGE 4-12% Bis-Tris gels and subsequently transferred onto PVDF membranes (Millipore, USA).…”

Integrins as the pivotal regulators of cisplatin response in tumor cells

“…ITGA5 knockdown in LSCC cells resulted in Jagged1 down regulation and Notch axis suppression. The mTORC1-mediated ITGA5 up regulation increased the levels of EFNB2 expressions via activation of the Jagged1/Notch axis in LSCC cells [ 81 ]. Neuropilin-1 (NRP1) acts as a co-receptor for VEGF during vascular formation and for class-3 semaphorin proteins through neuronal guidance [ 82 – 84 ].…”