Neuropilin-2 regulates α6β1 integrin in the formation of focal adhesions and signaling

Hira Lal Goel () 1 2 Bryan Pursell 1 2 Clive Standley 0 2 Kevin Fogarty 0 2 Arthur M. Mercurio 1 2 0 BioMedical Imaging Group, University of Massachusetts Medical School Worcester , MA 01605 , USA 1 Department of Cancer Biology, University of Massachusetts Medical School Worcester , MA 01605 , USA 2 Materials and Methods Reagents and antibodies Matrigel and collagen I were purchased from BD Biosciences (San Jose , CA); laminin-1 (LN-1) from Invitrogen (Carlsbad, CA), fibronectin from Sigma (St Louis, MO); VEGF-165 from Peprotech (Rocky Hill, NJ) and G06983 from Calbiochem (Darmstadt, Germany). Antibodies against the following proteins were used: a3 integrin (Millipore, Billerica, MA) used for immunoblotting, or P1B5 (Gibco, Invitrogen, used for immunofluorescence); a6 integrin (AA6A, provided by Anne Cress , University of Arizona Cancer Center , Tucson, AZ, USA; J8H, provided by Arnoud Sonnenberg , The Netherlands Cancer Center , Amsterdam , The Netherlands; and GoH3, purchased from Millipore); b1 integrin (AIIB2 , Developmental Studies Hybridoma Bank , Iowa); b4 integrin (439-9b , provided by Rita Falcioni (Regina Elena Cancer Institute , Rome, Italy); NRP2 (goat IgG, R&D, Minneapolis, MN; C9 and H300, Santa Cruz Biotechnology, Santa Cruz, CA); vinculin (Sigma); actin (Sigma); FAK-P (Y397) [mouse IgG (BD Bioscience) used for immunoblotting; rabbit IgG (AbCaM, Cambridge, MA, USA) used for immunofluorescence]; FLAG (Sigma); anti-rabbit-FITC; anti-goat FITC; anti-goat TRITC; rat IgG; mouse IgG (Jackson, West Grove, PA, USA); pan-phosphorylated-PKC S660; Src, phosphorylated-Src Summary The neuropilins (NRPs) contribute to the function of cancer cells in their capacity as VEGF receptors. Given that NRP2 is induced in breast cancer and correlates with aggressive disease, we examined the role of NRP2 in regulating the interaction of breast cancer cells with the ECM. Using epithelial cells from breast tumors, we defined NRP2high and NRP2low populations that differed in integrin expression and adhesion to laminin. Specifically, the NRP2high population adhered more avidly to laminin and expressed high levels of the a6b1 integrin than the NRP2low population. The NRP2high population formed numerous focal adhesions on laminin that were not seen in the NRP2low population. These results were substantiated using breast carcinoma cell lines that express NRP2 and a6b1 integrin. Depletion experiments revealed that adhesive strength on laminin but not collagen is dependent on NRP2, and that VEGF is needed for adhesion on laminin. A specific interaction between NRP2 and a6b1 integrin was detected by co-immunoprecipitation. NRP2 is ce necessary for focal adhesion formation on laminin and for the association of a6b1 integrin with the cytoskeleton. NRP2 also facilitates icne aT6hbe1m-inetcehgarni nis-mmebdyiatwedhiacchtivNaRtioPn2 orfegFuAlaKteasntdheSricn.teUrnacetxipoenctoefdlay6,bw1e idnitsecgorvinerewditthhaltaNmRinPin2 itsolofocarmtedfoincaflocaadlhaedshioenssioninsvoonlvleasmPinKinC. S activation. Together, our data reveal a new VEGF-NRP2 signaling pathway that activates the a6b1 integrin and enables it to form focal adhesions and signal. This pathway is important in the pathogenesis of breast cancer. - Introduction An emerging area of importance in cancer biology is the function of receptors for VEGF on tumor cells. Although most studies on VEGF receptors have focused on endothelial cells and their role in angiogenesis, it has become apparent that tumor cells also express specific VEGF receptors and that these receptors contribute to tumor initiation, migration, invasion and survival (Bachelder et al., 2001; Gray et al., 2008; Hu et al., 2007; Lichtenberger et al., 2010; Matsushita et al., 2007; Miao et al., 2000; Muders et al., 2009; Sulpice et al., 2008; Wang et al., 2007). The neuropilins (NRPs) are one class of VEGF receptors that are particularly interesting with respect to cancer biology. NRP1 and NRP2 were identified initially as neuronal receptors for semaphorins, which are axon guidance factors that function primarily in the developing nervous system (Uniewicz and Fernig, 2008). The seminal finding by Klagsbrun that neuropilins can also function as VEGF receptors and that they are expressed on endothelial and tumor cells launched studies aimed at understanding their function in angiogenesis and tumor biology (Soker et al., 1998). NRPs have the ability to interact with and modulate the function of tyrosine kinase VEGF receptors (VEGFR1 and VEGFR2), as well as other growth factor receptors (Neufeld et al., 2002; Sulpice et al., 2008). There is also evidence that NRPs can function independently of other receptors (Gray et al., 2005) and that they are valid targets for therapeutic inhibition of angiogenesis and cancer (Caunt et al., 2008; Gray et al., 2008; Pan et al., 2007). The observation that the expression of NRP2 is induced is some cancers such as breast cancer (Yasuoka et al., 2009) and that its expression correlates with aggressive disease and poor survival suggests that this NRP has a substantial influence on the behavior of breast carcinoma cells. The possibility that NRP2 influences the activation and function of specific integrins that contribute to tumor behavior merits consideration. Previous studies demonstrated that VEGF signaling activates specific integrins in endothelial cells (Byzova et al., 2000) and that integrins can be regulated by NRP1 (Valdembri et al., 2009). In this study we observed that loss of NRP2 expression in breast carcinoma cells impedes their ability to interact with laminin matrices. This later observation suggested that NRP2 regulates the a6 integrins, which function as laminin receptors. This hypothesis is compelling because the a6 integrins have been implicated in breast tumor formation and progression (Chung and Mercurio, 2004; Guo et al., 2006; Lipscomb et al., 2005), so we pursued this hypothesis in this study. Unexpectedly, we discovered that NRP2 is located in focal adhesions, that it associates with the a6b1 integrin specifically and that it regulates the ability of this integrin to form focal adhesions and signal. These studies add a new dimension to the function of the NRPs and their contribution to cell biology. Moreover, the fact that both NRP2 (Yasuoka et al., 2009) and the a6b1 integrin (Friedrichs et al., 1995; Wewer et al., 1997) have been implicated in aggressive breast cancer underscores the importance of NRP2-mediated regulation of a6b1 integrin function. Results Characterization of NRP2high and NRP2low populations of epithelial cells isolated from human breast tumors We isolated epithelial cells (EpCAM+) from human breast tumors to characterize the properties of NRP2-expressing cells. This approach is based on the report that NRP2 is expressed at low levels in normal breast epithelium and that its expression increases in breast cancer and correlates with aggressive disease (Yasuoka et al., 2009). Epithelial cells w (...truncated)