Applicants - Analyzing intra- and extracellular expression and function of NM23 (NME) homologs in metastatic processes of breast cancer

Functional and expressional analysis of NM23 homologs is a key element of our studies. One of the best characterized function linked to this protein family is its negative effect exerted on cell migration, however NM23 homologs are implicated in numerous other processes such as endosomal function, apoptotic engulfment, vesicular transport, cellular energetics, cytoskeletal and membrane rearrangement. Previously, we confirmed the inhibitory effect exerted on the migratory potential by overexpression of NM23-H1, H2 and their C. elegans counterpart NDK-1 in an invasive breast carcinoma cell line, MDA-MB231. We intend to examine the potential adhesional, metastatic and metabolic changes of these genetically modified cell lines in vitro and in vivo. We aim to explore how NM23-H1/H2 affect 1. the extracellular environment, in particular tumoral microenvironment, 2. metabolic changes in tumor cells related to therapeutic sensitivity 3. the protein level of metabolic regulators, mTORC1, C2.

First, we are interested to know whether extracellular NM23 appearing in the serum of cancer patients is indeed derived from the tumor cells. Second, the mechanism, which allows release of NM23 into the extracellular environment (exocytosis, exosome release, necrotic debris, apoptotic bodies) will be identified.
A sensitive ELISA system will be established to measure NM23-H1 and H2 levels derived from lysates and supernatants of different cell lines, fractionated exosome preparates from cell cultures and serum samples.

We plan to examine the level of NM23 released by tumor cells, the bioenergetic background of release, changes in metabolic profile and mTOR activity of tumor cells in response to various in vitro treatments (Rab27 silencing or pharmacological inhibition of exosome formation, Dynasore-exocytosis inhibition, medroxiprogesteron-to increase NM23 expression, preparates from NM23 containing supernatants).

To see how extracellular NM23 homologs might be relocalized into different other /microenvironmental cells, FLAG and MYC tags will help us to identify the fusion proteins in co-cultures (monocyte/macrophage, monocyte-leukemia cell lines, fibroblasts) and in in vitro xenograft models.

In parallel, NM23-H1/H2 expression will be analyzed in primary and metastatic breast cancer samples in paraffin-embedded, eventually frozen tissue blocks and in a new collection of breast cancer serum samples. Next, we intend to analyze the relationship between serum and tissue NM23-H1/H2 expression and clinicopathological data.