Research Article: Tyrosine 23 Phosphorylation-Dependent Cell-Surface Localization of Annexin A2 Is Required for Invasion and Metastases of Pancreatic Cancer

Date Published: April 29, 2011

Publisher: Public Library of Science

Author(s): Lei Zheng, Kelly Foley, Lanqing Huang, Ashley Leubner, Guanglan Mo, Kelly Olino, Barish H. Edil, Masamichi Mizuma, Rajni Sharma, Dung T. Le, Robert A. Anders, Peter B. Illei, Jennifer E. Van Eyk, Anirban Maitra, Daniel Laheru, Elizabeth M. Jaffee, Lin Zhang.

Abstract: The aggressiveness of pancreatic ductal adenocarcinoma (PDA) is characterized by
its high metastatic potential and lack of effective therapies, which is the
result of a lack of understanding of the mechanisms involved in promoting PDA
metastases. We identified Annexin A2 (ANXA2), a member of the Annexin family of
calcium-dependent phospholipid binding proteins, as a new molecule that promotes
PDA invasion and metastases. We found ANXA2 to be a PDA-associated antigen
recognized by post-treatment sera of patients who demonstrated prolonged
survival following treatment with a PDA-specific vaccine. Cell surface ANXA2
increases with PDA development and progression. Knockdown of ANXA2 expression by
RNA interference or blocking with anti-ANXA2 antibodies inhibits in
vitro invasion of PDA cells. In addition, post-vaccination patient
sera inhibits in vitro invasion of PDA cells, suggesting that
therapeutic anti-ANXA2 antibodies are induced by the vaccine. Furthermore,
cell-surface localization of ANXA2 is tyrosine 23 phosphorylation-dependent; and
tyrosine 23 phosphorylation is required for PDA invasion. We demonstrated that
tyrosine 23 phosphorylation resulting in surface expression of ANXA2 is required
for TGFβ-induced, Rho-mediated epithelial-mesenchymal transition (EMT),
linking the cellular function of ANXA2 which was previously shown to be
associated with small GTPase-regulated cytoskeletal rearrangements, to the EMT
process in PDA. Finally, using mouse PDA models, we showed that shRNA knock-down
of ANXA2, a mutation at tyrosine 23, or anti-ANXA2 antibodies,
inhibit PDA metastases and prolong mouse survival. Thus, ANXA2 is part of a
novel molecular pathway underlying PDA metastases and a new target for
development of PDA therapeutics.

Partial Text: Pancreatic ductal adenocarcinoma (PDA) remains a lethal cancer with an overall 5-year
survival rate of <5% [1]. Inability to diagnose early, high metastatic potential, and drug resistance account for its low survival rate. Although it is well-established that the pathogenesis of PDAs follows stepwise stages that display increasing cellular atypia and accumulate clonal mutations or aberrant expression of oncogenes or tumor suppressor genes such as K-Ras, p16, p53, and DPC4/SMAD4[2], drugs that target these molecular abnormalities have not yet translated into improved clinical responses [3]. The aggressive nature of PDA is featured by its high incidence of metastases at the time of initial diagnosis and high incidence of early metastases following surgical resection. However, little is known about the molecular mechanisms underlying its invasion and metastatic processes. A better understanding of these mechanisms is essential for the development of innovative and improved treatments for PDA. ANXA2 was brought to our attention as a target of vaccine induced immune responses identified on a proteomic screen of PDA proteins using immunized sera from patients who demonstrated prolonged DFS. A previously published study links ANXA2 overexpression in pancreatic cancer tissue with rapid recurrence after gemcitabine adjuvant chemotherapy in postoperative patients [26]. In this study, we demonstrate three new findings that elucidate the role of ANXA2 in PDA invasion and metastases. First, localization of ANXA2 expression on the cell surface is required for PDA invasion in vitro and metastases formation in vivo. Second, Tyr23-phosphorylation is required for localization of ANXA2 on the PDA cell surface and subsequent PDA invasion and metastases formation. Importantly, knockdown of ANXA2 or inhibition with ANXA2 antibody therapy inhibits the metastatic process. Third, loss of ANXA2 expression or phosphorylation at Tyr23 leads to loss of TGFβ-Rho-mediated EMT in PDA cells. Taken together, these findings identify a new role for phosphorylated ANXA2 in mediating PDA cell invasion via Rho-regulated EMT and facilitating PDA metastases. Source: