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 Ca-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 Silencing or pharmacologic inhibition of the two-pore channel TPC2 reduced lung metastasis of mammary mouse cancer cells. Disrupting TPC function halted trafficking of β1-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..

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-1a 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.

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. A better understanding of how viral vectors enter cells in vivo is critical to improve their therapeutic use. Adeno-associated virus 2 (AAV2) and adenovirus 5 (Ad5) vectors have shown promise in clinical trials for treatment of a wide variety of diseases (1, 2). Both vectors, when injected intravenously into mice, exhibit transgene expression in liver (3-5). Heparan sulfate proteoglycans (HSPGs) are the primary receptors currently thought to facilitate AAV2 and Ad5 entry into hepatocytes (6-8).HSPGs are present both on the cell surface and in the extracellular matrix (9, 10). They consist of a protein core posttranslationally modified to contain heparan sulfate (HS) chains (11). HS biosynthesis occurs by polymerization of alternating glucuronic acid and N-acetylglucosamine residues (12-14), catalyzed by an enzyme complex composed of EXT1 and EXT2 (15). EXT1 and EXT2 are essential molecules required for HS synthesis; cells lacking either molecule do not synthesize HS (16,49).AAV2 binds directly to cell surface HSPGs via an HS-binding motif on the virus capsid (3,17,18). AAV capsid modifications that alter the cluster of positive amino acids that constitute the HS binding motif abrogate liver transduction (3,19,20), suggesting that the ability of the capsid to bind to HS is critical for AAV2 liver transduction in vivo. In contrast, Ad5 binding to HSPGs requires the presence of blood coagulation factor X (FX), which binds to the Ad5 hexon when the virus comes in contact with blood (7,(21)(22)(23). The interaction of Ad.FX and HS is mediated by electrostatic interactions between the heparin binding exosite of the FX serine protease domain and the sulfate groups of HS (6,(23)(24)(25). FX is required for Ad5 transduction in vivo in wild-type mice. In the absence of FX, or when viruses with mutant hexon proteins unable to bind FX are used, Ad5 liver transduction is essentially completely abrogated (7, 21-23, 26, 27).

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.

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.