Most cancer patients with solid tumors who succumb to their illness die of metastatic disease. While early detection and improved treatment have led to reduced mortality, even for those with metastatic cancer, some patients still respond poorly to treatment. Understanding the mechanisms of metastasis is important to improve prognostication, to stratify patients for treatment, and to identify new targets for therapy. We have shown previously that expression of nephronectin (NPNT) is correlated with metastatic propensity in breast cancer cell lines. In the present study, we provide a comprehensive analysis of the expression pattern and distribution of NPNT in breast cancer tissue from 842 patients by immunohistochemical staining of tissue microarrays from a historic cohort. Several patterns of NPNT staining were observed. An association between granular cytoplasmic staining (in <10% of tumor cells) and poor prognosis was found. We suggest that granular cytoplasmic staining may represent NPNT-positive exosomes. We found that NPNT promotes adhesion and anchorage-independent growth via its integrin-binding and enhancer motifs and that enforced expression in breast tumor cells promotes their colonization of the lungs. We propose that NPNT may be a novel prognostic marker in a subgroup of breast cancer patients.

The extracellular matrix protein nephronectin plays an important regulatory role during embryonic development, controlling renal organogenesis through integrin α8β1 association. Nephronectin has three main domains: five N-terminal epidermal growth factor-like domains, a linker region harbouring two integrin-binding motifs (RGD and LFEIFEIER), and a C-terminal MAM domain. In this review, we look into the domain-related functions of nephronectin, and tissue distribution and expression. During the last two decades it has become evident that nephronectin also plays a role during cancer progression and in particular metastasis. Nephronectin is overexpressed in both human and mouse breast cancer compared to normal breast tissue where the protein is absent. Cancer cells expressing elevated levels of nephronectin acquire increased ability to colonise distant organs. In particular, the enhancer-motif (LFEIFEIER) which is specific to the integrin α8β1 association induces viability via p38 MAPK and plays a role in colonization. Integrins have long been desired as therapeutic targets, where low efficiency and receptor redundancy have been major issues. Based on the summarised publications, the enhancer-motif of nephronectin could present a novel therapeutic target.

Nephronectin (NPNT) is an extracellular matrix (ECM) protein involved in kidney development. We recently reported intracellular NPNT as a potential prognostic marker in breast cancer and that NPNT promotes metastasis in an integrin‐dependent manner. Here, we used reverse‐phase protein array (RPPA) to analyze NPNT‐triggered intracellular signaling in the 66cl4 mouse breast cancer cell line. The results showed that the integrin‐binding enhancer motif is important for the cellular effects upon NPNT interaction with its receptors, including phosphorylation of p38 mitogen‐activated protein kinase (MAPK). Furthermore, analysis using prediction tools suggests involvement of NPNT in promoting cell viability. In conclusion, our results indicate that NPNT, via its integrin‐binding motifs, promotes cell viability through phosphorylation of p38 MAPK.

Extracellular vesicles are emerging as biomarkers in breast cancer. Our recent report suggested that an intracellular granular staining pattern of the extracellular matrix protein Nephronectin (NPNT) in breast tumor sections correlated with poor prognosis. Furthermore, the results showed that NPNT is localized in extracellular vesicles derived from mouse breast cancer cells. In this study we performed proteomic analysis which reveals that several proteins, including tumor promoting molecules are differentially expressed in the cargo of small extracellular vesicles (sEVs) upon NPNT expression in mouse breast cancer cells. We identify three different forms of NPNT at 80, 60 and 20 kDa. We report that the native form of NPNT at 60 kDa gets further glycosylated and is detected as the 80 kDa NPNT, which may be processed by matrix metalloproteinases to a shorter form of around 20 kDa, not previously described. Although both 80 kDa and 20 kDa NPNT are detected in sEVs derived from breast cancer cells, the 20 kDa form of NPNT is concentrated in sEVs. In summary, we here show that a novel truncated form of NPNT is found in sEVs derived from breast cancer cells.