Canonical and Non Canonical Activation of Hedgehog Pathway in Multiple Myeloma
Authors: Blotta Simona , Podar Klaus , Tai Yu-Tzu , Roccaro Aldo M. , Gorgun Gullu , Franco Brunella , Mitsiades Constantine S. , Venuta Salvatore , Anderson Kenneth C. 
Published: April 2020

Abstract
To identify genes or Tumor-Associated Antigens (TAA) potentially involved in the transition from Monoclonal Gammopathy of Undetermined Significance (MGUS) to Multiple Myeloma (MM) and in the MM pathogenesis, we used SEREX and screened serum of MGUS and MM patients with a MM cDNA library. We identified 10 novel antigens and focused our attention on Oral-facial-digital syndrome 1 gene (Ofd1) due to a specific antibody response in MGUS (20.6%) as well as post-transplant MM patients (27.2%). Ofd1 is an embryonal antigen with an important role in the organogenesis and fetal survival; however, in the adult life its expression decreases significantly and is restricted to few tissues. Additionally it is a basal body/centrosome-related protein and a component of Tip60-HAT complex which is involved in cilia formation and functional regulation of Hedgehog (Hh)- and Wnt- pathways in mouse models. We first studied the expression of Ofd1 by western blotting analysis and observed that 11 MM cell lines and 4 primary MM patient cells express high levels compared with PBMCs. To define its role in MM pathogenesis, we specifically knocked-down Ofd1 using 100nM and 200nM of specific siRNA in MM1s cell line. Time-dependent but dose-independent inhibition of Ofd1 does not affect viability and proliferation of MM1s at 48h, as assayed by MTT and thymidine-uptake, respectively. Since mutations in proteins of cilia may block the Hh-signal transduction downstream to Smoothened (Smo) but upstream to Gli transcription factors, we next evaluated the effects of Ofd1 inhibition on the expression of Gli1 and Patched1 (Ptch1), hallmarks of Hh-activity. Western blotting analysis showed that specific knock-down of Ofd1 leads to down-regulation of Gli1 or Ptch1, and β-catenin. Therefore Ofd1 has a role in the functional regulation of Hh- and Wnt-pathway, and its overexpression might lead to constitutive activation of these tumorigenic pathways. These data strongly indicate the existence of an alternative and not canonical activation of Hh-pathway in MM. We also investigated if the canonical way is active in MM. To demonstrate that Hh-pathway is important in CD138+ MM patient cells, we used double staining with antibody anti-CD138 and anti-Sonic (Shh) to show that MM cells co-express Shh, the most common Hh-ligand, suggesting that MM cells might have autocrine and/or paracrine Hh-activity. We confirmed the expression of Gli1 and Ptch1, by western blotting, in 10 on 11 MM cell lines tested and using antibody anti-Gli1 we demonstrated, by immunofluorescence, predominant nuclear localization of Gli1 suggesting constitutive Hh-pathway activation. Treatment with 5μM of a specific Smo-inhibitor for 12–24 and 36 hours, induces down-regulation of Gli1 and Ptch1 in 50% MM cell lines. While down-regulation of Ptch1, a Gli-target gene, is an early event starting at 12 hours, down-regulation of Gli1 starts later at 36 hours. Therefore Gli1 is not the most important transducer of Hh-signaling in MM. In 50% MM cell lines, however, we didn’t observe any down-regulation of Gli1 and Ptch1 after treatment with Smo-inhibitor; in these MM cell lines, the predominant nuclear localization of Gli1 is completely abrogated by treatment for 24 hours with 10μM of forskolin, a compound inhibiting the nuclear translocation of Gli1 via PKA. Taken together our data show canonical as well as alternative not canonical activation of Hh-pathway associated with overexpression of ciliary protein Ofd1. Further delineation of the role of primary cilia in regulating cellular response to Hh-signaling in MM will both define their role in disease pathogenesis and support novel therapies targeting Hh-signaling to improve patient outcome in MM.