Membrane-bound full-length Sonic Hedgehog identifies cancer stem cells in human non-small cell lung cancer

Abstract

// Etienne Giroux Leprieur 1, 2, 3, * , Bhairavi Tolani 1, * , Hui Li 1, * , Fleur Leguay 1 , Ngoc T. Hoang 1 , Luis A. Acevedo 1 , Joy Q. Jin 1 , Hsin-Hui Tseng 1 , Dongsheng Yue 1, 4 , Il-Jin Kim 1 , Marie Wislez 5 , Changli Wang 4 , David M. Jablons 1 and Biao He 1 1 Thoracic Oncology Program, Department of Surgery, Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, CA, USA 2 Department of Respiratory Diseases and Thoracic Oncology, APHP - Ambroise Pare Hospital, Boulogne-Billancourt, France 3 EA 4340, UVSQ, Paris-Saclay University, Boulogne-Billancourt, France 4 Department of Lung Cancer, Lung Cancer Center, Tianjin Medical University Cancer Institute and Hospital, Tianjin, China 5 Sorbonne University, UPMC GRC-04 Theranoscan, Department of Respiratory Diseases, APHP - Tenon Hospital, Paris, France * These authors have contributed equally to this work Correspondence to: Biao He, email: biao.he@ucsfmedctr.org Keywords: non-small cell lung cancer (NSCLC), Sonic Hedgehog (Shh), cancer stem cells (CSCs), GDC0449, chemo-resistance Received: July 23, 2017      Accepted: September 05, 2017      Published: October 10, 2017 ABSTRACT The mechanism of Sonic Hedgehog (Shh) pathway activation in non-small cell lung cancer (NSCLC) is poorly described. Using an antibody against the Shh C-terminal domain, we found a small population of Shh-positive (Shh+) cells in NSCLC cells. The objective of this study was to characterize these Shh+ cells. Shh+ and Shh- cells were sorted by using Fluorescence Activated Cell Sorting (FACS) on 12 commercial NSCLC cell lines. Functional analyses on sorted cells were performed with gene expression assays (qRT-PCR and microarray) and cells were treated with cytotoxic chemotherapy and a targeted inhibitor of Shh signaling (GDC0449). We used in vivo models of nude mice inoculated with Shh+ and Shh- sorted cells and drug-treated cells. Finally, we confirmed our results in fresh human NSCLC samples (n=48) paired with normal lung tissue. We found that Shh+ cells produced an uncleaved, full-length Shh protein detected on the membranes of these cells. Shh+ cells exerted a paracrine effect on Shh- cells, inducing their proliferation and migration. Shh+ cells were chemo-resistant and showed features of cancer stem cells (CSCs) in vitro and in vivo . Pharmacological inhibition of the Shh pathway suppressed their CSC features. A high percentage of Shh+ cells was associated with poor prognosis in early-stage NSCLC patients. In conclusion, we describe for the first time the presence of an abnormal membrane-bound full-length Shh protein in human cancer cells that allows the identification of CSCs in vitro and in vivo .