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  • CSL is endowed with an intrinsic

    2022-07-04

    CSL is endowed with an intrinsic transcription-repressive function, and CAF-effector genes are induced as a consequence of CSL downmodulation (Hu et al., 2012, Procopio et al., 2015), as it can occur upon exposure to pro-carcinogenic stimuli such as UVA or smoke-derived compounds (Menietti et al., 2016). We show that loss of CSL transcription-repressive function is, by itself, not sufficient for induction of these genes and for the resulting tumor growth-enhancing activity of fibroblasts, for which GLI activation is needed. Aberrant activation of Hh/GLI signaling has been implicated in up to one third of all human cancers and is considered to be both a consequence and a driver of tumor-stromal interactions (Gupta et al., 2010, Ruiz i Altaba et al., 2002). Hh ligands have been implicated as paracrine mediators of cancer development (Rubin and de Sauvage, 2006). Earlier studies indicated that production of these molecules by colon, pancreatic, and ovarian cancer cells can promote tumor growth indirectly by activating Hh signaling in surrounding stroma (Theunissen and de Sauvage, 2009). However, recent evidence points to a restraining role of Hh stimulation in pancreatic and brdu cancer stroma (Özdemir et al., 2014, Rhim et al., 2014, Shin et al., 2014). In our own analysis of multiple HDFs, strains with or without CSL silencing, and skin-derived CAFs versus matched and unmatched controls, expression of Hh ligands was variously modulated. Similar variations were found in published profiles of CAFs from other cancer types. An attractive possibility is that, regardless of Hh levels, GLI activation in stromal fibroblasts can occur through increased activity of the ULK3 kinase, encoded by a direct CSL target gene upregulated by CSL downmodulation. ULK3 was recently proposed to fulfill the same central role in mammalian cells as the Drosophila Fused kinase in GLI2 activation (Maloverjan et al., 2010). Besides confirming these results, we found a detectable association of endogenous ULK3-GLI2 in HDFs and showed that increased ULK3 expression was both required and sufficient for GLI and CAF activation. Separately from this, we uncovered an interplay between CSL and ULK3 in control of autophagy that may apply, with context-dependent adjustments, to other cellular systems in which Notch signaling has been pharmacologically implicated (Barth and Köhler, 2014, Chen et al., 2016, Song et al., 2015, Yao et al., 2015). Importantly, while autophagy/mitophagy have been previously implicated in metabolic reprogramming of CAFs (Kim et al., 2011, Russell et al., 2013), our genetic evidence indicates that they are not significantly involved in the gene expression program leading to CAF activation. A much-needed development for cancer therapy is the identification of strategies affecting cancer stroma (Goruppi and Dotto, 2013). As in HDFs with CSL silencing and CAFs from skin SCCs, analysis of publically available gene expression profiles indicated that ULK3 expression is elevated in CAFs derived from several different cancer types, including HNSCC, prostate cancer, and breast cancer. Functionally, genetic suppression of ULK3 in patient-derived CAFs was sufficient to inhibit CAF-effector gene expression as well as GLI2 activation and suppressed the growth-enhancing and pro-tumorigenic properties of these cells on neighboring cancer cells. While similar effects were observed with a compound reported to inhibit ULK3 kinase activity, further translational studies await the identification of compounds with greater specificity. Thus, ULK3 kinase, by linking two key signaling pathways involved in CAF conversion, represents an interesting target for stroma-focused anti-cancer interventions.
    Experimental Procedures
    Author Contributions
    Acknowledgments We are grateful to Robert Lipinski for the MEFs gli1/2−/− and gli+/+ and to Noburo Mitzushima for the MEFs atg5−/− and atg5+/+. We thank Liuqing Yang for the P-GLI2 antibodies, Alessia Di Nardo for the shatg5 vector, Shanell Moijta for technical support brdu with histology, and Wolfram Hoetzenecker for providing SCC samples used in LCM. A.C. is supported by a Marie Curie fellowship from the Italian Association for Cancer Research and the European Union FP7 Marie Curie Program. This work was supported by grants from the NIH (R01AR039190 and R01AR064786; the content in this paper does not necessarily represent the official views of the NIH), the Swiss National Science Foundation (310030_156191/1), and the European Research Council (26075083) to G.P.D.