ABSTRACT
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.
Subject(s)
Biomarkers, Tumor/genetics , Breast Neoplasms/genetics , Extracellular Matrix Proteins/genetics , Proteomics , Animals , Breast Neoplasms/pathology , Disease Models, Animal , Extracellular Vesicles/genetics , Extracellular Vesicles/pathology , Female , Gene Expression Regulation, Neoplastic/genetics , Glycosylation , Humans , Mice , Protein Isoforms/geneticsABSTRACT
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.
ABSTRACT
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.
Subject(s)
Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Extracellular Matrix Proteins/metabolism , Integrins/metabolism , Neoplasm Metastasis/pathology , Aged , Animals , Biomarkers, Tumor/metabolism , Cell Line , Cytoplasm/metabolism , Exosomes/metabolism , Female , Humans , Lung/metabolism , Lung/pathology , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Mice , Mice, Inbred BALB C , PrognosisABSTRACT
BACKGROUND: Oral squamous cell carcinoma (OSCC) is associated with a poor 5-year survival rate. In general, patients diagnosed with small tumors have a fairly good prognosis, but some small tumors have an aggressive behavior leading to early death. There are at present no reliable prognostic biomarkers for oral cancers. Thus, to optimize treatment for the individual patient, there is a need for biomarkers that can predict tumor behavior. METHOD: In the present study the potential prognostic value of plectin was evaluated by a tissue microarray (TMA) based immunohistochemical analysis of primary tumor tissue obtained from a North Norwegian cohort of 115 patients diagnosed with OSCC. The expression of plectin was compared with clinicopathological variables and 5 year survival. RESULTS: The statistical analysis revealed that low expression of plectin in the tumor cells predicted a favorable outcome for patients with non-metastatic disease (p = 0.008). Furthermore, the expression of plectin was found to correlate (p = 0.01) with the expression of uPAR, which we have previously found to be a potential prognostic marker for T1N0 tumors. CONCLUSIONS: Our results indicate that low expression of plectin predicts a favorable outcome for patients with non-metastatic OSCC and the expression level of plectin may therefore be used in the treatment stratification for patients with early stage disease.
