Lung cancer is a critical health care problem among Veterans, who have a higher incidence and worse outcome. Most individuals with lung cancer die of their disease because of drug resistance, invasion and metastases, features closely linked to the epithelial-mesenchymal transition (EMT) and cancer stem cells (CSCs). Importantly, this resistant phenotype includes targeted agents (e.g., EGFR inhibitors), conventional chemotherapy, radiation, and escape from immune destruction by upregulation of PD-L1. The identification and therapeutic targeting of molecules contributing to the aggressive and resistant properties of lung cancer cells are key to tumor cell eradication and cure. Recently, we discovered that TGF?-mediated EMT involves upregulation of the cell surface receptor, Neuropilin-2 (NRP2). Importantly, NRP2 knockdown inhibits TGF?- mediated migration, invasion, metastasis and tumorsphere formation, a feature of CSCs. More recently, we discovered that TGF? induces the preferential translation of a previously uninvestigated isoform, NRP2b, which is identical to the prototype NRP2a in its extracellular domain, but diverges substantially before the transmembrane segment to encode a distinct, yet highly evolutionarily conserved, C-terminus. Our studies demonstrate that NRP2a and NRP2b have opposing effects on tumorigenesis, with NRP2b specifically promoting tumorigenesis, as well as signaling from the receptor tyrosine kinase, MET. The isoforms also differ in their binding to the PDZ domain protein, GIPC1, which is specific to NRP2a, and which is also known to function in receptor endocytosis and trafficking. Importantly, mutations that disrupt NRP2a-GIPC1 binding do not result in NRP2b-like activities, whereas C-terminal deletions of NRP2b attenuate its ability to enhance migration. These results suggest that the NRP2b C-terminus is critical to its pro-tumorigenic properties, which may result from differential interactions or trafficking of cell surface receptors, including MET. Understanding the mechanistic underpinnings of how NRP2b functions has therapeutic implications. This may come from identifying which cooperating receptors are most important, or elucidating the mechanisms by which NRP2b affects receptor trafficking, thus suggesting new therapeutic strategies.
Aim 1 will use genetic approaches to further examine the role of MET in NRP2b function and tumorigenesis, as well as determine if NRP2 isoforms differentially interact with MET, or affect its endocytosis, trafficking and recycling. In addition, we will examine patient samples for co-expression of NRP2b along with markers of MET activation and EMT.
Aim 2 will test the hypothesis that the effects of NRP2b on MET can be generalized to other receptors. Their contribution to NRP2b function and tumorigenesis will be examined.
In Aim 3, we will use site-specific mutagenesis and domain swaps to identify critical residues in the 93 amino acid NRP2b C-terminus (JM, TM and cytoplasmic segments) responsible for its distinct activity. The JM segment is unique, extracellular and predicted to be antigenic. We will make monoclonal antibodies against this segment and test them for specificity and inhibitory activity. NRP2b represents a highly innovative avenue of research, which can be exploited for therapeutic gain. In addition, EMT plays a major role in inflammation, fibrosis and normal development, where the role of NRP2b is unknown. Thus, our proposed studies should have broad applicability to lung cancer and other human diseases.
Lung cancer is a critical health care problem among veterans, who have a higher incidence and reported worse outcome from this disease. Lung cancer patients die because of invasion, metastasis and resistance to therapeutic agents. Two biologic processes, the epithelial-mesenchymal transition (EMT) and development of cancer stem cells underlie these aggressive features, as well as evasion of the immune system. Recently, we discovered that a cell surface receptor, NRP2, is required for EMT-associated invasion, metastasis, drug resistance, stem-cell properties and expression of the immune inhibitory ligand, PD-L1. Most recently, we discovered that a previously uninvestigated variant, NRP2b, was responsible for these pro-tumorigenic activities, as well as upregulated signaling from the pro-tumorigenic receptor, MET. Our proposed studies will examine how NRP2b affects MET mechanistically, the role of MET in NRP2b-dependent tumorigeneis and whether these findings can be generalized to other receptors.
|Gemmill, Robert M; Nasarre, Patrick; Nair-Menon, Joyce et al. (2017) The neuropilin 2 isoform NRP2b uniquely supports TGF?-mediated progression in lung cancer. Sci Signal 10:|