Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin's lymphoma, with multiple molecular subtypes. The activated B-cell-like DLBCL subtype accounts for roughly one-third of all the cases and has an inferior prognosis. There is a need to develop better class of therapeutics that could target molecular pathways in resistant DLBCLs; however, this requires DLBCLs to be studied in representative tumor microenvironments. The pathogenesis and progression of lymphoma has been mostly studied from the point of view of genetic alterations and intracellular pathway dysregulation. By comparison, the importance of lymphoma microenvironment in which these malignant cells arise and reside has not been studied in as much detail. We have recently elucidated the role of integrin signaling in lymphomas and demonstrated that inhibition of integrin-ligand interactions abrogated the proliferation of malignant cells in vitro and in patient-derived xenograft. Here we demonstrate the role of lymph node tissue stiffness on DLBCL in a B-cell molecular subtype specific manner. We engineered tunable bioartificial hydrogels that mimicked the stiffness of healthy and neoplastic lymph nodes of a transgenic mouse model and primary human lymphoma tumors. Our results demonstrate that molecularly diverse DLBCLs grow differentially in soft and high stiffness microenvironments, which further modulates the integrin and B-cell receptor expression level as well as response to therapeutics. We anticipate that our findings will be broadly useful to study lymphoma biology and discover new class of therapeutics that target B-cell tumors in physical environments. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1833-1844, 2017.

Epithelial to mesenchymal transition (EMT) is an essential differentiation program during tissue morphogenesis and remodeling. EMT is induced by soluble transforming growth factor β (TGF-β) family members, and restricted by vascular endothelial growth factor family members. While many downstream molecular regulators of EMT have been identified, these have been largely evaluated individually without considering potential crosstalk. In this study, we created an ensemble of dynamic mathematical models describing TGF-β induced EMT to better understand the operational hierarchy of this complex molecular program. We used ordinary differential equations (ODEs) to describe the transcriptional and post-translational regulatory events driving EMT. Model parameters were estimated from multiple data sets using multiobjective optimization, in combination with cross-validation. TGF-β exposure drove the model population toward a mesenchymal phenotype, while an epithelial phenotype was enhanced following vascular endothelial growth factor A (VEGF-A) exposure. Simulations predicted that the transcription factors phosphorylated SP1 and NFAT were master regulators promoting or inhibiting EMT, respectively. Surprisingly, simulations also predicted that a cellular population could exhibit phenotypic heterogeneity (characterized by a significant fraction of the population with both high epithelial and mesenchymal marker expression) if treated simultaneously with TGF-β and VEGF-A. We tested this prediction experimentally in both MCF10A and DLD1 cells and found that upwards of 45% of the cellular population acquired this hybrid state in the presence of both TGF-β and VEGF-A. We experimentally validated the predicted NFAT/Sp1 signaling axis for each phenotype response. Lastly, we found that cells in the hybrid state had significantly different functional behavior when compared to VEGF-A or TGF-β treatment alone. Together, these results establish a predictive mechanistic model of EMT susceptibility, and potentially reveal a novel signaling axis which regulates carcinoma progression through an EMT versus tubulogenesis response.

Foraminifera and radiolarians are closely related amoeboid protists (i.e., retarians) often characterized by their shells and pseudopodia. Previous studies hypothesized that the unusual "Type 2" β-tubulin (β2) is critically involved in forming helical filaments (HFs), a unique microtubule (MT) assembly/disassembly intermediate found in foraminiferan reticulopodia. Such noncanonical β-tubulin sequences have also been found in two radiolarian species and appear to be closely related to the foraminiferan β2. In this study, we report 119 new β-tubulin transcript sequences from six foraminiferans, four radiolarians, and a related non-retarian species. We found that foraminiferan and radiolarian β2-tubulins share some of the unusual substitutions in the structurally essential and usually conserved domains. In the β-tubulin phylogeny, retarian β2-tubulin forms a monophyletic clade, well separated from the canonical β-tubulin (β1) ubiquitous in eukaryotes. Furthermore, we found that foraminiferan and radiolarian β2-tubulin lineages were under positive selection, and used homology models for foraminiferan α- and β-tubulin hexamers to understand the structural effect of the positively selected substitutions. We suggest that the positively selected substitutions play key roles in the transition of MT to HF by altering the lateral and longitudinal interactions between α- and β-tubulin heterodimers. Our results indicate that the unusual β2-tubulin is a molecular synapomorphy of retarians, and the β-tubulin gene duplication occurred before the divergence of Foraminifera and radiolarians. The duplicates have likely been subjected to neofunctionalization responsible for the unique MT to HF assembly/disassembly dynamics, and/or other unknown physiological processes in retarian protists.

During valvulogenesis, cytoskeletal, secretory, and transcriptional events drive endocardial cushions growth and remodeling into thin fibrous leaflets. Genetic disorders play an important role in understanding valve malformations but only account for a minority of clinical cases. Mechanical forces are ever-present, but how they coordinate molecular and cellular decisions remains unclear. In this study, we used osmotic pressure to interrogate how compressive and tensile stresses influence valve growth and shaping maturation. We found that compressive stress drives a growth phenotype whereas tensile stress increases compaction. We identified a mechanically activated switch between valve growth and maturation, by which compression induces cushion growth via BMP-pSMAD1/5 while tension induces maturation via pSer-19 mediated MLC2 contractility. The compressive stress acts through BMP signaling to increase cell proliferation and decrease cell contractility, and MEK-ERK is essential for both compressive stress and BMP mediation of compaction. We further showed that the effects of osmotic stress are conserved through the condensation and elongation stages of development. Together, our results demonstrate that compressive/tensile stress regulation of BMP-pSMAD1/5 and MLC2 contractility orchestrates valve growth and remodeling.