Identification of 14-3-3ß gene as a novel miR-152 target using a proteome-based approach.

Recent studies demonstrated that miR-152 overexpression down-regulates the nonclassical human leukocyte antigen (HLA) class I molecule HLA-G in human tumors thereby contributing to their immune surveillance. Using two-dimensional gel electrophoresis followed by MALDI-TOF mass spectrometry, the protein expression profile of HLA-G(+), miR-152(low) cells, and their miR-152-overexpressing (miR(high)) counterparts was compared leading to the identification of 24 differentially expressed proteins. These were categorized according to their function and localization demonstrating for most of them an important role in the initiation and progression of tumors. The novel miR-152 target 14-3-3 protein ß/α/YWHAB (14-3-3ß) is down-regulated upon miR-152 overexpression, although its overexpression was often found in tumors of distinct origin. The miR-152-mediated reduction of the 14-3-3ß expression was accompanied by an up-regulation of BAX protein expression resulting in a pro-apoptotic phenotype. In contrast, the reconstitution of 14-3-3ß expression in miR-152(high) cells increased the expression of the anti-apoptotic BCL2 gene, enhances the proliferative activity in the presence of the cytostatic drug paclitaxel, and causes resistance to apoptosis induced by this drug. By correlating clinical microarray data with the patients' outcome, a link between 14-3-3ß and HLA-G expression was found, which could be associated with poor prognosis and overall survival of patients with tumors. Because miR-152 controls both the expression of 14-3-3ß and HLA-G, it exerts a dual role in tumor cells by both altering the immunogenicity and the tumorigenicity.

Reduced immunosuppressive properties of axitinib in comparison with other tyrosine kinase inhibitors.

The multikinase inhibitors sunitinib, sorafenib, and axitinib have an impact not only on tumor growth and angiogenesis, but also on the activity and function of immune effector cells. In this study, a comparative analysis of the growth inhibitory properties and apoptosis induction potentials of tyrosine kinase inhibitors on T cells was performed. Tyrosine kinase inhibitor treatment resulted in a dramatic decrease in T cell proliferation along with distinct impacts on the cell cycle progression. This was at least partially associated with an enhanced induction of apoptosis although triggered by distinct apoptotic mechanisms. In contrast to sunitinib and sorafenib, axitinib did not affect the mitochondrial membrane potential (Δψm) but resulted in an induction or stabilization of the induced myeloid leukemia cell differentiation protein (Mcl-1), leading to an irreversible arrest in the G2/M cell cycle phase and delayed apoptosis. Furthermore, the sorafenib-mediated suppression of immune effector cells, in particular the reduction of the CD8(+) T cell subset along with the down-regulation of key immune cell markers such as chemokine CC motif receptor 7 (CCR7), CD26, CD69, CD25, and CXCR3, was not observed in axitinib-treated immune effector cells. Therefore, axitinib rather than sorafenib seems to be suitable for implementation in complex treatment regimens of cancer patients including immunotherapy.

VHL-dependent alterations in the secretome of renal cell carcinoma: Association with immune cell response?

Secreted proteins could modulate the interaction between tumor, stroma and immune cells within the tumor microenvironment thereby mounting an immunosuppressive tumor microenvironment. In order to determine the secretome-mediated, von Hippel Lindau (VHL)-regulated cross-talk between tumor cells and T lymphocytes peripheral blood mononuclear cells (PBMC) from healthy donors were either cultured in conditioned media obtained from normoxic and hypoxic human VHL-deficient renal cell carcinoma (RCC) cell line (786-0VHL-) and its wild type (wt) VHL-transfected counterpart (786-0VHL+) or directly co-cultured with both cell lines. An increased T cell proliferation was detected in the presence of 786-0VHL+-conditioned medium. By applying a quantitative proteomic-based approach using differential gel electrophoresis followed by mass spectrometry fourteen proteins were identified to be differentially expressed within the secretome of 786-0VHL- cells when compared to that of 786-0VHL+ cells. All proteins identified were involved in multiple tumor-associated biological functions including immune responses. Functional studies on manganese superoxide dismutase 2 (MnSOD2) demonstrated that it was a regulator of T cell activation-induced oxidative signaling and cell death. Direct effects of soluble MnSOD2 on the growth properties and interleukin 2 (IL-2) secretion of T cells could be demonstrated underlining the critical role of extracellular MnSOD2 levels for T cell proliferation and activation.