Abstract 2484: Non-canonical Hedgehog/Gli1 signaling drives lung adenocarcinoma stem cells survival and its targeting inhibits CSC-derived tumors
Authors: Po Agnese , Silvano Marianna , Miele Evelina , Eramo Adriana , Todaro Matilde , Capalbo Carlo , Salvati Valentina , Sette Giovanni , Cucchi Danilo , Besharat Zein M. , Canettieri Gianluca , Di Marcotullio Lucia , Screpanti Isabella , Stassi Giorgio , De Maria Ruggero , Zeuner Ann , De Smaele Enrico , Ferretti Elisabetta 
Published: July 2016

Abstract
Introduction: Lung Adenocarcinoma (AC) is the most frequent lung cancer histological subtype and is a leading cause of cancer-related death worldwide. Hedgehog/Gli (Hh/Gli) signaling pathway regulates lung development and its aberrant activation contributes to tumor pathogenesis and play a role in cancer stem cells (CSC) control.
We investigated oncogenic Hh/Gli signaling in AC-CSC.
Methods: human AC-CSC were derived from primary tumors. For in vitro studies AC-CSC were maintained in serum-free medium supplemented with EGF/bFGF. For in vivo experiments, immunocompromised mice were injected with AC-CSC. Gli1 inhibitor GANT61 was used both in vitro and in vivo (IP 40 mg/kg twice/we) treatments.
Results: Using mRNA datasets and tissue microarray of Non-small Cell Lung Cancer (NCSLC) samples we found that in AC Gli1 is expressed independently of its canonical activator Smoothened (Smo) expression level. Similarly, CSC were Gli1 positive regardless of Smo expression levels. Indeed, we found that SMO regulatory region is highly methylated in AC cells where Smo mRNA and protein are low.
Looking for Smo-independent regulation of GLI activity we found that NRP2/Erk and IGF1R/Erk axes regulate Gli1 in AC-CSC. Suppression of Gli1 with GANT61 in CSC resulted in impairment of cell growth and stemness features. We performed in-vivo experiments: CSC-derived xenograft from GANT61 treated mice showed impaired tumor growth, reduced proliferation and increased apoptosis. We next highlighted a tumor-stroma crosstalk. A positive autocrine oncogenic feedback loop sustains Gli1 in AC-CSC since we found that the CSC expressed NRP2 and its ligands VEGF-A and VEGF-C. A paracrine positive loop also sustain Hh/Gli signaling in AC where Cancer-Associated Fibroblasts (CAFs) harbor Hh/Gli1 canonical pathway that regulates the expression of VEGF-A, VEGF-C and IGF2 ligands of NRP2 and IGF1R respectively.
Conclusion: Our findings suggest a non-canonical activation of Hh/Gli pathway in AC. In this context, GLI1 transcription factor is effectors of NRP2/Erk and/or IGF1R/Erk signaling inputs.
Our results have implications for the understanding of AC development and sustain the inclusion of target therapy acting downstream of Smo level in the design of AC treatment.
Citation Format: Agnese Po, Marianna Silvano, Evelina Miele, Adriana Eramo, Matilde Todaro, Carlo Capalbo, Valentina Salvati, Giovanni Sette, Danilo Cucchi, Zein M. Besharat, Gianluca Canettieri, Lucia Di Marcotullio, Isabella Screpanti, Giorgio Stassi, Ruggero De Maria, Ann Zeuner, Enrico De Smaele, Elisabetta Ferretti. Non-canonical Hedgehog/Gli1 signaling drives lung adenocarcinoma stem cells survival and its targeting inhibits CSC-derived tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2484.