Fibroblast-derived CXCL12 increases vascular permeability in a 3-D microfluidic model independent of extracellular matrix contractility.

Cancer-associated fibroblasts (CAFs) play an active role in remodeling the local tumor stroma to support tumor initiation, growth, invasion, metastasis, and therapeutic resistance. The CAF-secreted chemokine, CXCL12, has been directly implicated in the tumorigenic progression of carcinomas, including breast cancer. Using a 3-D in vitro microfluidic-based microtissue model, we demonstrate that stromal CXCL12 secreted by CAFs has a potent effect on increasing the vascular permeability of local blood microvessel analogues through paracrine signaling. Moreover, genetic deletion of fibroblast-specific CXCL12 significantly reduced vessel permeability compared to CXCL12 secreting CAFs within the recapitulated tumor microenvironment (TME). We suspected that fibroblast-mediated extracellular matrix (ECM) remodeling and contraction indirectly accounted for this change in vessel permeability. To this end, we investigated the autocrine effects of CXCL12 on fibroblast contractility and determined that antagonistic blocking of CXCL12 did not have a substantial effect on ECM contraction. Our findings indicate that fibroblast-secreted CXCL12 has a significant role in promoting a leakier endothelium hospitable to angiogenesis and tumor cell intravasation; however, autocrine CXCL12 is not the primary upstream trigger of CAF contractility.

Directional Migration of Breast Cancer Cells Hindered by Induced Electric Fields May Be Due to Accompanying Alteration of Metabolic Activity.

Background: Induced electric fields (iEFs) control directional breast cancer cell migration. While the connection between migration and metabolism is appreciated in the context of cancer and metastasis, effects of iEFs on metabolic pathways especially as they relate to migration, remain unexplored. Materials and Methods: Quantitative cell migration data in the presence and absence of an epidermal growth factor (EGF) gradient in the microfluidic bidirectional microtrack assay was retrospectively analyzed for additional effects of iEFs on cell motility and directionality. Surrogate markers of oxidative phosphorylation (succinate dehydrogenase [SDH] activity) and glycolysis (lactate dehydrogenase activity) were assessed in MDA-MB-231 breast cancer cells and normal MCF10A mammary epithelial cells exposed to iEFs and EGF. Results: Retrospective analysis of migration results suggests that iEFs increase forward cell migration speeds while extending the time cells spend migrating slowly in the reverse direction or remaining stationary. Furthermore, in the presence of EGF, iEFs differentially altered flux through oxidative phosphorylation in MDA-MB-231 cells and glycolysis in MCF10A cells. Conclusions: iEFs interfere with MDA-MB-231 cell migration, potentially, by altering mitochondrial metabolism, observed as an inhibition of SDH activity in the presence of EGF. The energy intensive process of migration in these highly metastatic breast cancer cells may be hindered by iEFs, thus, through hampering of oxidative phosphorylation.

Melanoma-associated mutants within the serine-rich domain of PAK5 direct kinase activity to mitogenic pathways.

The overexpression and hyperactivity of p21-activated serine/threonine kinases (PAKs) is known to facilitate tumorigenesis; however, the contribution of cancer-associated PAK mutations to tumor initiation and progression remains unclear. Here, we identify p21-activated serine/threonine kinase 5 (PAK5) as the most frequently altered PAK family member in human melanoma. More than 60% of melanoma-associated PAK5 gene alterations are missense mutations, and distribution of these variants throughout the protein coding sequence make it difficult to distinguish oncogenic drivers from passengers. To address this issue, we stably introduced the five most common melanoma-associated PAK5 missense mutations into human immortalized primary melanocytes (hMELTs). While expression of these mutants did not promote single-cell migration or induce temozolomide resistance, a subset of variants drove aberrant melanocyte proliferation. These mitogenic mutants, PAK5 S364L and D421N, clustered within an unstructured, serine-rich domain of the protein and inappropriately activated ERK and PKA through kinase-independent and -dependent mechanisms, respectively. Together, our findings establish the ability of mutant PAK5 to enhance PKA and MAPK signaling in melanocytes and localize the engagement of mitogenic pathways to a serine-rich region of PAK5.