Zinc finger E-box binding homeobox-1 (Zeb1) drives anterograde lysosome trafficking and tumor cell invasion via upregulation of Na+/H+ Exchanger-1 (NHE1).

Tumor cell invasion through the extracellular matrix is facilitated by the secretion of lysosome-associated proteases. As a common mechanism for secretion, lysosomes must first traffic to the cell periphery (anterograde trafficking), consistent with invasive cells often containing lysosomes closer to the plasma membrane compared to non-invasive cells. Epithelial to mesenchymal transition (EMT) is a transcriptionally driven program that promotes an invasive phenotype, and Zeb1 is one transcription factor that activates the mesenchymal gene expression program. The role of lysosome trafficking in EMT-driven invasion has not been previously investigated. We found that cells with increased levels of Zeb1 displayed lysosomes located closer to the cell periphery and demonstrated increased protease secretion and invasion in 3-dimensional (3D) cultures compared to their epithelial counterparts. Additionally, preventing anterograde lysosome trafficking via pharmacological inhibition of Na+/H+ exchanger 1 (NHE1) or shRNA depletion of ADP-ribosylation like protein 8b (Arl8b) reversed the invasive phenotype of mesenchymal cells, thus supporting a role for lysosome positioning in EMT-mediated tumor cell invasion. Immunoblot revealed that expression of Na+/H+ exchanger 1 correlated with Zeb1 expression. Furthermore, we found that the transcription factor Zeb1 binds to the Na+/H+ exchanger 1 promoter, suggesting that Zeb1 directly controls Na+/H+ transcription. Collectively, these results provide insight into a novel mechanism regulating Na+/H+ exchanger 1 expression and support a role for anterograde lysosome trafficking in Zeb1-driven cancer progression. © 2016 Wiley Periodicals, Inc.

Lysosome trafficking is necessary for EGF-driven invasion and is regulated by p38 MAPK and Na+/H+ exchangers.

BACKGROUND: Tumor invasion through a basement membrane is one of the earliest steps in metastasis, and growth factors, such as Epidermal Growth Factor (EGF) and Hepatocyte Growth Factor (HGF), stimulate this process in a majority of solid tumors. Basement membrane breakdown is one of the hallmarks of invasion; therefore, tumor cells secrete a variety of proteases to aid in this process, including lysosomal proteases. Previous studies demonstrated that peripheral lysosome distribution coincides with the release of lysosomal cathepsins. METHODS: Immunofluorescence microscopy, western blot, and 2D and 3D cell culture techniques were performed to evaluate the effects of EGF on lysosome trafficking and cell motility and invasion. RESULTS: EGF-mediated lysosome trafficking, protease secretion, and invasion is regulated by the activity of p38 mitogen activated protein kinase (MAPK) and sodium hydrogen exchangers (NHEs). Interestingly, EGF stimulates anterograde lysosome trafficking through a different mechanism than previously reported for HGF, suggesting that there are redundant signaling pathways that control lysosome positioning and trafficking in tumor cells. CONCLUSIONS: These data suggest that EGF stimulation induces peripheral (anterograde) lysosome trafficking, which is critical for EGF-mediated invasion and protease release, through the activation of p38 MAPK and NHEs. Taken together, this report demonstrates that anterograde lysosome trafficking is necessary for EGF-mediated tumor invasion and begins to characterize the molecular mechanisms required for EGF-stimulated lysosome trafficking.

Knockdown of core binding factorß alters sphingolipid metabolism.

Core binding factor (CBF) is a heterodimeric transcription factor containing one of three DNA-binding proteins of the Runt-related transcription factor family (RUNX1-3) and the non-DNA-binding protein, CBFß. RUNX1 and CBFß are the most common targets of chromosomal rearrangements in leukemia. CBF has been implicated in other cancer types; for example RUNX1 and RUNX2 are implicated in cancers of epithelial origin, including prostate, breast, and ovarian cancers. In these tumors, CBF is involved in maintaining the malignant phenotype and, when highly over-expressed, contributes to metastatic growth in bone. Herein, lentiviral delivery of CBFß-specific shRNAs was used to achieve a 95% reduction of CBFß in an ovarian cancer cell line. This drastic reduction in CBFß expression resulted in growth inhibition that was not associated with a cell cycle block or an increase in apoptosis. However, CBFß silencing resulted in increased autophagy and production of reactive oxygen species (ROS). Since sphingolipid and ceramide metabolism regulates non-apoptotic cell death, autophagy, and ROS production, fumonsin B1 (FB1), an inhibitor of ceramide synthase, was used to alter ceramide production in the CBFß-silenced cells. FB1 treatment inhibited the CBFß-dependent increase in autophagy and provided a modest increase in cell survival. To document alterations to sphingolipids in the CBFß-silenced cells, ceramide, and lactosylceramide levels were directly examined by mass spectrometry. Substantial increases in ceramide species and decreases in lactosylceramides were identified. Altogether, this report provides evidence that CBF transcriptional pathways control cellular survival, at least in part, through sphingolipid metabolism.

Thiazolidinediones induce Rab7-RILP-MAPK-dependent juxtanuclear lysosome aggregation and reduce tumor cell invasion.

Acidic extracellular pH (pHe) has been shown to stimulate peripheral lysosome trafficking, resulting in cathepsin B secretion and tumor invasion. In addition, inhibitors of sodium-proton exchangers (NHE) such as EIPA, cariporide and s3226, as well as the non-specific NHE inhibitor, troglitazone (Tro), blocked these changes. In this paper, we report a differential ability of the thiazolidinedione (TZD) family of compounds to induce a time-dependent retrograde aggregation of lysosomes over the microtubule-organizing center (MTOC) in tumor cells exposed to acidic pHe. This trafficking event depended on microtubules and the MAP-Kinase pathway, but was independent of Rho GTPase activity. Expression of shRNA implicated Rab7 in this process, and subcellular fractionation revealed that levels of Rab7, RILP and Erk1/2 were increased on lysosomes purified from cells treated with Tro. In addition, DN-RILP overexpression studies indicated that this Rab7 effector also played a role in TZD-induced retrograde trafficking. Tro was able to prevent acidic pHe-induced cell invasion. Finally, DU145 prostate tumor cells stably over-expressing WT-RILP, a condition where lysosomes aggregate to the MTOC in the absence of Tro, did not invade in response to acidic pHe, suggesting that the regulation of lysosome trafficking is an inherently important aspect of tumor cell invasion.

HGF-induced invasion by prostate tumor cells requires anterograde lysosome trafficking and activity of Na+-H+ exchangers.

Hepatocyte growth factor (HGF) is found in tumor microenvironments, and interaction with its tyrosine kinase receptor Met triggers cell invasion and metastasis. It was previously shown that acidic extracellular pH stimulated peripheral lysosome trafficking, resulting in increased cathepsin B secretion and tumor cell invasion, which was dependent upon sodium-proton exchanger (NHE) activity. We now demonstrate that HGF induced the trafficking of lysosomes to the cell periphery, independent of HGF-induced epithelial-mesenchymal transition. HGF-induced anterograde lysosome trafficking depended upon the PI3K pathway, microtubules and RhoA, resulting in increased cathepsin B secretion and invasion by the cells. HGF-induced NHE activity via increased net acid production, and inhibition of NHE activity with 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), or a combination of the NHE1-specific drug cariporide and the NHE3-specific drug s3226 prevented HGF-induced anterograde trafficking and induced retrograde trafficking in HGF-overexpressing cells. EIPA treatment reduced cathepsin B secretion and HGF-induced invasion by the tumor cells. Lysosomes were located more peripherally in Rab7-shRNA-expressing cells and these cells were more invasive than control cells. Overexpression of the Rab7 effector protein, RILP, resulted in a juxtanuclear location of lysosomes and reduced HGF-induced invasion. Together, these results suggest that the location of lysosomes is an inherently important aspect of invasion by tumor cells.

Na+/H+ exchangers and RhoA regulate acidic extracellular pH-induced lysosome trafficking in prostate cancer cells.

Acidic extracellular pH (pHe) is a common feature of the tumor microenvironment and has been implicated in tumor invasion through the induction of protease secretion.Since lysosomes constitute the major storehouse of cellular proteases, the trafficking of lysosomes to the cell periphery may be required in order to secrete proteases. We demonstrate that a pHe of 6.4-6.8 induced the trafficking of lysosomes to membrane protrusions in the cell periphery. This trafficking event depended upon the PI3K pathway, the GTPase RhoA and sodium-proton exchange activity, resulting in lysosomal exocytosis. Acidic pHe induced a cytoplasmic acidification (although cytoplasmic acidification was not sufficient for acidic pHe-induced lysosome trafficking and exocytosis) and inhibition of NHE activity with the amiloride derivative, EIPA or the anti-diabetic agent troglitazone prevented lysosome trafficking to the cell periphery. Interestingly, using the more specific NHE1 and NHE3 inhibitors, cariporide and s3226 respectively, we show that multiple NHE isoforms are involved in acidic pHe-induced lysosome trafficking and exocytosis. Moreover, in cells expressing NHE1 shRNA, although basal NHE activity was decreased, lysosomes still underwent acidic pHe-induced trafficking,suggesting compensation by other NHE family members.Together these data implicate proton exchangers, especially NHE1 and NHE3, in acidic pHe-induced lysosome trafficking and exocytosis.

The polyphenol epigallocatechin-3-gallate affects lipid rafts to block activation of the c-Met receptor in prostate cancer cells.

The HGF/c-Met pathway is an important regulator of signaling pathways responsible for invasion and metastasis of most human cancers, including prostate cancer. Exposure of DU145 prostate tumor cells to HGF stimulates the PI3-kinase and MAPK pathways, leading to increased scattering, motility, and invasion, which was prevented by the addition of EGCG. EGCG acted at the level of preventing phosphorylation of tyrosines 1234/1235 in the kinase domain of the c-Met receptor without effecting dimerization. HGF-induced changes were independent of the formation of reactive oxygen species, suggesting that EGCG functioned independent of its antioxidant ability. ECG, another tea polyphenol, was as effective as EGCG, while EGC and EC were less effective. EGCG added up to 4 h after the addition of HGF still blocked cell scattering and reduced the HGF-induced phosphorylation of c-Met, Akt, and Erk, suggesting that EGCG could act both by preventing activation of c-Met by HGF and by attenuating the activity of pathways already induced by HGF. HGF did not activate the MAPK and PI3-K pathways in cells treated with methyl-beta-cyclodextrin (mCD) to remove cholesterol. Furthermore, subcellular fractionation approaches demonstrated that only phosphorylated c-Met accumulated in Triton X-100 membrane insoluble fractions, supporting a role for lipid rafts in regulating c-Met signaling. Finally, EGCG treatment inhibited DiIC16 incorporation into membrane lipid ordered domains, and cholesterol partially inhibited the EGCG effects on signaling. Together, these results suggest that green tea polyphenols with the R1 galloyl group prevent activation of the c-Met receptor by altering the structure or function of lipid rafts.

Inhibition of fatty acid synthase by luteolin post-transcriptionally down-regulates c-Met expression independent of proteosomal/lysosomal degradation.

The hepatocyte growth factor (HGF)/c-Met signaling pathway is involved in the progression of several cancers and associated with increased tumor invasion and metastatic potential. We determined previously that the polyphenol epigallocatechin-3-gallate inhibited HGF-induced c-Met phosphorylation in a variety of tumor cell lines in part by disrupting lipid rafts. Fatty acid synthase (FASN) is implicated in cancer progression and may regulate lipid raft function. We therefore examined the effects of luteolin, a potent FASN inhibitor, on c-Met signaling. Luteolin blocked HGF-induced c-Met phosphorylation and scattering of DU145 prostate cancer cells, but inhibition required at least a 4 h preincubation time. Western blot analysis indicated that inhibition of HGF-induced scattering by luteolin occurred coincident with reduction of total c-Met protein in DU145 cells. In addition, luteolin-induced c-Met down-regulation was mimicked by a pharmacologic inhibitor of FASN, C75, or short hairpin RNA knockdown of FASN. Consistent with a role for FASN, loss of c-Met in cells treated with C75 or luteolin was prevented by exogenous addition of palmitate. Luteolin-induced loss of c-Met primarily occurred at a post-transcriptional level and involved cell surface internalization but did not involve translation inhibition, nor was it dependent on the activity of the 26S proteosome or acidic lysosomes. Taken together, our study shows a novel connection between FASN activity and c-Met protein expression and suggests that luteolin could act as a novel HGF/c-Met inhibitor by reducing expression of this receptor.

TIMP-1 overexpression promotes tumorigenesis of MDA-MB-231 breast cancer cells and alters expression of a subset of cancer promoting genes in vivo distinct from those observed in vitro.

TIMP-1 (Tissue inhibitor of matrix metalloproteinase-1) is typically associated with inhibition of matrix metalloproteinases (MMP) induced invasion. However, TIMP-1 is overexpressed in many malignancies and is associated with poor prognosis in breast cancer. The mechanisms by which TIMP-1 promotes tumorigenesis are unclear. Reduced levels of TIMP-1 mediated by shRNA in MDA-MB-231 breast cancer cells had no effect on cellular physiology in vitro or tumor growth in SCID mice compared to vector control MDA-MB-231 cells. However, overexpression of TIMP-1 in MDA-MB-231 cells resulted in inhibition of cell invasion and enhanced phosphorylation of p38 MAPK and AKT in vitro. Additionally, treatment of parental MDA-MB-231 cells with purified TIMP-1 protein led to activation of p38 MAPK and MKK 3/6. cDNA array analysis demonstrated that high expression of TIMP-1 in MDA-MB-231 cells resulted in alterations in expression of approximately 200 genes, 1.5 fold or greater compared to vector control cells (P < 0.1). Real-time RT-PCR confirmed changes in expression of several genes associated with cancer progression including DAPK1, FGFR4 and MAPK13. In vivo, high TIMP-1 expression induced tumor growth in SCID mice compared to vector control cells and increased tumor vessel density. Affymetrix array analysis of vector control and TIMP-1 MDA-MB-231 xenograft tumors revealed that TIMP-1 altered expression of approximately 600 genes in vivo, including MMP1, MMP13, S100A14, S100P, Rab25 and ID4. These combined observations suggest that the effects of TIMP-1 differ significantly in a 2-D environment compared to the 3-D environment and that TIMP-1 stimulates tumor growth.

Helicobacter pylori induces cancer cell motility independent of the c-Met receptor.

BACKGROUND: The hepatocyte growth factor (HGF) receptor, c-Met, is strongly implicated in late-stage cancer progression and poor patient prognosis. The stomach pathogen, Helicobacter pylori ( H. pylori ), was recently proposed to stimulate c-Met phosphorylation dependent upon interaction of c-Met with the bacterial CagA protein required for H. pylori -induced cancer cell motility and invasion. MATERIALS AND METHODS: In this report, we employed short hairpin RNA (shRNA), western blot analysis using antibodies recognizing phosphorylation at discrete c-Met residues, and immunofluorescence microscopy to investigate the CagA-c-Met interaction. RESULTS: The data showed that shRNA-mediated c-Met knockdown did not reduce H. pylori -induced cell motility, suggesting that c-Met was not required for motility. Surprisingly, c-Met knockdown did not reduce the level of an H. pylori -induced protein recognized by a phospho-c-Met antibody. This 125 kD protein was 10 kD smaller than c-Met, suggesting that H. pylori did not phosphorylate c-Met but cross-reacted with another protein. This hypothesis was confirmed when c-Met phosphorylation inhibitors did not lower the levels of the bacteria-induced 125 kD protein, and c-Met immunoprecipitation (IP) did not detect this 125 kD protein from H. pylori -treated lysates. This protein was identified as a product of antibody cross reactivity with phosphorylated CagA. We also confirmed that CagA interacts with c-Met, but this interaction may have caused previous authors to misinterpret phosphorylated CagA as c-Met phosphorylation. Finally, pretreatment with the proteasomal inhibitor, lactacystin, caused prolonged HGF-induced c-Met phosphorylation and facilitated a CagA-negative H. pylori to stimulate AGS cell motility, suggesting that sustained c-Met phosphorylation compensates for the loss of CagA-dependent signaling. CONCLUSIONS: These data demonstrate that H. pylori stimulates cancer cell motility independent of the c-Met receptor. We further hypothesize that although H. pylori does not target c-Met, the bacteria may still utilize c-Met effector signaling to stimulate CagA-independent cancer cell motility, which may provide a further mechanism of H. pylori -dependent gastric cancer progression.

Induction of guanylate binding protein 5 by gamma interferon increases susceptibility to Salmonella enterica serovar Typhimurium-induced pyroptosis in RAW 264.7 cells.

The regulation of caspase-1 activation in macrophages plays a central role in host defense against bacterial pathogens. The activation of caspase-1 by the detection of bacterial products through Nod-like receptors leads to the secretion of mature interleukin-1beta (IL-1beta) and IL-18 and the induction of rapid host cell death (pyroptosis). Here, we report that pyroptosis induced by Salmonella enterica serovar Typhimurium can be positively regulated by prior gamma interferon (IFN-gamma) stimulation of RAW 264.7 cells. This increase in cell death is dependent on both caspase-1 activation and, in part, Salmonella pathogenicity island 1 (SPI-1) expression by Salmonella. Furthermore, the exogenous expression of the IFN-gamma-induced protein guanylate binding protein 5 (GBP-5) is sufficient to induce a heightened susceptibility of RAW 264.7 cells to Salmonella-induced pyroptosis, and the endogenous expression of GBP-5 is important for this phenomenon. RAW 264.7 cells with decreased expression of GBP-5 mRNA (inhibited by short hairpin RNA against GBP-5) release twofold less lactate dehydrogenase (a marker of membrane permeability) upon infection by invasive S. enterica serovar Typhimurium than do infected control cells. Importantly, 3x FLAG-tagged GBP-5 is localized to membrane ruffles, which contact invasive Salmonella, and is found on the membranes of spacious phagosomes containing Salmonella (although it is also found in the cytoplasm and on other cellular membranes), placing 3x FLAG GBP-5 at the interface of secreted SPI-1 effectors and host protein machinery. The regulation of pyroptosis by the IFN-gamma-induced protein GBP-5 may play an important role in the host defense against Salmonella enterica serovar Typhimurium and perhaps other invasive bacterial pathogens.

Large-scale purification of latex bead phagosomes from mouse macrophage cell lines and subsequent preparation for high-throughput quantitative proteomics.

Phagocytosis involves the engagement of a diverse array of cell surface receptors whose signal must be integrated on the membrane of the forming phagosomal cup. This method enables the quantitative proteomic analysis of phagosome fractions derived from phagocytes stimulated under two different conditions, thus allowing the complexity of phagosomal signaling to be analyzed in terms of the quantitative changes in phagosomal fraction protein content.

GLUT1CBP(TIP2/GIPC1) interactions with GLUT1 and myosin VI: evidence supporting an adapter function for GLUT1CBP.

We identified a novel interaction between myosin VI and the GLUT1 transporter binding protein GLUT1CBP(GIPC1) and first proposed that as an adapter molecule it might function to couple vesicle-bound proteins to myosin VI movement. This study refines the model by identifying two myosin VI binding domains in the GIPC1 C terminus, assigning respective oligomerization and myosin VI binding functions to separate N- and C-terminal domains, and defining a central region in the myosin VI tail that binds GIPC1. Data further supporting the model demonstrate that 1) myosin VI and GIPC1 interactions do not require a mediating protein; 2) the myosin VI binding domain in GIPC1 is necessary for intracellular interactions of GIPC1 with myosin VI and recruitment of overexpressed myosin VI to membrane structures, but not for the association of GIPC1 with such structures; 3) GIPC1/myosin VI complexes coordinately move within cellular extensions of the cell in an actin-dependent and microtubule-independent manner; and 4) blocking either GIPC1 interactions with myosin VI or GLUT1 interactions with GIPC1 disrupts normal GLUT1 trafficking in polarized epithelial cells, leading to a reduction in the level of GLUT1 in the plasma membrane and concomitant accumulation in internal membrane structures.

Development and in-vitro characterization of nanoemulsions loaded with paclitaxel/γ-tocotrienol lipid conjugates.

Vitamin E TPGS is a tocopherol (α-T) based nonionic surfactant that was used in the formulation of the Tocosol™ paclitaxel nanoemulsion, which was withdrawn from phase III clinical trials. Unlike tocopherols, however, the tocotrienol (T3) isomers of vitamin E were found to have innate anticancer activity and were shown to potentiate the antitumor activity of paclitaxel. The primary objective of the present study was therefore to develop a paclitaxel nanoemulsions by substituting α-T oil core of Tocosol™ with γ-T3 in, and vitamin E TPGS with PEGylated γ-T3 as the shell, and test the nanoemulsions against Bx-PC-3 and PANC-1 pancreatic tumor cells. A secondary objective was to test the activity of paclitaxel when directly conjugated with the γ-T3 isomer of vitamin E. The synthesis of the conjugates was confirmed by NMR and mass spectroscopy. Developed nanoemulsions were loaded with free or lipid conjugated paclitaxel. Nanoemulsions droplets were <300 nm with fastest release observed with formulations loaded with free paclitaxel when γ-T3 was used as the core. Substituting α-T with γ-T3 was also found to potentiate the anticancer activity of the nanoemulsions. Although marginal increase in activity was observed when nanoemulsions were loaded with free paclitaxel, a significant increase in activity was observed when lipid conjugates were used. The results from this study suggest that the developed paclitaxel nanoemulsions with either γ-T3, PEGylated γ-T3, or paclitaxel lipid conjugates may represent a more promising option for paclitaxel delivery in cancer chemotherapy.

Correction: Modulating lysosomal function through lysosome membrane permeabilization or autophagy suppression restores sensitivity to cisplatin in refractory non-small-cell lung cancer cells.

[This corrects the article DOI: 10.1371/journal.pone.0184922.].

Human papillomavirus type 16 E5-mediated upregulation of Met in human keratinocytes.

Human papillomaviruses (HPVs) cause benign lesions that can lead to malignancy. How cellular changes induced by viral oncogenes contribute to the progeny virion production is not always clear. Stromally-derived growth factors and their receptors are critical for development of malignancy, but their impact on the pre-malignant HPV life cycle is unknown. We show that HPV16 increases levels of Met, a growth factor receptor critical for tumor cell invasion, motility, and cancer metastasis. The viral oncogene E5 is primarily responsible for Met upregulation, with E6 playing a minor role. Met induction by E5 requires the epidermal growth factor receptor, which is also increased by E5 at the mRNA level. E5-induced Met contributes motility of HPV-containing cells. Finally, Met signaling is necessary for viral gene expression, particularly in the differentiation-dependent phase of the viral life cycle. These studies show a new role for E5 in epithelial-stromal interactions, with implications for cancer development.

Dictyostelium LvsB mutants model the lysosomal defects associated with Chediak-Higashi syndrome.

Chediak-Higashi syndrome is a genetic disorder caused by mutations in a gene encoding a protein named LYST in humans ("lysosomal trafficking regulator") or Beige in mice. A prominent feature of this disease is the accumulation of enlarged lysosome-related granules in a variety of cells. The genome of Dictyostelium discoideum contains six genes encoding proteins that are related to LYST/Beige in amino acid sequence, and disruption of one of these genes, lvsA (large volume sphere), results in profound defects in cytokinesis. To better understand the function of this family of proteins in membrane trafficking, we have analyzed mutants disrupted in lvsA, lvsB, lvsC, lvsD, lvsE, and lvsF. Of all these, only lvsA and lvsB mutants displayed interesting phenotypes in our assays. lvsA-null cells exhibited defects in phagocytosis and contained abnormal looking contractile vacuole membranes. Loss of LvsB, the Dictyostelium protein most similar to LYST/Beige, resulted in the formation of enlarged vesicles that by multiple criteria appeared to be acidic lysosomes. The rates of endocytosis, phagocytosis, and fluid phase exocytosis were normal in lvsB-null cells. Also, the rates of processing and the efficiency of targeting of lysosomal alpha-mannosidase were normal, although lvsB mutants inefficiently retained alpha-mannosidase, as well as two other lysosomal cysteine proteinases. Finally, results of pulse-chase experiments indicated that an increase in fusion rates accounted for the enlarged lysosomes in lvsB-null cells, suggesting that LvsB acts as a negative regulator of fusion. Our results support the notion that LvsB/LYST/Beige function in a similar manner to regulate lysosome biogenesis.

Modulating lysosomal function through lysosome membrane permeabilization or autophagy suppression restores sensitivity to cisplatin in refractory non-small-cell lung cancer cells.

Lung cancer is the leading cause of cancer-related deaths. Most patients develop resistance to platinum within several months of treatment. We investigated whether triggering lysosomal membrane permeabilization (LMP) or suppressing autophagy can restore cisplatin susceptibility in lung cancer with acquired chemoresistance. Cisplatin IC50 in A549Pt (parental) and A549cisR (cisplatin resistant) cells was 13 µM and 47 µM, respectively. Following cisplatin exposure, A549cisR cells failed to elicit an apoptotic response. This was manifested by diminished Annexin-V staining, caspase 3 and 9, BAX and BAK activation in resistant but not in parental cells. Chloroquine preferentially promoted LMP in A549cisR cells, revealed by leakage of FITC-dextran into the cytosol as detected by immunofluorescence microscopy. This was confirmed by increased cytosolic cathepsin D signal on Immunoblot. Cell viability of cisplatin-treated A549cisR cells was decreased when co-treated with chloroquine, corresponding to a combination index below 0.8, suggesting synergism between the two drugs. Notably, chloroquine activated the mitochondrial cell death pathway as indicated by increase in caspase 9 activity. Interestingly, inhibition of lysosomal proteases using E64 conferred cytoprotection against cisplatin and chloroquine co-treatment, suggesting that chloroquine-induced cell death occurred in a cathepsin-mediated mechanism. Likewise, blockage of caspases partially rescued A549cisR cells against the cytotoxicity of cisplatin and chloroquine combination. Cisplatin promoted a dose-dependent autophagic flux induction preferentially in A549cisR cells, as evidenced by a surge in LC3-II/α-tubulin following pre-treatment with E64 and increase in p62 degradation. Compared to untreated cells, cisplatin induced an increase in cyto-ID-loaded autophagosomes in A549cisR cells that was further amplified by chloroquine, pointing toward autophagic flux activation by cisplatin. Interestingly, this effect was less pronounced in A549Pt cells. Blocking autophagy by ATG5 depletion using siRNA markedly enhances susceptibility to cisplatin in A549cisR cells. Taken together, our results underscore the utility of targeting lysosomal function in overcoming acquired cisplatin refractoriness in lung cancer.

Identification of small molecules that disrupt vacuolar function in the pathogen Candida albicans.

The fungal vacuole is a large acidified organelle that performs a variety of cellular functions. At least a sub-set of these functions are crucial for pathogenic species of fungi, such as Candida albicans, to survive within and invade mammalian tissue as mutants with severe defects in vacuolar biogenesis are avirulent. We therefore sought to identify chemical probes that disrupt the normal function and/or integrity of the fungal vacuole to provide tools for the functional analysis of this organelle as well as potential experimental therapeutics. A convenient indicator of vacuolar integrity based upon the intracellular accumulation of an endogenously produced pigment was adapted to identify Vacuole Disrupting chemical Agents (VDAs). Several chemical libraries were screened and a set of 29 compounds demonstrated to reproducibly cause loss of pigmentation, including 9 azole antifungals, a statin and 3 NSAIDs. Quantitative analysis of vacuolar morphology revealed that (excluding the azoles) a sub-set of 14 VDAs significantly alter vacuolar number, size and/or shape. Many C. albicans mutants with impaired vacuolar function are deficient in the formation of hyphal elements, a process essential for its pathogenicity. Accordingly, all 14 VDAs negatively impact C. albicans hyphal morphogenesis. Fungal selectivity was observed for approximately half of the VDA compounds identified, since they did not alter the morphology of the equivalent mammalian organelle, the lysosome. Collectively, these compounds comprise of a new collection of chemical probes that directly or indirectly perturb normal vacuolar function in C. albicans.

Palmitoylation regulates the intracellular trafficking and stability of c-Met.

c-Met is a receptor tyrosine kinase whose activity can promote both mitogenic and motogenic phenotypes involved in tissue development and cancer progression. Herein, we report the first evidence that c-Met is palmitoylated and that palmitoylation facilitates its trafficking and stability. Inhibition of palmitoylation reduced the expression of c-Met in multiple cancer cell lines post-transcriptionally. Using surface biotinylation, confocal microscopy, and metabolic labeling we determined that inhibition of palmitoylation reduces the stability of newly synthesized c-Met and causes accumulation at the Golgi. Acyl-biotin exchange and click chemistry-based palmitate labeling indicated the c-Met ß-chain is palmitoylated, and site-directed mutagenesis revealed two likely cysteine palmitoylation sites. Moreover, by monitoring palmitoylation kinetics during the biosynthesis and trafficking of c-Met, we revealed that stable palmitoylation occurs in the endoplasmic reticulum prior to cleavage of the 170 kDa c-Met precursor to the mature 140 kDa form. Our data suggest palmitoylation is required for egress from the Golgi for transport to the plasma membrane. These findings introduce palmitoylation as a critical modification of c-Met, providing a novel therapeutic target for c-Met-driven cancers.