Blocking Hedgehog release from pancreatic cancer cells increases paracrine signaling potency

Abstract

Members of the Hedgehog (Hh) family of morphogens play crucial roles in development but are also involved in the progression of certain types of cancer. Despite being synthesized as hydrophobic dually lipid-modified molecules, and thus being strongly membraneassociated, Hh ligands are able to spread through tissues and act on target cells several cell diameters away. Various mechanisms that mediate Hh release have been discussed in recent years; however, little is known about dispersion of this ligand from cancer cells. Using co-culture models in conjunction with a newly developed reporter system, we were able to show that different members of the ADAM family of metalloproteinases strongly contribute to the release of endogenous bioactive Hh from pancreatic cancer cells, but that this solubilization decreases the potency of cancer cells to signal to adjacent stromal cells in direct co-culture models. These findings imply that under certain conditions, cancer-cell-tethered Hh molecules are the more potent signaling activators and that retaining Hh on the surface of cancer cells can unexpectedly increase the effective signaling range of this ligand, depending on tissue context.

Sonic hedgehog processing and release are regulated by glypican heparan sulfate proteoglycans

“…S3). This observation is in line with the described shedding of endogenous Shh from the PANC1 cell surface (Damhofer et al, 2015). We confirmed Shh-HA shedding from PANC1 cells by the nonphysiological and physiological release factors MβCD and Scube2, as indicated by enhanced electrophoretic product mobility in comparison to the cell-bound protein precursor (Fig.…”

“…2A). We and others have shown that proteolytic removal of N-and C-terminal lipidated peptides (called shedding) releases bioactive Shh morphogens from the surface of producing cells in vitro (Damhofer et al, 2015;Dierker et al, 2009;Jakobs et al, 2014;Ohlig et al, 2011). We further showed that because of the lack of the N-terminal Hhat acceptor cysteine (C25S or C25A in mouse Shh) (Hardy and Resh, 2012) non-palmitoylated Hh proteins require only C-terminal shedding for their solubilization, whereas most Shh N-termini are left intact (Jakobs et al, 2014).…”

“…Secreted lipidated Shh (nucleotides 1-1314, corresponding to amino acids 1-438) was generated in Bosc23 cells [a human 293 T derivative, an embryonic kidney cell line (Zeng et al, 2001)], PANC1 cells (Damhofer et al, 2015) and CHO cells affected in their ability to synthesize heparan sulfate. Secreted unlipidated ShhN (nucleotides 1-594, corresponding to amino acid 1-198 of murine Shh) was also produced.…”

Phenotypic drug profiling in droplet microfluidics for better targeting of drug-resistant tumors

“…Signaling through paracrine mechanisms between homotypic and heterotypic cells results in increasing cancer survival and therapeutic resistance. 32,33 Furthermore, apoptosis-related cellular processes can initiate or deactivate responses in surrounding cells. 34,35 Droplet microfluidics provides a suitable platform for characterizing cytotoxic drug uptake and apoptosis in single cells without affecting the functionality of adjacent cells.…”

Hedgehog Signaling Non-Canonical Activated by Pro-Inflammatory Cytokines in Pancreatic Ductal Adenocarcinoma

“…So, GLI1 expression leads to a positive feed-back loop, and it acts as a constitutive activator24. In most cases, GLI1 overexpression is considered as a symbol of HH pathway activation32. Other than canonical HH pathway, the non-canonical activation pathways are defined as any form of signaling relating to HH pathway components that differs from the usual HH pathway paradigm27.…”