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

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 contributes to invasive motility in human breast cancer cells.

Cancer metastasis involves multiple steps including detachment of the metastatic cells from neighboring cells, the acquisition of motility and invasion to other tissue. All of these steps require the reorganization of the actin cytoskeleton. In this study, we found that the protein palladin, a molecular scaffold with an important function in actin organization, is expressed at higher overall levels in tumors compared with benign breast tissue, and also expressed significantly higher in four invasive breast cancer cell lines when compared with four non-invasive cell lines. In addition, we found that palladin plays a key role in the formation of podosomes. Podosomes are actin-rich structures that function in adhesion and matrix degradation, and have been found in many invasive cell types. Our results show that phorbol ester treatment stimulated the formation of palladin-containing podosomes in invasive, but not in non-invasive cell lines. More importantly, palladin knockdown resulted in decreased podosome formation and a significant reduction in transwell migration and invasive motility. Palladin overexpression induced podosome formation in the non-invasive MCF7 cells, which are otherwise unable to form podosomes, suggesting that palladin plays a critical role in the assembly of podosomes. Overall, these results indicate that palladin overexpression contributes to the invasive behavior of metastatic cells.

Influence of protein nutrition on calpain activity of mouse kidney: modulation by calpastatin.

The effect of protein depletion and refeeding with a normal diet on calpain activity was examined in mouse kidney soluble homogenate. In terms of units per gram of protein, it increased 2.9 times with depletion and decreased upon refeeding. After a DEAE-Sephacel chromatography, the homogenate yielded three enzymatic activities. Their sum, assessed as total calpain activity, was higher than the activity measured before fractionation and did not appreciably change during protein depletion and refeeding. Because the proportion of total activity displayed by the complete homogenate increased with depletion and decreased with refeeding, a low calpastatin content in depleted kidney was envisaged. This was confirmed by direct estimations: depleted kidney had 6 times less calpastatin compared to both normal and 16 h refed tissue. We concluded that a decrease in calpastatin content contributes to an increased calpain activity related to degradable protein in protein depleted kidney. In view of this, it seems not unlikely that the in vivo rate of protein breakdown depicted by kidney during protein depletion and refeeding is in part effected through modulation of the calpain proteolytic system.

Effect of dietary level of protein on the activity of mouse liver calpain.

The effect of protein depletion and refeeding with a normal diet on mouse liver soluble homogenate calpain activity was studied. It was unchanged when expressed in terms of whole liver (units/liver). However, when expressed in terms of degradable protein (units/mg protein) it increased with depletion and decreased with refeeding. DEAE Sephacel chromatography of soluble homogenate yielded three calpain activities which were eluted at 0.04, 0.16 and 0.23 M NaCl, respectively. On the basis of whole liver, they decreased with depletion and increased with refeeding. Immunochemical analysis revealed similar changes in the mass of the calpain eluted with 0.16 M NaCl. The sum of these three activities (total liver calpain activity) was higher than the activity displayed by the soluble homogenate, indicating that they were separated from calpastatin. Furthermore, the percentage of total calpain activity displayed by soluble homogenate increased with depletion and decreased with refeeding, suggesting that depleted liver had the lowest calpastatin content. This was confirmed by direct measurements which indicated that depleted homogenate had in average 5.5 and 3.1 times less calpastatin compared to normal and 16 hours refed liver, respectively. It is concluded that a remarkable decrease in calpastatin content maintained unchanged whole liver soluble homogenate calpain activity during protein depletion and refeeding and contributes to an increased calpain activity related to degradable protein in depleted livers. This increase is in accordance with the high in vivo rate of protein breakdown depicted by these livers.