Palladin promotes invasion of pancreatic cancer cells by enhancing invadopodia formation in cancer-associated fibroblasts

Abstract

The stromal compartment surrounding epithelial-derived pancreatic tumors is thought to have a key role in the aggressive phenotype of this malignancy. Emerging evidence suggests that cancer-associated fibroblasts (CAFs), the most abundant cells in the stroma of pancreatic tumors, contribute to the tumor’s invasion, metastasis and resistance to therapy, but the precise molecular mechanisms that regulate CAFs behavior are poorly understood. In this study, we utilized immortalized human pancreatic CAFs to investigate molecular pathways that control the matrix-remodeling and invasion-promoting activity of CAFs. We showed previously that palladin, an actin-associated protein, is expressed at high levels in CAFs of pancreatic tumors and other solid tumors, and also in an immortalized line of human CAFs. In this study, we found that short-term exposure of CAFs to phorbol esters reduced the number of stress fibers and triggered the appearance of individual invadopodia and invadopodial rosettes in CAFs. Molecular analysis of invadopodia revealed that their composition resembled that of similar structures (that is, invadopodia and podosomes) described in other cell types. Pharmacological inhibition and small interfering RNA knockdown experiments demonstrated that protein kinase C, the small GTPase Cdc42 and palladin were necessary for the efficient assembly of invadopodia by CAFs. In addition, GTPase activity assays showed that palladin contributes to the activation of Cdc42. In mouse xenograft experiments using a mixture of CAFs and tumor cells, palladin expression in CAFs promoted the rapid growth and metastasis of human pancreatic tumor cells. Overall, these results indicate that high levels of palladin expression in CAFs enhance their ability to remodel the extracellular matrix by regulating the activity of Cdc42, which in turn promotes the assembly of matrix-degrading invadopodia in CAFs and tumor cell invasion. Together, these results identify a novel molecular signaling pathway that may provide new molecular targets for the inhibition of pancreatic cancer metastasis.

Palladin promotes assembly of non-contractile dorsal stress fibers through VASP recruitment

“…Palladin is a large, multidomain protein, which has an important role in the assembly of actin-rich structures, such as podosomes, invadopodia and stress fibers (Parast and Otey, 2000;Goicoechea et al, 2009;Goicoechea et al, 2013). Consequently, palladin regulates cell migration to drive tumor invasion and metastasis (Goicoechea et al, 2009;Goicoechea et al, 2010); however, the mechanisms by which palladin contributes to the assembly and/or dynamics of cellular actin filament arrays has remained elusive.…”

The microenvironment controls invadosome plasticity

“…Thus, owing to the dynamic nature of this complex, identifying the full molecular identity of these structures is challenging (Artym et al, 2015;Beaty et al, 2013;Sharma et al, 2013;Valenzuela-Iglesias et al, 2015). Currently, there is no study that is able to define the invadosome proteome as accurately as has been done for focal adhesions (Goicoechea et al, 2014;Robertson et al, 2015). In addition, there are only few studies describing the very existence and role of invadopodia in vivo.…”

“…Some non-hematopoietic cells, including endothelial cells, can also form invadosomes upon appropriate stimulation, such as expression of a constitutively active form of Cdc42 (Moreau et al, 2003), upon c-Src activation or following treatment with phorbol esters or sodium fluoride (Goicoechea et al, 2014;Kaverina et al, 2003;Tatin et al, 2010Tatin et al, , 2006, as well as after treatment with various cytokines, such as transforming growth factor β (TGF-β), vascular endothelial growth factor (VEGF) and tumour necrosis factor α (TNFα, also known as TNF) (Osiak et al, 2005;Varon et al, 2006b). Similarly, cancer cells can exhibit either constitutive or inducible invadosomes (Saltel et al, 2011).…”

Differential Quantitative Proteomics of Human Microvascular Endothelial Cells 1 by iTRAQ Reveals Palladin to be a New Biomarker During TGF-?1 Induced Endothelial Mesenchymal Transition

“…Palladin levels are up-regulated in metastatic cancer cells and play an important role in organizing actin arrays within migrating cells and in invasive motility [32]. The palladin gene was found within a cluster of invasion-specific genes in pancreatic and colorectal cancers [40][41][42]. The palladin protein levels increase significantly in human dermal fibroblasts in response to TGF-β 1 treatment, and during myofibroblast differentiation in the presence of TGF-β1 [39].…”