Expression of PTRF in PC-3 Cells Modulates Cholesterol Dynamics and the Actin Cytoskeleton Impacting Secretion Pathways

Abstract

Expression of caveolin-1 is up-regulated in prostate cancer metastasis and is associated with aggressive recurrence of the disease. Intriguingly, caveolin-1 is also secreted from prostate cancer cell lines and has been identified in secreted prostasomes. Caveolin-1 is the major structural component of the plasma membrane invaginations called caveolae. Co-expression of the coat protein Polymerase I and transcript release factor (PTRF) is required for caveolae formation. We recently found that expression of caveolin-1 in the aggressive prostate cancer cell line PC-3 is not accompanied by PTRF, leading to noncaveolar caveolin-1 lipid rafts. Moreover, ectopic expression of PTRF in PC-3 cells sequesters caveolin-1 into caveolae. Here we quantitatively analyzed the effect of PTRF expression on the PC-3 proteome using stable isotope labeling by amino acids in culture and subcellular proteomics. We show that PTRF reduced the secretion of a subset of proteins including secreted proteases, cytokines, and growth regulatory proteins, partly via a reduction in prostasome secretion. To determine the cellular mechanism accounting for the observed reduction in secreted proteins we analyzed total membrane and the detergent-resistant membrane fractions. Our data show that PTRF expression selectively impaired the recruitment of actin cytoskeletal proteins to the detergentresistant membrane, which correlated with altered cholesterol distribution in PC-3 cells expressing PTRF. Consistent with this, modulating cellular cholesterol altered the actin cytoskeleton and protein secretion in PC-3 cells. Intriguingly, several proteins that function in ER to Golgi trafficking were reduced by PTRF expression. Taken together, these results suggest that the noncaveolar caveolin-1 found in prostate cancer cells generates a lipid raft microenvironment that accentuates secretion pathways, possibly at the step of ER sorting/exit. Importantly, these effects could be modulated by PTRF expression. Molecular & Cellular Proteomics 11: 10.1074/mcp.M111.012245, 1-13, 2012.Prostate cancer is the most commonly diagnosed cancer in men and the second leading cause of cancer related deaths in developed countries. Although localized prostate cancer is treatable, metastatic recurrence, together with development of androgen-independence, leads to advanced prostate cancer, which currently has a low survival rate. Caveolin-1, a cholesterol-binding integral membrane protein, has been shown to be up-regulated in prostate cancer metastasis and is associated with androgen-independence and aggressive recurrence of the disease (1). A prostate cancer mouse model showed that genetic ablation of caveolin-1 delays the onset of advanced prostate cancer (2), underscoring its importance in prostate cancer progression. Hence understanding caveolin-1 action in prostate cancer progression is crucial for the design of novel intervention strategies to manage this devastating disease.Caveolin-1 is a major structural component of caveolae, specialized lipid raft microdomains of the pl...

Caveolae and cancer: A new mechanical perspective

“…[123,126] These effects are mediated through reduced expression of matrix metalloproteases and altered secretion of proteases, cytokines and growth regulatory proteins. [127,128] Furthermore, re-expression of Cavin1 in PC3 cells (human prostate adenocarcinoma) that have lost Cavin1 expression reduces the migratory capacity of these cells and tumor growth and metastasis in vivo. [126,129] Similarly, Cavin1 expression is decreased in lung cancer.…”

Reducing the cost of semi-automated in-gel tryptic digestion and GeLC sample preparation for high-throughput proteomics

“…We have utilized this protocol for GeLC-MS/MS in several scientific studies [10][11][12][13][14][15][16][17][18]; here we report the results of a series of experiments conducted to determine the reproducibility, degree of contamination and time savings of the semi-automated protocols for IGD and GeLC. For these direct comparisons, the protocols were performed with the same incubation times, consumables and reagents.…”

A computational approach to uncover cholesterol-rich membrane micro-domain function in health and disease

“…One thousand protein-protein interaction (PPI) networks were generated by fetching experimentally validated interactions between 230 randomly sampled proteins (the total number of proteins identified in ovarian raft) from multiple datasets: the complete human proteome, human raft proteins (from RaftProt) 238 along with cancer raft datasets belonging to RCC 243 and prostate cancer lipid raft proteome. 20 Each of the thousand networks was built by retrieving interactions associated with each random protein set from the PINA2 human interactome. For each PPI network, I calculated the total number of nodes, total number of edges and average degree distribution using the igraph package in R (www.r-project.org) 378 .…”

“…Lipid rafts were prepared using the detergent resistant membrane (DRM) extraction method as previously described 20,106,144,258,[368][369][370] . Equal amount of DRM proteins prepared from each cell line was combined to make a Triplex SILAC mixed sample.…”

“…The formation of caveolae is regulated by proteins of the caveolin and cavin families [19][20] . They can respond to plasma membrane stresses and external stimuli and therefore considered as sensors of plasma membrane.…”