Jason J. David

Serum response factor neutralizes Purα- and Purβ-mediated repression of the fetal vascular smooth muscle α-actin gene in stressed adult cardiomyocytes

Mouse hearts subjected to repeated transplant surgery and ischemia-reperfusion injury develop substantial interstitial and perivascular fibrosis that was spatially associated with dysfunctional activation of fetal smooth muscle alpha-actin (SM alpha A) gene expression in graft ventricular cardiomyocytes. Compared with cardiac fibroblasts in which nuclear levels of the Sp1 and Smad 2/3 transcriptional-activating proteins increased markedly after transplant injury, the most abundant SM alpha A gene-activating protein in cardiomyocyte nuclei was serum response factor (SRF). Additionally, cardiac intercalated discs in heart grafts contained substantial deposits of Pur alpha, an mRNA-binding protein and known negative modulator of SRF-activated SM alpha A gene transcription. Activation of fetal SM alpha A gene expression in perfusion-isolated adult cardiomyocytes was linked to elevated binding of a novel protein complex consisting of SRF and Pur alpha to a purine-rich DNA element in the SM alpha A promoter called SPUR, previously shown to be required for induction of SM alpha A gene transcription in injury-activated myofibroblasts. Increased SRF binding to SPUR DNA plus one of two nearby CArG box consensus elements was observed in SM alpha A-positive cardiomyocytes in parallel with enhanced Pur alpha:SPUR protein:protein interaction. The data suggest that de novo activation of the normally silent SM alpha A gene in reprogrammed adult cardiomyocytes is linked to elevated interaction of SRF with fetal-specific CArG and injury-activated SPUR elements in the SM alpha A promoter as well as the appearance of novel Pur alpha protein complexes in both the nuclear and cytosolic compartments of these cells.

Y-box binding protein-1 implicated in translational control of fetal myocardial gene expression after cardiac transplant.

Peri-transplant surgical trauma and ischemia/reperfusion injury in accepted murine heterotopic heart grafts has been associated with myofibroblast differentiation, cardiac fibrosis and biomechanical-stress activation of the fetal myocardial smooth muscle α-actin (SMαA) gene. The wound-healing agonists, transforming growth factor β1 and thrombin, are known to coordinate SMαA mRNA transcription and translation in activated myofibroblasts by altering the subcellular localization and mRNA-binding affinity of the Y-box binding protein-1 (YB-1) cold-shock domain (CSD) protein that governs a variety of cellular responses to metabolic stress. YB-1 accumulated in polyribosome-enriched regions of the sarcoplasm proximal to cardiac intercalated discs in accepted heart grafts. YB-1 binding to a purine-rich motif in exon 3 of SMαA mRNA that regulates translational efficiency increased substantially in perfusion-isolated, rod-shaped adult rat cardiomyocytes during phenotypic de-differentiation in the presence of serum-derived growth factors. Cardiomyocyte de-differentiation was accompanied by the loss of a 60 kDa YB-1 variant that was highly expressed in both adult myocardium and freshly isolated myocytes and replacement with the 50 kDa form of YB-1 (p50) typically expressed in myofibroblasts that demonstrated sequence-specific interaction with SMαA mRNA. Accumulation of p50 YB-1 in reprogrammed, de-differentiated myocytes was associated with a 10-fold increase in SMαA protein expression. Endomyocardial biopsies collected from patients up to 14 years after heart transplant showed variable yet coordinately elevated expression of SMαA and p50 YB-1 protein and demonstrable p50 YB-1:SMαA mRNA interaction. The p60 YB-1 variant in human heart graft samples, but neither mouse p60 nor mouse or human p50, reacted with an antibody specific for the phosphoserine 102 modification in the YB-1 CSD. Modulation of YB-1 subcellular compartmentalization and mRNA-binding activity may be linked with reprogramming of contractile protein gene expression in ventricular cardiomyocytes that could contribute to maladaptive remodeling in accepted, long-term heart grafts.

Autophagy Induction Results in Enhanced Anoikis Resistance in Models of Peritoneal Disease

Peritoneal carcinomatosis and peritoneal sarcomatosis is a potential complication of nearly all solid tumors and results in profoundly increased morbidity and mortality. Despite the ubiquity of peritoneal carcinomatosis/peritoneal sarcomatosis, there are no clinically relevant targeted therapies for either its treatment or prevention. To identify potential therapies, we developed in vitro models of peritoneal carcinomatosis/peritoneal sarcomatosis using tumor cell lines and patient-derived spheroids (PDS) that recapitulate anoikis resistance and spheroid proliferation across multiple cancer types. Epithelial- and mesenchymal-derived cancer cell lines (YOU, PANC1, HEYA8, CHLA10, and TC71) were used to generate spheroids and establish growth characteristics. Differential gene expression analyses of these spheroids to matched adherent cells revealed a consensus spheroid signature. This spheroid signature discriminates primary tumor specimens from tumor cells found in ascites of ovarian cancer patients and in our PDS models. Key in this gene expression signature is BNIP3 and BNIP3L, known regulators of autophagy and apoptosis. Elevated BNIP3 mRNA expression is associated with poor survival in ovarian cancer patients and elevated BNIP3 protein, as measured by IHC, and is also associated with higher grade tumors and shorter survival. Pharmacologic induction of autophagy with rapamycin significantly increased spheroid formation and survival while decreasing the induction of apoptosis. In contrast, the autophagy inhibitor hydroxychloroquine abrogated spheroid formation with a clear increase in apoptosis. Modulation of BNIP3 and the critical autophagy gene Beclin-1 (BECN1) also caused a significant decrease in spheroid formation. Combined, these data demonstrate how modulation of BNIP3-related autophagy, in PDS and in vitro spheroid models, alters the survival and morphology of spheroids. Implications: Development of BNIP3/BNIP3L-targeting agents or autophagy-targeting agents may reduce morbidity and mortality associated with peritoneal carcinomatosis and sarcomatosis. Mol Cancer Res; 15(1); 26–34. ©2016 AACR.

Transglutaminase-2 mediates calcium-regulated crosslinking of the Y-Box 1 (YB-1) translation-regulatory protein in TGFβ1-activated myofibroblasts

Myofibroblast differentiation is required for wound healing and accompanied by activation of smooth muscle α‐actin (SMαA) gene expression. The stress‐response protein, Y‐box binding protein‐1 (YB‐1) binds SMαA mRNA and regulates its translational activity. Activation of SMαA gene expression in human pulmonary myofibroblasts by TGFβ1 was associated with formation of denaturation‐resistant YB‐1 oligomers with selective affinity for a known translation‐silencer sequence in SMαA mRNA. We have determined that YB‐1 is a substrate for the protein‐crosslinking enzyme transglutaminase 2 (TG2) that catalyzes calcium‐dependent formation of covalent γ‐glutamyl‐isopeptide linkages in response to reactive oxygen signaling. TG2 transamidation reactions using intact cells, cell lysates, and recombinant YB‐1 revealed covalent crosslinking of the 50 kDa YB‐1 polypeptide into protein oligomers that were distributed during SDS–PAGE over a 75–250 kDa size range. In vitro YB‐1 transamidation required nanomolar levels of calcium and was enhanced by the presence of SMαA mRNA. In human pulmonary fibroblasts, YB‐1 crosslinking was inhibited by (a) anti‐oxidant cystamine, (b) the reactive‐oxygen antagonist, diphenyleneiodonium, (c) competitive inhibition of TG2 transamidation using the aminyl‐surrogate substrate, monodansylcadaverine, and (d) transfection with small‐interfering RNA specific for human TG2 mRNA. YB‐1 crosslinking was partially reversible as a function of oligomer‐substrate availability and TG2 enzyme concentration. Intracellular calcium accumulation and peroxidative stress in injury‐activated myofibroblasts may govern SMαA mRNA translational activity during wound healing via TG2‐mediated crosslinking of the YB‐1 mRNA‐binding protein. J. Cell. Biochem. 114: 2753–2769, 2013.

Abstract C124: Proteomic and phenotype derived model for selection of combinations of targeted therapies in ovarian cancer.

Combinations of molecular targeted therapies may be more effective due to collective inhibition at multiple points within a signaling network, but selecting the right targets has been elusive. We have used a partial least squares regression (PLS) statistical model to identify key functional proteins in four ovarian cancer cell lines (HEYA8, OVCAR8, CAOV3, SKOV3). We measured the changes in 85 proteins by reverse phase protein arrays (RPPA) when these four cell lines were treated with seven different kinase inhibitors (AZD6244, XL147, vandetinib, dasatinib, rapamycin, PF0573228, sunitinib). We also measured how these drugs modulated five different phenotypic endpoints: (1) a novel optimized co-culture differentiation assay with tumor cells and primary human microvascular endothelial cells (HMVECs), (2) mitochondrial activity as a surrogate for cytotoxicity using an XTT assay, and the production of (3) VEGF, (4) bFGF, and (5) EGF by tumor cells. We then applied PLS regression to the relationship between the proteins measured by RPPA and in vitro phenotypes to define important driver signaling proteins. We identified 14 proteins in this analysis: EGFR, p27, mTOR, p38, HER3, ERalpha, JNK, PRAS40, CHK2, TAZ, YAP, CHK1, AKT, and AMPK. Nine of these proteins have readily available inhibitors in various stages of clinical development: EGFR (BIBW2992), mTOR (MLN0128), p38 (SB203580), HER3 (AZD8931), ERalpha (tamoxifen), JNK (SP600125), CHK2 (AZD7762), CHK1 (LY2603618), AKT (MK2206). We examined these nine agents against all four cell lines with XTT assays. The mean IC50 was significantly less for the inhibitors targeting PLS selected driving proteins when compared to the original seven inhibitors tested across all four cell lines (Mean IC50 2.2x10−5M vs. 3.2 x 10−4M, p=0.0025). Similarly, while TORC1 inhibition with temsirolimus has been clinically tested in ovarian cancer, the PLS algorithm suggested that inhibitors targeting both TORC1 and TORC2 signaling might be more effective. MLN0128 (TORC1/2 inhibitor) was significantly more effective than sirolimus in HEYA8 (>5x10−4M vs. 8.9x10−7M, p=0.002), SKOV3 (1.4x10−5M vs 1.4 x10−8M, p=0.0006), CAOV3 (5.2x10−6M vs. 8.3x10−8M, p=0.018) and OVCAR8 (8.8x10−7M vs. 5.8x10−8M, p=0.0012) cell lines when comparing IC50 values for an XTT assay. The four most active compounds (MLN0128, AZD7762, LY2603618 and MK2206) across all four cell lines were selected for further testing. These compounds demonstrate good single agent activity as well as pair-wise synergism in all four cell lines. The PLS method provides a unique and predictive algorithm to examine interactions between proteomics and phenotypic endpoints and has yielded driver signaling events that will guide novel target combinations for phase I examination in ovarian cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C124. Citation Format: John L. Hays, Eric C. Polley, Jason J. David, Joyce Lu, Anna M. Leone, Saawan Mehta, Elise C. Kohn. Proteomic and phenotype derived model for selection of combinations of targeted therapies in ovarian cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C124.