Lanqing Huang

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

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.

Ectopic Expression of Vascular Cell Adhesion Molecule-1 as a New Mechanism for Tumor Immune Evasion

Immune escape is an important reason why the immune system cannot control tumor growth, but how escape variants emerge during immunotherapy remains poorly understood. Here, we identify a new mechanism of tumor immune escape using an in vivo selection strategy. We generated a highly immune-resistant cancer cell line (P3) by subjecting a susceptible cancer cell line (P0/TC-1) to multiple rounds of in vivo immune selection. Microarray analysis of P0 and P3 revealed that vascular cell adhesion molecule-1 (VCAM-1) is up-regulated in the P3-resistant variant. Retroviral transfer of VCAM-1 into P0 significantly increased its resistance against a vaccine-induced immune response. Analysis of tumors showed a dramatic decrease in the number of tumor-infiltrating cluster of differentiation 8(+) (CD8(+)) T cells in the tumors expressing VCAM-1. In vitro transwell migration assays showed that VCAM-1 can promote the migration of CD8(+) T cells through its interaction with the alpha(4)beta(1) integrin. Site-directed mutagenesis of VCAM-1 at amino acid residues required for interaction with alpha(4)beta(1) integrin completely abolished the immune resistance conferred by VCAM-1 in vivo. Surface staining showed that most renal cell carcinomas (RCC) express VCAM-1, whereas an RCC that responded to vaccination was VCAM-1 negative. These data provide evidence that tumor expression of VCAM-1 represents a new mechanism of immune evasion and has important implications for the development of immunotherapy for human RCC.

B7-H3 Promotes Pathogenesis of Autoimmune Disease and Inflammation by Regulating the Activity of Different T Cell Subsets

B7-H3 is a cell surface molecule in the immunoglobulin superfamily that is frequently upregulated in response to autoantigens and pathogens during host T cell immune responses. However, B7-H3s role in the differential regulation of T cell subsets remains largely unknown. Therefore, we constructed a new B7-H3 deficient mouse strain (B7-H3 KO) and evaluated the functions of B7-H3 in the regulation of Th1, Th2, and Th17 subsets in experimental autoimmune encephalomyelitis (EAE), experimental asthma, and collagen-induced arthritis (CIA); these mouse models were used to predict human immune responses in multiple sclerosis, asthma, and rheumatoid arthritis, respectively. Here, we demonstrate that B7-H3 KO mice have significantly less inflammation, decreased pathogenesis, and limited disease progression in both EAE and CIA mouse models when compared with littermates; these results were accompanied by a decrease in IFN-γ and IL-17 production. In sharp contrast, B7-H3 KO mice developed severe ovalbumin (OVA)-induced asthma with characteristic infiltrations of eosinophils in the lung, increased IL-5 and IL-13 in lavage fluid, and elevated IgE anti-OVA antibodies in the blood. Our results suggest B7-H3 has a costimulatory function on Th1/Th17 but a coinhibitory function on Th2 responses. Our studies reveal that B7-H3 could affect different T cell subsets which have important implications for regulating pathogenesis and disease progression in human autoimmune disease.

Nondominant CD8 T Cells Are Active Players in the Vaccine-Induced Antitumor Immune Response

We previously reported that CD8+ T cells are directed predominantly toward the immunodominant Her-2/neu (neu) epitope RNEU420–429 in nontolerized FVB/N but not tolerized HER-2/neu (neu-N) mice. In this study, we screened overlapping peptides of the entire neu protein and identified six new epitopes recognized by vaccine-induced neu-N–derived T cells. Evaluation of individual nondominant responses by tetramer staining and IFN-γ secretion demonstrate that this repertoire is peripherally tolerized. To address the role that the complete CD8+ T cell repertoire plays in vaccine-induced antitumor immunity, we created a whole-cell vaccine-expressing neu cDNA that has been mutated at the RNEU420–429 anchor residue, thereby abrogating activation of immunodominant epitope responses. Studies comparing the mutated and nonmutated vaccines indicate that nondominant CD8+ T cells can induce antitumor immunity when combined with regulatory T cell-depleting agents in both neu-N and FVB/N mice. Collectively, these studies demonstrate that the neu-directed T cell repertoire is not intrinsically incapable of eradicating tumors. Rather, they are suppressed by mechanisms of peripheral tolerance. Thus, these studies provide new insights into the function of the complete T cell repertoire directed toward a clinically relevant tumor Ag in tumor-bearing hosts.

Abstract B59: Regulation of tumor-specific CD8 T cells via interactions between galectin-3 and surface glycoproteins.

This study is testing the hypothesis that galectin-3, a 31 KDa carbohydrate-binding lectin, is involved in regulating T cell function within the tumor micro-environment by interacting with oligosaccharide chains on cell surface glycoproteins. Our interest in galectin-3 arose when we found that patients responding to a granulocyte-macrophage colony-stimulating factor (GM-CSF) secreting allogeneic pancreatic tumor vaccine developed post immunization antibody responses to galectin-3 on a proteomic screen. Oligosaccharide chains on plasma membrane proteins form a thick coat known as the glycocalyx that surrounds the entire cell. The glycocalyx has been shown to be important in regulating a diverse array of cellular processes such as adhesion, growth, and trafficking via its interactions with ligands on neighboring cells or in the extracellular environment. Lectin mediated clustering of receptors can lead to activation of downstream signaling proteins or even impact the half-life of a receptor on the cell-surface, thus affecting the duration of signaling. Changes in oligosaccharide structure through differential expression of enzymes involved in the glycosylation pathway may therefore modulate cellular activity by altering the availability and structure of binding motifs for glycan-binding proteins. Thus, the importance of lectin-glycan interactions in tumor development and the corresponding anti-tumor immune response cannot be ignored. Galectin-3 is over-expressed in several different malignancies. It has also been shown to modulate T cell responses through a diverse array of mechanisms including induction of apoptosis, TCR cross linking in CD8+ T cells, and T cell receptor (TCR) down regulation in CD4+ T cells. We have used the HER-2/neu (neu-N) transgenic mouse model to study galectin-3 binding on adoptively transferred high avidity neu-specific CD8+ T cells derived from TCR transgenic mice. Galectin-3 binding was found to be associated with increased expression of programmed death 1 (PD1) and apoptosis markers in vivo, and diminished cytokine production in vitro. We also observed that galectin-3 binding to the T cell surface occurs in an autocrine manner, and is dependant on antigen stimulation as well as sialylation of terminal galactose residues. Microarray analyses of galectin-3 bound CD8+ tumor infiltrating lymphocytes (TILs) have identified CEACAM1 (CD66) and TIM-3 as possible targets through which galectin-3 can inhibit T cell function. These studies provide a potential new mechanism by which the function of antigen-specific T cells is down-regulated after trafficking into the tumor micro-environment. Citation Format: Theodore S. Kouo, Lanqing Huang, Todd Armstrong, Elizabeth M. Jaffee. Regulation of tumor-specific CD8 T cells via interactions between galectin-3 and surface glycoproteins. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B59.

Abstract 3935: AnnexinA2 regulates invasion and metastasis of pancreatic ductal adenocarcinoma through Semaphorin3d.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Pancreatic ductal adenocarcinoma (PDA), a devastating disease with a median survival of less than six months, is characterized by its high metastatic ability and a lack of effective therapies. The mechanisms involved in promoting metastasis of PDA have yet to be elucidated. We recently identified AnnexinA2 (AnxA2) as a PDA-associated antigen. Tyr23 phosphorylated AnxA2 promotes metastasis of PDA cells, while shRNA knockdown, knockout or therapeutic inhibition of AnxA2 prevents metastasis to the liver in a mouse model. To identify downstream mediators of AnxA2 signaling, we evaluated gene expression differences in cell lines derived from the primary tumors of mice that express AnxA2 (KPC) and are metastatic or lack AnxA2 expression (KPCA) and are unable to metastasize. Using microarray analysis, we observed that expression of the ligand-receptor pair Semaphorin3d (Sema3d) and PlexinD1 (PlxnD1) was significantly down regulated in the non-metastatic KPCA cells. Sema3d and PlxnD1 were first identified as axon guidance genes. However, recently, axon guidance genes, including Sema3d and PlxnD1, were recently found by the International Cancer Genome Consortium to be among the cellular pathways that are most frequently altered by genetic mutations and amplification in human PDA (Biankin 2012). Therefore, we hypothesized that AnxA2 interacts with Sema3d and PlxnD1 in PDA to induce metastasis. We confirmed by RT-PCR and immunofluorescence analysis in multiple independent tumors from KPC and KPCA mice that Sema3d and PlxnD1 expression is down regulated in the absence of AnxA2 expression. AnxA2, Sema3d and PlxnD1 colocalize at the cell surface during pancreatic tumor development when AnxA2 is expressed. Interestingly, co-immunoprecipitation analysis demonstrated a direct interaction between Sema3d and AnxA2 in PDA cells supporting our hypothesis that AnxA2 regulates the secretion of Sema3d from PDA cells to enable its paracrine interaction with PlxnD1 resulting in invasion of PDA cells. Clinically, we have found that Sema3d expression correlates with increased lymphovascular invasion, increased perineural invasion, poor survival and increased metastatic potential in human PDA patients. Therefore, understanding the pathways that lead to metastasis of PDA will enable the development of more effective therapeutics and better clinical outcomes. Citation Format: Kelly Foley, Donger Zhou, Qian Xiao, Agnieszka Rucki, Guanglan Mo, Lanqing Huang, Anirban Maitra, Elizabeth Jaffee, Lei Zheng. AnnexinA2 regulates invasion and metastasis of pancreatic ductal adenocarcinoma through Semaphorin3d. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3935. doi:10.1158/1538-7445.AM2013-3935