Carbonic anhydrase XII expression is linked to suppression of Sonic hedgehog ligand expression in triple negative breast cancer cells G. Guerrini a , J. Durivault c , I. Filippi a , M. Criscuoli a , S. Monaci a , J. Pouyssegur c, d , A. Naldini a , F. Carraro b , S.K. Parks c, * a Department of Molecular and Developmental Medicine, Cellular and Molecular Physiology Unit, University of Siena, Siena, Italy b Department of Medical Biotechnologies, University of Siena, Siena, Italy c Biomedical Department, Centre Scientifique de Monaco, Monaco, Principality of Monaco d Universit eC^ ote D'Azur (UCA), Nice, France article info Article history: Received 3 June 2019 Accepted 7 June 2019 Available online xxx Keywords: Hedgehog (Hh) pathway CAXII GLI1 Sonic hedgehog ligand Tumor aggressiveness abstract Aberrant activity of the hedgehog (Hh) pathway is prevalent in pathologies such as cancer. Improved understanding of Hh activity in the aggressive tumor cell phenotype is being pursued for development of targeted therapies. Recently, we described a link between Hh activity and carbonic anhydrase XII (CAXII) expression. Extracellular facing CAs (IX/XII) are highly expressed in hypoxia, contribute to tumor pH regulation and are thus of clinical interest. Here we have extended the investigation of potential in- teractions between Hh activity and CAXII utilizing genomic disruption/knockout of either GLI1 (the main transcriptional factor induced with Hh activity) or CAXII in the triple negative breast cancer cell lines MDA-MB-231 and BT-549. Knockout of GLI1 and CAXII significantly decreased hallmarks of tumor aggressiveness including proliferation and migration. Most intriguingly, CAXII knockout caused a massive induction of the Sonic hedgehog (Shh) ligand expression (gene and protein). This novel finding indicates that CAXII plays a potential role in suppression of Shh and may act in a feedback loop to regulate overall Hh activity. Enhanced knowledge of these CA-Hh interactions in future studies may be of value in understanding this currently ‘incurable’ subclass of breast cancer. © 2019 Elsevier Inc. All rights reserved. 1. Introduction Aberrant reactivation of developmental pathways such as the hedgehog (Hh) pathway often results in pathologies including growth defects and cancer. The canonical Hh pathway involves binding of the Sonic hedgehog (Shh) ligand to the receptor Patched (PTCH). This triggers the activation of Smoothened (SMO), the main transducer of the pathway, leading to activation of the Glioma- associated oncogene (GLI1-3) transcription factors and induction of Hh target genes. Many other oncogenic pathways that trigger proliferation, cytoskeleton reorganization, migration and Warburg metabolism are referred to as non-canonical Hh pathways because they are modulated by one of the components of the Hh pathway but differ from the canonical one [1 ,2] and can be SMO-dependent or -independent. Carbonic anhydrases (CAs) are a class of metallo-enzymes that catalyze the reaction CO 2 þH 2 O % HCO 3 -þ H þ and extracellular facing CAs play an important role in tumor pH regulation [3]. In addition to promoting cell survival and migration, CAs have been implicated in the priming of the cancer stem cell niche [4]. Mem- brane bound CAIX and XII expression is triggered mainly by hyp- oxia in tumor cells, in order to maintain a reverse pH gradient (alkaline intracellular and acidic extracellular) [5], which contrib- utes to the metastatic phenotype. Breast cancers (BCs) account for 25.1% of all cancers worldwide [6] and exhibit several sub- classifications. Although ER/PR positive BCs respond to hormone therapies resulting in a better prognosis for patients, triple negative breast cancers (TNBCs) lack any effective treatment strategies. TNBCs are highly invasive (mainly to bone, lungs, liver and brain) and metastases can be already present at the first stage of BC. The Hh pathway is reactivated in BC compared to the normal mammary gland epithelium [7 ,8] and GLI1 overexpression is associated with poor overall survival [9]. Moreover, the Hh pathway is often aber- rantly activated in the progression of metastatic TNBCs, in a ligand- dependent manner that requires the secreted Shh ligand, which in * Corresponding author. E-mail address: sparks@centrescientifique.mc (S.K. Parks). Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc https://doi.org/10.1016/j.bbrc.2019.06.040 0006-291X/© 2019 Elsevier Inc. All rights reserved. Biochemical and Biophysical Research Communications xxx (xxxx) xxx Please cite this article as: G. Guerrini et al., Carbonic anhydrase XII expression is linked to suppression of Sonic hedgehog ligand expression in triple negative breast cancercells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.06.040