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 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

Mechanical properties of 67NR tumours are broadly independent of the immune status of the host mouse

Tumour stiffness is increasingly seen as a clinically relevant parameter, with increased stiffness being linked to poorer prognosis, but it is also one that is not inherently easy to measure either in vivo or ex vivo. We have established a testing protocol for fresh ex vivo tumour tissue from tumour bearing mice using a Stable Micro Systems texture analyser fitted with a 2mm diameter cylindrical probe. The data demonstrate that, in this tumour model, changes in tumour growth time alter the tumour stiffness, but the presence or absence of T-cells does not significantly alter tumour stiffness. However, there may be more substantial differences if a more immunogenic tumour model was employed

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

Targeting the non-canonical roles of PCNA modifies and increases the response to targeted anti-cancer therapy

Receptor tyrosine kinases (RTKs), such as HER2 and/or EGFR are important therapeutic targets in multiple cancer cells. Low and/or short response to targeted therapies are often due to activation of compensatory signaling pathways, and therefore a combination of kinase inhibitors with other anti-cancer therapies have been proposed as promising strategies. PCNA is recently shown to have non-canonical cytosolic roles, and targeting PCNA with a cell-penetrating peptide containing the PCNA-interacting motif APIM is shown to mediate changes in central signaling pathways such as PI3K/Akt and MAPK, acting downstream of multiple RTKs. In this study, we show how targeting PCNA increased the anti-cancer activity of EGFR/HER2/VEGFR inhibition in vitro as well as in vivo. The combination treatment resulted in reduced tumor load and increased the survival compared to either single agent treatments. The combination treatment affected multiple cellular signaling responses not seen by EGFR/HER2/VEGFR inhibition alone, and changes were seen in pathways determining protein degradation, ER-stress, apoptosis and autophagy. Our results suggest that targeting the non-canonical roles of PCNA in cellular signaling have the potential to improve targeted therapies

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

Salt-inducible kinase 1 (SIK1) is induced by gastrin and inhibits migration of gastric adenocarcinoma cells.

Salt-inducible kinase 1 (SIK1/Snf1lk) belongs to the AMP-activated protein kinase (AMPK) family of kinases, all of which play major roles in regulating metabolism and cell growth. Recent studies have shown that reduced levels of SIK1 are associated with poor outcome in cancers, and that this involves an invasive cellular phenotype with increased metastatic potential. However, the molecular mechanism(s) regulated by SIK1 in cancer cells is not well explored. The peptide hormone gastrin regulates cellular processes involved in oncogenesis, including proliferation, apoptosis, migration and invasion. The aim of this study was to examine the role of SIK1 in gastrin responsive adenocarcinoma cell lines AR42J, AGS-GR and MKN45. We show that gastrin, known to signal through the Gq/G11-coupled CCK2 receptor, induces SIK1 expression in adenocarcinoma cells, and that transcriptional activation of SIK1 is negatively regulated by the Inducible cAMP early repressor (ICER). We demonstrate that gastrin-mediated signalling induces phosphorylation of Liver Kinase 1B (LKB1) Ser-428 and SIK1 Thr-182. Ectopic expression of SIK1 increases gastrin-induced phosphorylation of histone deacetylase 4 (HDAC4) and enhances gastrin-induced transcription of c-fos and CRE-, SRE-, AP1- and NF-κB-driven luciferase reporter plasmids. We also show that gastrin induces phosphorylation and nuclear export of HDACs. Next we find that siRNA mediated knockdown of SIK1 increases migration of the gastric adenocarcinoma cell line AGS-GR. Evidence provided here demonstrates that SIK1 is regulated by gastrin and influences gastrin elicited signalling in gastric adenocarcinoma cells. The results from the present study are relevant for the understanding of molecular mechanisms involved in gastric adenocarcinomas