Nephronectin as a Matrix Effector in Cancer.

The extracellular matrix protein nephronectin plays an important regulatory role during embryonic development, controlling renal organogenesis through integrin α8ß1 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 α8ß1 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 promotes breast cancer brain metastatic colonization via its integrin-binding domains.

This study demonstrates a role for the extracellular matrix protein nephronectin (NPNT) in promoting experimental breast cancer brain metastasis, possibly through enhanced binding to- and migration through brain endothelial cells. With the introduction of more targeted breast cancer treatments, a prolonged survival has resulted during the last decade. Consequently, an increased number of patients develop metastasis in the brain, a challenging organ to treat. We recently reported that NPNT was highly expressed in primary breast cancer and associated with unfavourable prognosis. The current study addresses our hypothesis that NPNT promotes brain metastases through its integrin-binding motifs. SAGE-sequencing revealed that NPNT was significantly up-regulated in human breast cancer tissue compared to pair-matched normal breast tissue. Human brain metastatic breast cancers expressed both NPNT and its receptor, integrin α8ß1. Using an open access repository; BreastMark, we found a correlation between high NPNT mRNA levels and poor prognosis for patients with the luminal B subtype. The 66cl4 mouse cell line was used for expression of wild-type and mutant NPNT, which is unable to bind α8ß1. Using an in vivo model of brain metastatic colonization, 66cl4-NPNT cells showed an increased ability to form metastatic lesions compared to cells with mutant NPNT, possibly through reduced endothelial adhesion and transmigration.

A Novel Truncated Form of Nephronectin Is Present in Small Extracellular Vesicles Isolated from 66cl4 Cells.

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 a 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 that revealed that several proteins, including tumor-promoting molecules, are differentially expressed in the cargo of small extracellular vesicles (sEVs) derived from NPNT-expressing mouse breast cancer cells. We also identified three different forms of NPNT at 80, 60, and 20 kDa. We report that the native form of NPNT at 60 kDa becomes 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, which has not previously been described. Although both 80 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 show that a novel truncated form of NPNT is found in sEVs derived from breast cancer cells.

Nephronectin mediates p38 MAPK-induced cell viability via its integrin-binding enhancer motif.

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.

Nephronectin is Correlated with Poor Prognosis in Breast Cancer and Promotes Metastasis via its Integrin-Binding Motifs.

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.

Bias in chemokine receptor signalling.

Chemokine receptors are widely expressed on a variety of immune cells and play a crucial role in normal physiology as well as in inflammatory and infectious diseases. The existence of 23 chemokine receptors and 48 chemokine ligands guarantees a tight control and fine-tuning of the immune system. Here, we discuss the multiple regulatory mechanisms of chemokine signalling at a systemic, cellular, and molecular level. In particular, we focus on the impact of biased signalling at the receptor level; an emerging concept in molecular pharmacology. An improved understanding of these mechanisms may provide a framework for more effective drug discovery and development at a target class that is so relevant for immune function.