Receptor Tyrosine Kinase-like Orphan Receptor 2 (Ror2) Expression Creates a Poised State of Wnt Signaling in Renal Cancer

Expression of the receptor tyrosine kinase-like orphan receptor 2 (Ror2) has been identified in an increasing array of tumor types and is known to play a role as an important mediator of Wnt signaling cascades. In this study, we aimed to clarify Ror2 interactions with the Wnt pathways within the context of renal cell carcinoma (RCC). An examination of Ror2 expression in primary human RCC tumors showed a significant correlation with several Wnt signaling genes, including the classical feedback target gene Axin2. We provide evidence that Ror2 expression results in a partially activated state for canonical Wnt signaling through an increased signaling pool of β-catenin, leading to an enhancement of downstream target genes following Wnt3a stimulation in both renal and renal carcinoma-derived cells. Additionally, inhibition of low-density lipoprotein receptor-related protein 6 (LRP6) with either siRNA or dickkopf decreased the response to Wnt3a stimulation, but no change was seen in the increased β-catenin pool associated with Ror2 expression, suggesting that LRP6 cofactor recruitment is necessary for a Wnt3a-induced signal but that it does not participate in the Ror2 effect on β-catenin signaling. These results highlight a new role for Ror2 in conveying a tonic signal to stabilize soluble β-catenin and create a poised state of enhanced responsiveness to Wnt3a exogenous signals in RCC.     

Pan-Mammalian Target of Rapamycin (mTOR) Inhibitor AZD8055 Primes Rhabdomyosarcoma Cells for ABT-737-induced Apoptosis by Down-regulating Mcl-1 Protein

The PI3K/mammalian Target of Rapamycin (mTOR) pathway is often aberrantly activated in rhabdomyosarcoma (RMS) and represents a promising therapeutic target. Recent evaluation of AZD8055, an ATP-competitive mTOR inhibitor, by the Preclinical Pediatric Testing Program showed in vivo antitumor activity against childhood solid tumors, including RMS. Therefore, in the present study, we searched for AZD8055-based combination therapies. Here, we identify a new synergistic lethality of AZD8055 together with ABT-737, a BH3 mimetic that antagonizes Bcl-2, Bcl-x_L, and Bcl-w but not Mcl-1. AZD8055 and ABT-737 cooperate to induce apoptosis in alveolar and embryonal RMS cells in a highly synergistic fashion (combination index < 0.2). Synergistic induction of apoptosis by AZD8055 and ABT-737 is confirmed on the molecular level, as AZD8055 and ABT-737 cooperate to trigger loss of mitochondrial membrane potential, activation of caspases, and caspase-dependent apoptosis that is blocked by the pan-caspase inhibitor Z-VAD-fmk. Similar to AZD8055, the PI3K/mTOR inhibitor NVP-BEZ235, the PI3K inhibitor NVP-BKM120 and Akt inhibitor synergize with ABT-737 to trigger apoptosis, whereas no cooperativity is found for the mTOR complex 1 inhibitor RAD001. Interestingly, molecular studies reveal a correlation between the ability of different PI3K/mTOR inhibitors to potentiate ABT-737-induced apoptosis and to suppress Mcl-1 protein levels. Importantly, knockdown of Mcl-1 increases ABT-737-induced apoptosis similar to AZD8055/ABT-737 cotreatment. This indicates that AZD8055-mediated suppression of Mcl-1 protein plays an important role in the synergistic drug interaction. By identifying a novel synergistic interaction of AZD8055 and ABT-737, our findings have important implications for the development of molecular targeted therapies for RMS.     

LY2228820 Dimesylate,a Selective Inhibitor of p38 Mitogen-activated Protein Kinase,Reduces Angiogenic Endothelial Cord Formation in Vitro and in Vivo

LY2228820 dimesylate is a highly selective small molecule inhibitor of p38α and p38β mitogen-activated protein kinases (MAPKs) that is currently under clinical investigation for human malignancies. p38 MAPK is implicated in a wide range of biological processes, in particular those that support tumorigenesis. One such process, angiogenesis, is required for tumor growth and metastasis, and many new cancer therapies are therefore directed against the tumor vasculature. Using an in vitro co-culture endothelial cord formation assay, a surrogate of angiogenesis, we investigated the role of p38 MAPK in growth factor- and tumor-driven angiogenesis using LY2228820 dimesylate treatment and by shRNA gene knockdown. p38 MAPK was activated in endothelial cells upon growth factor stimulation, with inhibition by LY2228820 dimesylate treatment causing a significant decrease in VEGF-, bFGF-, EGF-, and IL-6-induced endothelial cord formation and an even more dramatic decrease in tumor-driven cord formation. In addition to involvement in downstream cytokine signaling, p38 MAPK was important for VEGF, bFGF, EGF, IL-6, and other proangiogenic cytokine secretion in stromal and tumor cells. LY2228820 dimesylate results were substantiated using p38α MAPK-specific shRNA and shRNA against the downstream p38 MAPK effectors MAPKAPK-2 and HSP27. Using in vivo models of functional neoangiogenesis, LY2228820 dimesylate treatment reduced hemoglobin content in a plug assay and decreased VEGF-A-stimulated vascularization in a mouse ear model. Thus, p38α MAPK is implicated in tumor angiogenesis through direct tumoral effects and through reduction of proangiogenic cytokine secretion via the microenvironment.     

Nanoscale mapping and organization analysis of target proteins on cancer cells from B-cell lymphoma patients

CD20, a membrane protein highly expressed on most B-cell lymphomas, is an effective target demonstrated in clinical practice for treating B-cell non-Hodgkin's lymphoma (NHL). Rituximab is a monoclonal antibody against CD20. In this work, we applied atomic force microscopy (AFM) to map the nanoscale distribution of CD20 molecules on the surface of cancer cells from clinical B-cell NHL patients under the assistance of ROR1 fluorescence recognition (ROR1 is a specific cell surface marker exclusively expressed on cancer cells). First, the ROR1 fluorescence labeling experiments showed that ROR1 was expressed on cancer cells from B-cell lymphoma patients, but not on normal cells from healthy volunteers. Next, under the guidance of ROR1 fluorescence, the rituximab-conjugated AFM tips were moved to cancer cells to image the cellular morphologies and detect the CD20-rituximab interactions on the cell surfaces. The distribution maps of CD20 on cancer cells were constructed by obtaining arrays of (16×16) force curves in local areas (500×500 nm²) on the cell surfaces. The experimental results provide a new approach to directly investigate the nanoscale distribution of target protein on single clinical cancer cells.     

Structure-based design of HSPA5 inhibitors: From peptide to small molecule inhibitors

We identified nine small-molecule hit compounds of Heat shock 70 kDa protein 5 (HSPA5) from cascade in silico screening based on the binding modes of the tetrapeptides derived from the peptide substrate or inhibitors of Escherichia coli HSP70. Two compounds exhibit promising inhibition activities from cancer cell viability and tumor inhibition assays. The binding modes of the hit compounds provide a platform for development of selective small molecule inhibitors of HSPA5.