Serglycin Is Involved in TGF-ß Induced Epithelial-Mesenchymal Transition and Is Highly Expressed by Immune Cells in Breast Cancer Tissue.

Serglycin is a proteoglycan highly expressed by immune cells, in which its functions are linked to storage, secretion, transport, and protection of chemokines, proteases, histamine, growth factors, and other bioactive molecules. In recent years, it has been demonstrated that serglycin is also expressed by several other cell types, such as endothelial cells, muscle cells, and multiple types of cancer cells. Here, we show that serglycin expression is upregulated in transforming growth factor beta (TGF-ß) induced epithelial-mesenchymal transition (EMT). Functional studies provide evidence that serglycin plays an important role in the regulation of the transition between the epithelial and mesenchymal phenotypes, and it is a significant EMT marker gene. We further find that serglycin is more expressed by breast cancer cell lines with a mesenchymal phenotype as well as the basal-like subtype of breast cancers. By examining immune staining and single cell sequencing data of breast cancer tissue, we show that serglycin is highly expressed by infiltrating immune cells in breast tumor tissue.

Validation of Selected Head and Neck Cancer Prognostic Markers from the Pathology Atlas in an Oral Tongue Cancer Cohort.

The Pathology Atlas is an open-access database that reports the prognostic value of protein-coding transcripts in 17 cancers, including head and neck cancer. However, cancers of the various head and neck anatomical sites are specific biological entities. Thus, the aim of the present study was to validate promising prognostic markers for head and neck cancer reported in the Pathology Atlas in oral tongue squamous cell carcinoma (OTSCC). We selected three promising markers from the Pathology Atlas (CALML5, CD59, LIMA1), and analyzed their prognostic value in a Norwegian OTSCC cohort comprising 121 patients. We correlated target protein and mRNA expression in formalin-fixed, paraffin-embedded cancer tissue to five-year disease-specific survival (DSS) in univariate and multivariate analyses. Protein expression of CALML5 and LIMA1 were significantly associated with five-year DSS in the OTSCC cohort in univariate analyses (p = 0.016 and p = 0.043, respectively). In multivariate analyses, lymph node metastases, tumor differentiation, and CALML5 were independent prognosticators. The prognostic role of the other selected markers for head and neck cancer patients identified through unbiased approaches could not be validated in our OTSCC cohort. This underlines the need for subsite-specific analyses for head and neck cancer.

Molecular Interactions Stabilizing the Promatrix Metalloprotease-9·Serglycin Heteromer.

Previous studies have shown that THP-1 cells produced an SDS-stable and reduction-sensitive complex between proMMP-9 and a chondroitin sulfate proteoglycan (CSPG) core protein. The complex could be reconstituted in vitro using purified serglycin (SG) and proMMP-9 and contained no inter-disulfide bridges. It was suggested that the complex involved both the FnII module and HPX domain of proMMP-9. The aims of the present study were to resolve the interacting regions of the molecules that form the complex and the types of interactions involved. In order to study this, we expressed and purified full-length and deletion variants of proMMP-9, purified CSPG and SG, and performed in vitro reconstitution assays, peptide arrays, protein modelling, docking, and molecular dynamics (MD) simulations. ProMMP-9 variants lacking both the FnII module and the HPX domain did not form the proMMP-9∙CSPG/SG complex. Deletion variants containing at least the FnII module or the HPX domain formed the proMMP-9∙CSPG/SG complex, as did the SG core protein without CS chains. The interacting parts covered large surface areas of both molecules and implicated dynamic and complementary ionic, hydrophobic, and hydrogen bond interactions. Hence, no short single interacting linear motifs in the two macromolecules could explain the strong SDS-stable and reduction-sensitive binding.

Generation of a PAX6 knockout glioblastoma cell line with changes in cell cycle distribution and sensitivity to oxidative stress.

BACKGROUND: The transcription factor PAX6 is expressed in various cancers. In anaplastic astrocytic glioma, PAX6 expression is inversely related to tumor grade, resulting in low PAX6 expression in Glioblastoma, the highest-grade astrocytic glioma. The aim of the present study was to develop a PAX6 knock out cell line as a tool for molecular studies of the roles PAX6 have in attenuating glioblastoma tumor progression. METHODS: The CRISPR-Cas9 technique was used to knock out PAX6 in U251 N cells. Viral transduction of a doxycycline inducible EGFP-PAX6 expression vector was used to re-introduce (rescue) PAX6 expression in the PAX6 knock out cells. The knock out and rescued cells were rigorously characterized by analyzing morphology, proliferation, colony forming abilities and responses to oxidative stress and chemotherapeutic agents. RESULTS: The knock out cells had increased proliferation and colony forming abilities compared to wild type cells, consistent with clinical observations indicating that PAX6 functions as a tumor-suppressor. Cell cycle distribution and sensitivity to H2O2 induced oxidative stress were further studied, as well as the effect of different chemotherapeutic agents. For the PAX6 knock out cells, the percentage of cells in G2/M phase increased compared to PAX6 control cells, indicating that PAX6 keeps U251 N cells in the G1 phase of the cell cycle. Interestingly, PAX6 knock out cells were more resilient to H2O2 induced oxidative stress than wild type cells. Chemotherapy treatment is known to generate oxidative stress, hence the effect of several chemotherapeutic agents were tested. We discovered interesting differences in the sensitivity to chemotherapeutic drugs (Temozolomide, Withaferin A and Sulforaphane) between the PAX6 expressing and non-expressing cells. CONCLUSIONS: The U251 N PAX6 knock out cell lines generated can be used as a tool to study the molecular functions and mechanisms of PAX6 as a tumor suppressor with regard to tumor progression and treatment of glioblastoma.

Both binding and activation of p38 mitogen-activated protein kinase (MAPK) play essential roles in regulation of the nucleocytoplasmic distribution of MAPK-activated protein kinase 5 by cellular stress.

The p38 mitogen-activated protein kinase (MAPK) pathway is an important mediator of cellular responses to environmental stress. Targets of p38 include transcription factors, components of the translational machinery, and downstream serine/threonine kinases, including MAPK-activated protein kinase 5 (MK5). Here we have used enhanced green fluorescent protein fusion proteins to analyze the subcellular localization of MK5. Although this protein is predominantly nuclear in unstimulated cells, MK5 shuttles between the nucleus and the cytoplasm. Furthermore, we have shown that the C-terminal domain of MK5 contains both a functional nuclear localization signal (NLS) and a leucine-rich nuclear export signal (NES), indicating that the subcellular distribution of this kinase reflects the relative activities of these two signals. In support of this, we have shown that stress-induced activation of the p38 MAPK stimulates the chromosomal region maintenance 1 protein-dependent nuclear export of MK5. This is regulated by both binding of p38 MAPK to MK5, which masks the functional NLS, and stress-induced phosphorylation of MK5 by p38 MAPK, which either activates or unmasks the NES. These properties may define the ability of MK5 to differentially phosphorylate both nuclear and cytoplasmic targets or alternatively reflect a mechanism whereby signals initiated by activation of MK5 in the nucleus may be transmitted to the cytoplasm.