Two-Pore Channel Function Is Crucial for the Migration of Invasive Cancer Cells.

Metastatic invasion is the major cause of cancer-related deaths. In this study, we introduce two-pore channels (TPC), a recently described class of NAADP- and PI(3,5)P2-sensitive Ca2+-permeable cation channels in the endolysosomal system of cells, as candidate targets for the treatment of invasive cancers. Inhibition of the channel abrogated migration of metastatic cancer cells in vitro Silencing or pharmacologic inhibition of the two-pore channel TPC2 reduced lung metastasis of mammary mouse cancer cells. Disrupting TPC function halted trafficking of ß1-integrin, leading to its accumulation in EEA1-positive early endosomes. As a consequence, invasive cancer cells were no longer able to form leading edges, which are required for adequate migration. Our findings link TPC to cancer cell migration and provide a preclinical proof of concept for their candidacy as targets to treat metastatic cancers. Cancer Res; 77(6); 1427-38. ©2017 AACR.

MDM2 antagonist nutlin-3a sensitizes tumors to V-ATPase inhibition.

Treating cancer is one of the big challenges of this century and it has become evident that single chemotherapeutic treatment is rarely effective. As tumors often carry multiple mutations using combination therapy which addresses different targets seems therefore more beneficial. One of the most frequently mutated genes in tumors is the tumor suppressor p53. Significant work has been put in the development of p53 activators, which are now in clinical studies against diverse cancers. Recently, we could show that inhibition of V-ATPase, a multisubunit proton pump, by archazolid induces p53 protein levels in cancer cells. In this study, we provide evidence that the combination of archazolid with the p53 activator nutlin-3a is synergistically inducing cell death in different p53 wild type tumor cell lines. Mechanistically, this effect could presumably be attributed to reduction of glycolysis as TIGAR mRNA levels were increased and glucose uptake and Glut1 protein levels were reduced. In addition, combination treatment highly activated pro-apoptotic pathways including IGFBP3 and Bax inducing caspase-9 and PARP cleavage. Remarkably, combination of archazolid and nutlin-3a was more efficient in reducing tumor growth compared to single dose treatment in a U87MG mouse model in vivo. Hence, our findings suggest the combination of archazolid and nutlin-3a as a highly promising strategy for the treatment of p53 wild type tumors.

Hepatocyte Heparan Sulfate Is Required for Adeno-Associated Virus 2 but Dispensable for Adenovirus 5 Liver Transduction In Vivo.

UNLABELLED: Adeno-associated virus 2 (AAV2) and adenovirus 5 (Ad5) are promising gene therapy vectors. Both display liver tropism and are currently thought to enter hepatocytes in vivo through cell surface heparan sulfate proteoglycans (HSPGs). To test directly this hypothesis, we created mice that lack Ext1, an enzyme required for heparan sulfate biosynthesis, in hepatocytes. Ext1(HEP) mutant mice exhibit an 8-fold reduction of heparan sulfate in primary hepatocytes and a 5-fold reduction of heparan sulfate in whole liver tissue. Conditional hepatocyte Ext1 gene deletion greatly reduced AAV2 liver transduction following intravenous injection. Ad5 transduction requires blood coagulation factor X (FX); FX binds to the Ad5 capsid hexon protein and bridges the virus to HSPGs on the cell surface. Ad5.FX transduction was abrogated in primary hepatocytes from Ext1(HEP) mice. However, in contrast to the case with AAV2, Ad5 transduction was not significantly reduced in the livers of Ext1(HEP) mice. FX remained essential for Ad5 transduction in vivo in Ext1(HEP) mice. We conclude that while AAV2 requires HSPGs for entry into mouse hepatocytes, HSPGs are dispensable for Ad5 hepatocyte transduction in vivo. This study reopens the question of how adenovirus enters cells in vivo. IMPORTANCE: Our understanding of how viruses enter cells, and how they can be used as therapeutic vectors to manage disease, begins with identification of the cell surface receptors to which viruses bind and which mediate viral entry. Both adeno-associated virus 2 and adenovirus 5 are currently thought to enter hepatocytes in vivo through heparan sulfate proteoglycans (HSPGs). However, direct evidence for these conclusions is lacking. Experiments presented herein, in which hepatic heparan sulfate synthesis was genetically abolished, demonstrated that HSPGs are not likely to function as hepatocyte Ad5 receptors in vivo. The data also demonstrate that HSPGs are required for hepatocyte transduction by AAV2. These results reopen the question of the identity of the Ad5 receptor in vivo and emphasize the necessity of demonstrating the nature of the receptor by genetic means, both for understanding Ad5 entry into cells in vivo and for optimization of Ad5 vectors as therapeutic agents.

Anti-leukemic effects of the V-ATPase inhibitor Archazolid A.

Prognosis for patients suffering from T-ALL is still very poor and new strategies for T-ALL treatment are urgently needed. Our study shows potent anti-leukemic effects of the myxobacterial V-ATPase inhibitor Archazolid A. Archazolid A reduced growth and potently induced death of leukemic cell lines and human leukemic samples. By inhibiting lysosomal acidification, Archazolid A blocked activation of the Notch pathway, however, this was not the mechanism of V-ATPase inhibition relevant for cell death induction. In fact, V-ATPase inhibition by Archazolid A decreased the anti-apoptotic protein survivin. As underlying mode of action, this work is in line with recent studies from our group demonstrating that Archazolid A induced S-phase cell cycle arrest by interfering with the iron metabolism in leukemic cells. Our study provides evidence for V-ATPase inhibition as a potential new therapeutic option for T-ALL.

Vacuolar-ATPase Inhibition Blocks Iron Metabolism to Mediate Therapeutic Effects in Breast Cancer.

Generalized strategies to improve breast cancer treatment remain of interest to develop. In this study, we offer preclinical evidence of an important metabolic mechanism underlying the antitumor activity of inhibitors of the vacuolar-type ATPase (V-ATPase), a heteromultimeric proton pump. Specifically, our investigations in the 4T1 model of metastatic breast cancer of the V-ATPase inhibitor archazolid suggested that its ability to trigger metabolic stress and apoptosis associated with tumor growth inhibition related to an interference with hypoxia-inducible factor-1α signaling pathways and iron metabolism. As a consequence of disturbed iron metabolism, archazolid caused S-phase arrest, double-stranded DNA breaks, and p53 stabilization, leading to apoptosis. Our findings link V-ATPase to cell-cycle progression and DNA synthesis in cancer cells, and highlight the basis for the clinical exploration of V-ATPase as a potentially generalizable therapy for breast cancer.