Pancreatic secretory trypsin inhibitor causes autocrine-mediated migration and invasion in bladder cancer and phosphorylates the EGF receptor, Akt2 and Akt3, and ERK1 and ERK2.

Abstract

Pancreatic secretory trypsin inhibitor (PSTI) is expressed in most bladder carcinomas, where its pathophysiological relevance is unclear. Using recombinant normal sequence PSTI/tumor-associated trypsin inhibitor (TATI), a variant associated with familial pancreatitis (N34S), an active site-inactivated variant (R18/V19), and immunoneutralization and RNA interference-mediated knockdown techniques, we investigated the actions of PSTI/TATI on cell migration (wounding monolayers), collagen invasion (gel invasion assays), and proliferation (Alamar blue) on 253J, RT4, and HT1376 human bladder carcinoma cell lines. All three forms of PSTI/TATI stimulated migration twofold, and normal sequence PSTI/TATI showed synergistic promigratory effects when added with EGF. Addition of structurally unrelated soybean trypsin inhibitor had no promigratory activity. Similar results were seen using collagen invasion assays, although the active site mutated variant had no proinvasive activity, probably due to reduced Akt2 activation. PSTI/TATI did not stimulate proliferation despite acting, at least partially, through the EGF receptor, as effects of PSTI/TATI were truncated by the addition of an EGF receptor blocking antibody or the tyrosine kinase inhibitor tyrphostin. Cell lines produced endogenous PSTI/TATI, and PSTI/TATI RNA interference knockdown or the addition of PSTI/TATI, EGF receptor, or tyrphostin blocking agents reduced migration and invasion below baseline. PSTI/TATI induced phosphorylation of the EGF receptor, ERK1 and ERK2, Akt2 and Akt3, JNK1, MKK3, and ribosomal protein S6 kinase 1. This profile was more limited than that induced by EGF and did not include Akt1, probably explaining the lack of proproliferative activity. Our findings of autocrine stimulation and synergistic responses between EGF and PSTI/TATI at concentrations found in urine and tissue suggest that PSTI/TATI has pathophysiological relevance.

• Authors: Tania Marchbank, Asif Mahmood, Raymond J Playford
• Journal: American journal of physiology. Renal physiology (Am J Physiol Renal Physiol) Vol. 305 Issue 3 Pg. F382-9 (Aug 01 2013) ISSN: 1522-1466 [Electronic] United States
• PMID: 23698120 (Publication Type: Journal Article)

Chemical References

• Carrier Proteins
• RNA, Messenger
• Recombinant Proteins
• SPINK1 protein, human
• Trypsin Inhibitor, Kazal Pancreatic
• Collagen
• ErbB Receptors
• AKT2 protein, human
• AKT3 protein, human
• Proto-Oncogene Proteins c-akt
• MAPK1 protein, human
• Mitogen-Activated Protein Kinase 1
• Mitogen-Activated Protein Kinase 3

Topics

• Autocrine Communication (drug effects)
• Blotting, Western
• Carrier Proteins (pharmacology)
• Cell Line, Tumor
• Cell Movement (drug effects)
• Cell Proliferation
• Collagen (metabolism)
• Electrophoresis, Polyacrylamide Gel
• ErbB Receptors (metabolism)
• Female
• Humans
• Male
• Middle Aged
• Mitogen-Activated Protein Kinase 1 (metabolism)
• Mitogen-Activated Protein Kinase 3 (metabolism)
• Neoplasm Invasiveness
• Phosphorylation
• Proto-Oncogene Proteins c-akt (metabolism)
• RNA, Messenger (biosynthesis, genetics)
• Recombinant Proteins (biosynthesis)
• Trypsin Inhibitor, Kazal Pancreatic
• Urinary Bladder Neoplasms (pathology)