PDF - 86K, Legends for Supplementary Figures 1 through 10.

PDF file 1545 K. Methods and Materials Cell culture, Scratch Assay/Immunfluorescence, siRNA transfection Fig. S1: Archazolid inhibits the acidification of lysosomes. Fig. S2: Archazolid inhibits cancer cell migration. Fig. S3: EGF-R and Her2 expression on the cell surface after archazolid treatment. Fig. S4: EGF-R plays a role in cell polarization. Fig.S5: The downregulation of the V-ATPase subunit c affects the cell migration process also in L3.6pl cells. Fig. S6: Rac1-GFP is less localized in Rab5-induced enlarged endosomes of archazolid treated cells. Fig. S7: Dissemination of 4T1-Luc breast cancer cells is decreased in archazolid A (1mg/kg) pretreated mice. Fig. S8: Scheme proposing the impact of V-ATPase inhibition on cell migration. Fig.S9: Beside the localization in lysosomes the V-ATPase is also localized at the plasma membrane. Fig. S10: Archazolid affects cellular recycling.

PDF - 300K, Figure S1A: Chemical structure of archazolid A; S1B: Alkalization of lysosomes by archazolid treatment; Figure S2A: T24 cells stimulated with archazolid for 24h show no sign of apoptosis; S2B: Concanamycin impairs colony formation in T24 cells; Figure S3: Adherent T24 cells stimulated with archazolid for 48h are highly apoptotic; Figure S4: 4T1 cells show no cell death induction after 24h archazolid treatment; Figure S5: BIM levels after archazolid treatment in MDA-MB-231 and MCF7 cells; Figure S6: Quantification of BIM rescue by proteasome inhibitors; Figure S7A: ROS induction in floating 4T1 cells; S7B: ROS levels of archazolid treated, floating cells reached ROS levels of untreated adherent cells; Figure S8: Phosphorylation of Akt and ERK is increased early after detachment but decreases again after 24h; Figure S9: The inhibitors LY-294002 (Akt), PD 98059 (ERK) or Saracatinib (c-Scr) induce apoptosis in floating cancer cells; Figure S10: Anoikis resistance and archazolid action on anoikis resistant cancer cells.

Background A major player in the process of metastasis is the actin cytoskeleton as it forms key structures in both invasion mechanisms, mesenchymal and amoeboid migration. We tested the actin binding compound Chondramide as potential anti-metastatic agent. Methods In vivo, the effect of Chondramide on metastasis was tested employing a 4T1-Luc BALB/c mouse model. In vitro, Chondramide was tested using the highly invasive cancer cell line MDA-MB-231 in Boyden-chamber assays, fluorescent stainings, Western blot and Pull down assays. Finally, the contractility of MDA-MB-231 cells was monitored in 3D environment and analyzed via PIV analysis. Results In vivo, Chondramide treatment inhibits metastasis to the lung and the migration and invasion of MDA-MB-231 cells is reduced by Chondramide in vitro. On the signaling level, RhoA activity is decreased by Chondramide accompanied by reduced MLC-2 and the stretch induced guanine nucleotide exchange factor Vav2 activation. At same conditions, EGF-receptor autophosphorylation, Akt and Erk as well as Rac1 are not affected. Finally, Chondramide treatment disrupted the actin cytoskeleton and decreased the ability of cells for contraction. Conclusions Chondramide inhibits cellular contractility and thus represents a potential inhibitor of tumor cell invasion.

The myxobacterial agent archazolid inhibits the vacuolar proton pump V-ATPase. V-ATPases are ubiquitously expressed ATP-dependent proton pumps, which are known to regulate the pH in endomembrane systems and thus play a crucial role in endo- and exocytotic processes of the cell. As cancer cells depend on a highly active secretion of proteolytic proteins in order to invade tissue and form metastases, inhibition of V-ATPase is proposed to affect the secretion profile of cancer cells and thus potentially abrogate their metastatic properties. Archazolid is a novel V-ATPase inhibitor. Here, we show that the secretion pattern of archazolid treated cancer cells includes various prometastatic lysosomal proteins like cathepsin A, B, C, D and Z. In particular, archazolid induced the secretion of the proforms of cathepsin B and D. Archazolid treatment abrogates the cathepsin B maturation process leading to reduced intracellular mature cathepsin B protein abundance and finally decreased cathepsin B activity, by inhibiting mannose-6-phoshate receptor-dependent trafficking. Importantly, in vivo reduced cathepsin B protein as well as a decreased proteolytic cathepsin B activity was detected in tumor tissue of archazolid-treated mice. Our results show that inhibition of V-ATPase by archazolid reduces the activity of prometastatic proteases like cathepsin B in vitro and in vivo.

Abstract Fighting metastasis is a major challenge in cancer therapy and novel therapeutic targets and drugs are highly appreciated. Resistance of invasive cells to anoikis, a particular type of apoptosis induced by loss of cell–matrix contact, is a major prerequisite for their metastatic spread. Inducing anoikis in metastatic cancer cells is therefore a promising therapeutic approach. The vacuolar-ATPase (V-ATPase), a proton pump located at the membrane of acidic organelles, has recently come to focus as an antimetastatic cancer target. As V-ATPase inhibitors have shown to prevent invasion of tumor cells and are able to induce apoptosis, we proposed that V-ATPase inhibition induces anoikis-related pathways in invasive cancer cells. We used the V-ATPase inhibitor archazolid to investigate the mechanism of anoikis induction in various metastatic cancer cells (T24, MDA-MB-231, 4T1, 5637) in vitro. Anoikis induction by archazolid was characterized by decreased c-FLIP expression and caspase-8 activation as well as reduction of active integrin-β1 and an early increase of the proapoptotic protein BIM. However, we observed that archazolid also induces mechanisms opposing anoikis such as degradation of BIM mediated by extracellular signal-regulated kinase (ERK), Akt and Src kinases at later time points and induction of reactive oxygen species. Still, intravenous injection of archazolid-treated 4T1-Luc2 mouse breast cancer cells resulted in reduced metastasis in mouse lungs. Thus, V-ATPase inhibition is not only an interesting option to reduce cancer metastasis, but also to better understand anoikis resistance and to find choices to fight against it. Mol Cancer Ther; 13(4); 926–37. ©2014 AACR.

Nine sequence-defined, polycationic oligomers were synthesized containing motifs of three consecutive tyrosines (Y3) as stabilizing components for pDNA and siRNA polyplex assembly. For pDNA, a combination of terminal oligotyrosines and cysteines was necessary and sufficient for stable polyplex formation. Stable siRNA binding required a combination of terminal cysteines and oligotyrosines, as well as a central hydrophobic modification (oligotyrosines or fatty acids). The phenolic group within the aromatic amino acids of Y3 containing oligomers further increased the endosomal buffer capacity. As a result, the new class of oligotyrosine containing oligomers was efficient in pDNA and siRNA transfection, in most cases superior to a previously established cysteine-containing, dioleic acid modified oligomer without the Y3 motif. Additionally, increased serum stability of the new oligomers with terminal Y3 motifs was demonstrated by gel shift and fluorescence correlations spectroscopy (FCS). In vivo stability and biodistribution was monitored by intravenous administration of chemically stabilized Cy7 siRNA either as free form, or complexed with the nine Y3 containing oligomers or control oligomers. Oligomer 332, with the overall most beneficial in vitro and in vivo characteristics, was applied in RAN siRNA polyplexes for intratumoral treatment of neuroblastoma-bearing mice. This resulted in significantly reduced tumor growth compared to animal treated with control siRNA polyplexes.

The low molecular weight of many chemotherapeutics causes their untargeted distribution in the body and fast renal clearance, which leads to a loss of therapeutic activity and to unspecific toxic side effects. Therefore, there is a growing interest in conjugating anticancer drugs to water soluble polymers and thus, take advantage of the 'enhanced permeability and retention' (EPR) effect in tumors. In this study, water soluble polyphosphoesters were used as polymer carriers of melphalan hydrochloride (hydrochloride of p-bis(2-chloroethyl)amino-L-phenylalanine), which is a multifunctional alkylating agent. Melphalan was chemically immobilized by covalent bonding to poly(oxyethylene H-phosphonate) under Atherton-Todd reaction conditions. Novel polymer-melphalan complexes with ionic and hydrogen bonds were designed as controls, basing on two other biodegradable polyphosphoesters: poly(hydroxyoxyethylene phosphate) and poly(methyloxyethylene phosphate). The structure of the formed products was elucidated by (1)H, (13)C, (31)P NMR and FT-IR spectroscopy. The cytotoxic effect of the melphalan formulations was evaluated on different tumor cell lines. The novel polymer formulations showed a concentration dependent antitumoral activity, comparable to the effect of unmodified melphalan. The polymer-melphalan conjugate was also evaluated in vivo in the human hepatocellular carcinoma HuH7 xenograft mouse model. It improved the therapeutic efficacy of pure melphalan without causing side effects.

Although our understanding of RNAi and our knowledge on designing and synthesizing active and safe siRNAs significantly increased during the past decade, targeted delivery remains the major limitation in the development of siRNA therapeutics. On one hand, practical considerations dictate robust chemistry reproducibly providing precise carrier molecules. On the other hand, the multistep delivery process requires dynamic multifunctional carriers of substantial complexity. We present a monodisperse and multifunctional carrier system, synthesized by solid phase supported chemistry, for siRNA delivery in vitro and in vivo. The sequence-defined assembly includes a precise cationic (oligoethanamino)amide core, terminated at the ends by two cysteines for bioreversible polyplex stabilization, at a defined central position attached to a monodisperse polyethylene glycol chain coupled to a terminal folic acid as cell targeting ligand. Complexation with an endosomolytic influenza peptides-iRNA conjugate results in nanosized functional polyplexes of 6 nm hydrodynamic diameter. The necessity of each functional substructure of the carrier system for a specific and efficient gene silencing was confirmed. The nanosized polyplexes showed stability in vivo, receptor-specific cell targeting, and silencing of the EG5 gene in receptor-positive tumors. The nanosized appearance of these particles can be precisely controlled by the oligomer design (from 5.8 to 8.8 nm diameter). A complete surface charge shielding together with the high stability result in good tolerability in vivo and the absence of accumulation in nontargeted tissues such as liver, lung, or spleen. Due to their small size, siRNA polyplexes are efficiently cleared by the kidney.

Abstract The abundance of the multimeric vacuolar ATP-dependent proton pump, V-ATPase, on the plasma membrane of tumor cells correlates with the invasiveness of the tumor cell, suggesting the involvement of V-ATPase in tumor metastasis. V-ATPase is hypothesized to create a proton efflux leading to an acidic pericellular microenvironment that promotes the activity of proinvasive proteases. An alternative, not yet explored possibility is that V-ATPase regulates the signaling machinery responsible for tumor cell migration. Here, we show that pharmacologic or genetic reduction of V-ATPase activity significantly reduces migration of invasive tumor cells in vitro. Importantly, the V-ATPase inhibitor archazolid abrogates tumor dissemination in a syngeneic mouse 4T1 breast tumor metastasis model. Pretreatment of cancer cells with archazolid impairs directional motility by preventing spatially restricted, leading edge localization of epidermal growth factor receptor (EGFR) as well as of phosphorylated Akt. Archazolid treatment or silencing of V-ATPase inhibited Rac1 activation, as well as Rac1-dependent dorsal and peripheral ruffles by inhibiting Rab5-mediated endocytotic/exocytotic trafficking of Rac1. The results indicate that archazolid effectively decreases metastatic dissemination of breast tumors by impairing the trafficking and spatially restricted activation of EGFR and Rho-GTPase Rac1, which are pivotal for directed movement of cells. Thus, our data reveals a novel mechanism underlying the role of V-ATPase in tumor dissemination. Cancer Res; 72(22); 5976–87. ©2012 AACR.