Invadopodia formation in nasopharyngeal carcinoma

Abstract

Cancer metastasis is a major cause of mortality, accounting for approximately 90% of cancer-related deaths. Cancer cells migrate to important organs and interrupt their normal functions. Emerging evidences suggest that tumor microenvironment plays an indispensable role in cancer metastasis. Recruitment of innate and adaptive immune cells to the tumor results in chronic inflammation which favors tumor growth and progression. Tumor associated macrophages (TAMs), in particular M2 macrophages, are found to promote the initiation, growth, and metastasis in several types of cancers. Nasopharyngeal carcinoma (NPC) is one of the highly invasive and metastatic cancers which is closely associated with EBV infection. The aim of this study is to examine the postulation role of TAMs in increasing the invasiveness of NPC cells by enhancing the formation of invadopodia, which are membranous actin-rich protrusions with digestive ability of extra-cellular matrix. In this study, monocyte THP-1 cells were treated with PMA/IL-4, and co-cultured with nasopharyngeal cells in a contained chamber-insert. The stimulated nasopharyngeal cells formed massive numbers of invadopodia and digested larger area of gelatin compared with the unstimulated counterparts. A cytokine antibody array was used to identify the upregulated cytokines in the PMA/IL-4-treated THP-1 (PItT) cultured medium and the NPC cells. Tumor necrosis factor alpha (TNFα) was revealed to be the responsible cytokine to induce the formation of invadopodia and digestion of gelatin. Neutralizing the TNFα in the PItT-conditioned medium abrogated the generation of invadopodia in the treated NPC cells. Western blot analysis identified that several actin-associated proteins such as EGFR, Src, ERK, and cortactin were involved in the PItT cells- and TNFα-mediated invadopodia formation. Interestingly, the expression of latent membrane protein 1 (LMP1), a commonly encoded EBV-encoded protein which is well-known for promoting invasion could be induced in NPC cells after co-culturing with the PItT-conditioned medium . Knockdown of LMP1 in the EBV-infected NPC cells partially suppressed the formation of PItT cells-mediated invadopodia, suggesting the involvement of this viral protein to the activation of invadopodia generation in NPC cells. Through transient transfection and stable expression of LMP1 in NPC cells, the LMP1 was shown to promote formation of invadopodia. The LMP1 was found to induce invadopodia formation through the Cdc42/N-WASP signaling axis, but less dependent on cortactin activation. By using the wildtype and truncated mutants of LMP1 with deletion of different domains, the C-terminal of LMP1 was shown to be responsible for the activation of Cdc42/N-WASP signaling axis. Surprisingly, the LMP1-expressing cells were more responsive to TNFα treatment and generated more invadopodia than the control cells transfected with empty vector. The synergistic effect of LMP1 and TNFα to induce invadopodia formation in NPC cells suggests the interplay of EBV-infected cancer cells with the inflammatory tumor microenvironment in enforcing the invasiveness of NPC cells and their ability to degrade extra-cellular matrix. Taken together, therapeutic strategies targeting LMP1 may have selective advantages to eliminate NPC cells with enhanced invasive and metastatic potentials. (478 words)