Chiara Cavallini

A bstract: Tumor-associated macrophages (TAM) enhance cancer progression by promoting angiogenesis, extracellular matrix (ECM) remodeling, and immune suppression. Nerve infiltration is a hallmark of various cancers and is known to directly contribute to tumor growth. However, the role of TAM in promoting intratumoral nerve growth remains poorly understood. In this study, we demonstrate that TAM expressed a distinct “neural growth” gene signature. TAM actively enhance neural growth within tumors and directly promote neurites outgrowth. We identify secreted phosphoprotein 1 (Spp1) as a key mediator of TAM-driven neural growth activity, which triggers neuronal mTORC2 signaling. Leveraging this new neural growth function, which added to the TAM wound healing properties, we explored TAM potential to repair central nervous system. Adoptive transfer of in vitro -generated TAM in a severe complete-compressive-contusive spinal cord injury (scSCI) model, not only repaired the damaged neural parenchyma by improving tissue oxygenation, ECM remodeling, and dampening chronic inflammation, but also resulted in neural regrowth and partial functional motor recovery. Proteomic analysis and subsequent functional validation confirmed that TAM-induced spinal cord regeneration is mediated through the activation of neural mTORC2 signaling pathways. Collectively, our data unveil a previously unrecognized role of TAM in tumor innervation, neural growth, and neural tissue repair.

YAP and TAZ are central determinants of malignancy; however, their functions remain still undruggable. We identified TGFβ-activated kinase 1 (TAK1) as a central hub integrating the most relevant signals sustaining pancreatic cancer aggressiveness and chemoresistance. Glycogen synthase kinase (GSK)3 is known to stabilize TAK1, and its inhibition causes a reduction in TAK1 levels. Here, we hypothesized that TAK1 could sustain YAP/TAZ program, and thus, modulation of TAK1 expression through the inhibition of GSK3 could impair YAP/TAZ functions in pancreatic cancer.Differentially expressed transcripts between pancreatic cancer cells expressing scramble or TAK1-specific shRNA were annotated for functional interrelatedness by ingenuity pathway analysis. TAK1 expression was modulated by using different GSK3 inhibitors, including LY2090314. In vivo activity of LY2090314 alone or in combination with nab-paclitaxel was evaluated in an orthotopic nude mouse model.Differential gene expression profiling revealed significant association of TAK1 expression with HIPPO and ubiquitination pathways. We measured a significant downregulation of YAP/TAZ and their regulated genes in shTAK1 cells. TAK1 prevented YAP/TAZ proteasomal degradation in a kinase independent manner, through a complex with TRAF6, thereby fostering their K63-ubiquitination versus K48-ubiquitination. Pharmacologic modulation of TAK1 by using GSK3 inhibitors significantly decreased YAP/TAZ levels and suppressed their target genes and oncogenic functions. In vivo, LY2090314 plus nab-paclitaxel significantly prolonged mice survival duration.Our study demonstrates a unique role for TAK1 in controlling YAP/TAZ in pancreatic cancer. LY2090314 is a novel agent that warrants further clinical development in combination with nab-paclitaxel for the treatment of pancreatic cancer.

Figure S3 shows: (A) Immunoblot showing total K63-linked ubiquitination levels in AsPC1, Panc1 and MDA-Panc28 ctrl and shTAK1 cell lines. (B) Immunoblot analysis of YAP/TAZ immunoprecipitated complexes using the indicated antibodies.

Figure S6 shows: (A) Anchorage-independent cell growth of AsPC1shTAK1, Panc1shTAK1 and MDA-Panc28shTAK1 cells either treated or not with LY2090314 3μM for 72 hours, and allowed to form spheroids in ultra-low attachment plates. Histograms show spheroids diameter.(B) AsPC1shTAK1, Panc1shTAK1 and MDA-Panc28shTAK1 cells were seeded and either treated or not with LY2090314 3μM for 72 hours. Cells were then counted and seeded into the upper chamber of a transwell. Histograms show the percentage of migrated cells.

Figure S6 shows: (A) Anchorage-independent cell growth of AsPC1shTAK1, Panc1shTAK1 and MDA-Panc28shTAK1 cells either treated or not with LY2090314 3μM for 72 hours, and allowed to form spheroids in ultra-low attachment plates. Histograms show spheroids diameter.(B) AsPC1shTAK1, Panc1shTAK1 and MDA-Panc28shTAK1 cells were seeded and either treated or not with LY2090314 3μM for 72 hours. Cells were then counted and seeded into the upper chamber of a transwell. Histograms show the percentage of migrated cells.

Figure S4 shows: (A) Immunoblot analysis of the indicated cell lines treated with increasing amounts of LiCl for 72h. (B) mRNA expression levels of the indicated genes over β-actin in the indicated cell lines treated with NaCl or LiCl 20mM for 72h. (C) The indicated cell lines were treated with 3μM LY2090314 for 72 hours and cellular extracts were subjected to immunoblot with the indicated antibodies.

Figure S5 shows: (A) Immunoblot analyses of FLO-1 and KYAE1 esophageal cancer cell lines treated with the GSK3 inhibitor LY2090314 3μM for 72 hours. Cell extracts were subjected to immunoblot with the indicated antibodies. (B) mRNA expression levels of the indicated YAP/TAZ target genes over β-actin from cells treated as in (A).

Figure S2 shows: (A) Immunoblot analyses of AsPC1 cells treated with the TAK1 kinase inhibitor (5Z)-7-Oxozeaenol for 48 hours. Cell extracts were subjected to immunoblot with the indicated antibodies. (B) The indicated cell lines were treated with the proteasome inhibitor MG-132 for 24 hours. Cell extracts were subjected to immunoblot with the indicated antibodies.

Figure S5 shows: (A) Immunoblot analyses of FLO-1 and KYAE1 esophageal cancer cell lines treated with the GSK3 inhibitor LY2090314 3μM for 72 hours. Cell extracts were subjected to immunoblot with the indicated antibodies. (B) mRNA expression levels of the indicated YAP/TAZ target genes over β-actin from cells treated as in (A).

Fig.S1 shows: (A) Chemical structure of the indicated compounds. (B) Immunoblot on cellular extracts from cells stably silenced for TAK1 expression. (C) Immunoblot on cellular extracts from cells transiently transfected with the indicated siRNA