Nephronectin in breast cancer progression and metastasis

Breast cancer is life threatening due to its ability to spread and invade other tissues. The best approach in solving a problem of this sort is to be able to reason backwards. Tumors appear chaotic as they are composed of multiple abnormal cell types and a complex matrix of proteins that cushions the tumor cells extracellularly. In fact, some characteristics of tumor development resemble those seen in developing organs. Nephronectin (NPNT) is identified in several developing organs, but it is absent in normal healthy breast tissue. However for breast cancer progression and metastasis, NPNT appears to play a significant role, as elaborated in this thesis. Our results show that 596 out of 842 breast cancer cases stain positive for NPNT and the cytoplasmic granular staining pattern in less than 10% of the tumor cells correlates with poor prognosis. This granular staining pattern could indicate the involvement of vesicular communication. Extracellular vesicles derived from tumor cells facilitate intercellular communication both locally and systemically in the body. These vesicles are packed with oncogenic traits that can influence cancer progression, and metastasis. As per our investigation, NPNT is one the signaling molecules packed in the extracellular vesicles derived from breast cancer cells. Interestingly, the truncated form of NPNT was concentrated in these vesicles. We further show that the protein of small extracellular vesicles is altered upon NPNT expression in 66cl4 mouse breast cancer cells. Breast cancer cells mainly spread to bones, liver, lungs and brain. It is challenging for the cancer cells to survive and adapt to a distant tissue microenvironment which is different compared to the primary tumor. In this thesis we highlight several oncogenic properties which are enhanced in presence of NPNT. We found that NPNT promotes viability, adhesion and anchorage-independent growth via its integrin-binding motifs. These oncogenic properties bestowed on tumor cells by NPNT enables them to colonize the lungs more efficiently. Different ligands can activate different intracellular signaling pathways, although binding to the same receptor. Therefore, it is important to investigate and document key signaling molecules triggered by different ligands in specific cell types. Our results indicate that NPNT induces phosphorylation of p38 MAPK via its enhancer motif to promote viability in 66cl4 cells. Therefore, we suggest that targeting both the enhancer and the RGD motif simultaneously would be more effective in rendering NPNT protein inactive

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 as a matrix effector in cancer

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

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

Peter Watkins' Culloden (1964)

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 α8β1. 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 α8β1. 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