A new series of fluorogen-activating proteins for quantitative protein trafficking and co-localization studies in S. cerevisiae.

Spatial and temporal protein tracking in live cells permits proteome remodeling in response to extracellular cues. Historically, protein dynamics during trafficking have been visualized using constitutively active fluorescent proteins (FPs) fused to proteins of interest. While powerful, such FPs label all cellular pools of a protein, potentially masking the dynamics of select subpopulations. To help study protein subpopulations, bioconjugate tags, including the fluorogen activation proteins (FAPs), were developed. FAPs are comprised of two components: a single chain antibody (SCA) fused to the protein of interest and a malachite-green (MG) derivative, which fluoresces only when bound to the SCA. Importantly, the MG derivatives can be either cell-permeant or -impermeant, thus permitting isolated detection of SCA-tagged proteins at the cell surface and facilitating quantitative endocytic measures. To expand FAP use in yeast, we optimized the SCA for yeast expression, created FAP-tagging plasmids, and generated FAP-tagged organelle markers. To demonstrate FAP efficacy, we coupled the SCA to the yeast G-protein coupled receptor Ste3. We measured Ste3 endocytic dynamics in response to pheromone and characterized cis- and trans-acting regulators of Ste3. Our work significantly expands FAP technology for varied applications in S. cerevisiae.

Changing course: Glucose starvation drives nuclear accumulation of Hexokinase 2 in S. cerevisiae.

Glucose is the preferred carbon source for most eukaryotes, and the first step in its metabolism is phosphorylation to glucose-6-phosphate. This reaction is catalyzed by hexokinases or glucokinases. The yeast Saccharomyces cerevisiae encodes three such enzymes, Hxk1, Hxk2, and Glk1. In yeast and mammals, some isoforms of this enzyme are found in the nucleus, suggesting a possible moonlighting function beyond glucose phosphorylation. In contrast to mammalian hexokinases, yeast Hxk2 has been proposed to shuttle into the nucleus in glucose-replete conditions, where it reportedly moonlights as part of a glucose-repressive transcriptional complex. To achieve its role in glucose repression, Hxk2 reportedly binds the Mig1 transcriptional repressor, is dephosphorylated at serine 15 and requires an N-terminal nuclear localization sequence (NLS). We used high-resolution, quantitative, fluorescent microscopy of live cells to determine the conditions, residues, and regulatory proteins required for Hxk2 nuclear localization. Countering previous yeast studies, we find that Hxk2 is largely excluded from the nucleus under glucose-replete conditions but is retained in the nucleus under glucose-limiting conditions. We find that the Hxk2 N-terminus does not contain an NLS but instead is necessary for nuclear exclusion and regulating multimerization. Amino acid substitutions of the phosphorylated residue, serine 15, disrupt Hxk2 dimerization but have no effect on its glucose-regulated nuclear localization. Alanine substation at nearby lysine 13 affects dimerization and maintenance of nuclear exclusion in glucose-replete conditions. Modeling and simulation provide insight into the molecular mechanisms of this regulation. In contrast to earlier studies, we find that the transcriptional repressor Mig1 and the protein kinase Snf1 have little effect on Hxk2 localization. Instead, the protein kinase Tda1 regulates Hxk2 localization. RNAseq analyses of the yeast transcriptome dispels the idea that Hxk2 moonlights as a transcriptional regulator of glucose repression, demonstrating that Hxk2 has a negligible role in transcriptional regulation in both glucose-replete and limiting conditions. Our studies define a new model of cis- and trans-acting regulators of Hxk2 dimerization and nuclear localization. Based on our data, the nuclear translocation of Hxk2 in yeast occurs in glucose starvation conditions, which aligns well with the nuclear regulation of mammalian orthologs. Our results lay the foundation for future studies of Hxk2 nuclear activity.

Non-Essential Amino Acid Availability Influences Proteostasis and Breast Cancer Cell Survival During Proteotoxic Stress.

Protein homeostasis (proteostasis) regulates tumor growth and proliferation when cells are exposed to proteotoxic stress, such as during treatment with certain chemotherapeutics. Consequently, cancer cells depend to a greater extent on stress signaling, and require the integrated stress response (ISR), amino acid metabolism, and efficient protein folding and degradation pathways to survive. To define how these interconnected pathways are wired when cancer cells are challenged with proteotoxic stress, we investigated how amino acid abundance influences cell survival when Hsp70, a master proteostasis regulator, is inhibited. We previously demonstrated that cancer cells exposed to a specific Hsp70 inhibitor induce the ISR via the action of two sensors, GCN2 and PERK, in stress-resistant and sensitive cells, respectively. In resistant cells, the induction of GCN2 and autophagy supported resistant cell survival, yet the mechanism by which these events were induced remained unclear. We now report that amino acid availability reconfigures the proteostasis network. Amino acid supplementation, and in particular arginine addition, triggered cancer cell death by blocking autophagy. Consistent with the importance of amino acid availability, which when limited activates GCN2, resistant cancer cells succumbed when challenged with a potentiator for another amino acid sensor, mTORC1, in conjunction with Hsp70 inhibition. IMPLICATIONS: These data position amino acid abundance, GCN2, mTORC1, and autophagy as integrated therapeutic targets whose coordinated modulation regulates the survival of proteotoxic-resistant breast cancer cells.

Dihydroartemisinin Inhibits mTORC1 Signaling by Activating the AMPK Pathway in Rhabdomyosarcoma Tumor Cells.

Dihydroartemisinin (DHA), an anti-malarial drug, has been shown to possess potent anticancer activity, partly by inhibiting the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) signaling. However, how DHA inhibits mTORC1 is still unknown. Here, using rhabdomyosarcoma (RMS) as a model, we found that DHA reduced cell proliferation and viability in RMS cells, but not those in normal cells, which was associated with inhibition of mTORC1. Mechanistically, DHA did not bind to mTOR or FK506 binding protein 12 (FKBP12). In addition, DHA neither inhibited insulin-like growth factor-1 receptor (IGF-1R), phosphoinositide 3-kinase (PI3K), and extracellular signal-regulated kinase ½ (Erk1/2), nor activated phosphatase and tensin homolog (PTEN) in the cells. Rather, DHA activated AMP-activated protein kinase (AMPK). Pharmacological inhibition of AMPK, ectopic expression dominant negative or kinase-dead AMPK, or knockdown of AMPKa attenuated the inhibitory effect of DHA on mTORC1 in the cells. Additionally, DHA was able to induce dissociation of regulatory-associated protein of mTOR (raptor) from mTOR and inhibit mTORC1 activity. Moreover, treatment with artesunate, a prodrug of DHA, dose-dependently inhibited tumor growth and concurrently activated AMPK and suppressed mTORC1 in RMS xenografts. The results indicated that DHA inhibits mTORC1 by activating AMPK in tumor cells. Our finding supports that DHA or artesunate has a great potential to be repositioned for treatment of RMS.

Iron chelation inhibits mTORC1 signaling involving activation of AMPK and REDD1/Bnip3 pathways.

The mammalian target of rapamycin (mTOR) functions as two complexes (mTORC1 and mTORC2), regulating cell growth and metabolism. Aberrant mTOR signaling occurs frequently in cancers, so mTOR has become an attractive target for cancer therapy. Iron chelators have emerged as promising anticancer agents. However, the mechanisms underlying the anticancer action of iron chelation are not fully understood. Particularly, reports on the effects of iron chelation on mTOR complexes are inconsistent or controversial. Here, we found that iron chelators consistently inhibited mTORC1 signaling, which was blocked by pretreatment with ferrous sulfate. Mechanistically, iron chelation-induced mTORC1 inhibition was not related to ROS induction, copper chelation, or PP2A activation. Instead, activation of AMPK pathway mainly and activation of both HIF-1/REDD1 and Bnip3 pathways partially contribute to iron chelation-induced mTORC1 inhibition. Our findings indicate that iron chelation inhibits mTORC1 via multiple pathways and iron is essential for mTORC1 activation.

The Human Cytomegalovirus Nonstructural Glycoprotein UL148 Reorganizes the Endoplasmic Reticulum.

Human cytomegalovirus (HCMV) encodes an endoplasmic reticulum (ER)-resident glycoprotein, UL148, which activates the unfolded protein response (UPR) but is fully dispensable for viral replication in cultured cells. Hence, its previously ascribed roles in immune evasion and modulation of viral cell tropism are hypothesized to cause ER stress. Here, we show that UL148 is necessary and sufficient to drive the formation of prominent ER-derived structures that on average occupy 5% of the infected cell cytoplasm. The structures are sites where UL148 coalesces with cellular proteins involved in ER quality control, such as HRD1 and EDEM1. Electron microscopy revealed that cells infected with wild-type but not UL148-null HCMV show prominent accumulations of densely packed ruffled ER membranes which connect to distended cisternae of smooth and partially rough ER. During ectopic expression of UL148-green fluorescent protein (GFP) fusion protein, punctate signals traffic to accumulate at conspicuous structures. The structures exhibit poor recovery of fluorescence after photobleaching, which suggests that their contents are poorly mobile and do not efficiently exchange with the rest of the ER. Small-molecule blockade of the integrated stress response (ISR) prevents the formation of puncta, leading to a uniform reticular fluorescent signal. Accordingly, ISR inhibition during HCMV infection abolishes the coalescence of UL148 and HRD1 into discrete structures, which argues that UL148 requires the ISR to cause ER reorganization. Given that UL148 stabilizes immature forms of a receptor binding subunit for a viral envelope glycoprotein complex important for HCMV infectivity, our results imply that stress-dependent ER remodeling contributes to viral cell tropism.IMPORTANCE Perturbations to endoplasmic reticulum (ER) morphology occur during infection with various intracellular pathogens and in certain genetic disorders. We identify that a human cytomegalovirus (HCMV) gene product, UL148, profoundly reorganizes the ER during infection and is sufficient to do so when expressed on its own. Our results reveal that UL148-dependent reorganization of the ER is a prominent feature of HCMV-infected cells. Moreover, we find that this example of virally induced organelle remodeling requires the integrated stress response (ISR), a stress adaptation pathway that contributes to a number of disease states. Since ER reorganization accompanies roles of UL148 in modulation of HCMV cell tropism and in evasion of antiviral immune responses, our results may have implications for understanding the mechanisms involved. Furthermore, our findings provide a basis to utilize UL148 as a tool to investigate organelle responses to stress and to identify novel drugs targeting the ISR.

Ciclopirox activates ATR-Chk1 signaling pathway leading to Cdc25A protein degradation.

Ciclopirox olamine (CPX), an off-patent anti-fungal drug, has been found to inhibit the G1-cyclin dependent kinases partly by increasing the phosphorylation and degradation of Cdc25A. However, little is known about the molecular target(s) of CPX responsible for Cdc25A degradation. Here, we show that CPX induced the degradation of Cdc25A neither by increasing CK1α or decreasing DUB3 expression, nor via activating GSK3ß, but through activating Chk1 in rhabdomyosarcoma (Rh30) and breast carcinoma (MDA-MB-231) cells. This is strongly supported by the findings that inhibition of Chk1 with TCS2312 or knockdown of Chk1 profoundly attenuated CPX-induced Cdc25A degradation in the cells. Furthermore, we observed that CPX caused DNA damage, which was independent of reactive oxygen species (ROS) induction, but related to iron chelation. CPX treatment resulted in the activation of ataxia telangiectasia mutated (ATM) and ATM-and RAD3-related (ATR) kinases. Treatment with Ku55933 (a selective ATM inhibitor) failed to prevent CPX-induced Chk1 phosphorylation and Cdc25A degradation. In contrast, knockdown of ATR conferred high resistance to CPX-induced Chk1 phosphorylation and Cdc25A degradation. Therefore, the results suggest that CPX-induced degradation of Cdc25A is attributed to the activation of ATR-Chk1 signaling pathway, a consequence of iron chelation-induced DNA damage.

SKLB188 inhibits the growth of head and neck squamous cell carcinoma by suppressing EGFR signalling.

BACKGROUND: Overexpression of epidermal growth factor receptor (EGFR) occurs in approximately 90% of head and neck squamous cell carcinoma (HNSCC), and is correlated with poor prognosis. Thus, targeting EGFR is a promising strategy for treatment of HNSCC. Several small molecule EGFR inhibitors have been tested in clinical trials for treatment of HNSCC, but none of them are more effective than the current chemotherapeutic drugs. Thus, it is urgently needed to develop novel EGFR inhibitors for HNSCC treatment. METHODS: By screening an in-house focused library containing approximately 650 000 known kinase inhibitors and kinase inhibitor-like compounds containing common kinase inhibitor core scaffolds, we identified SKLB188 as a lead compound for inhibition of EGFR. The anticancer effects of SKLB188 on HNSCC cells were investigated by in vitro cell growth, cell cycle and apoptosis assays, as well as in vivo FaDu xenograft mouse model. Molecular docking, in vitro kinase profiling and western blotting were performed to characterise EGFR as the molecular target. RESULTS: SKLB188 inhibited HNSCC cell proliferation by inducing G1 cell cycle arrest, which was associated with downregulating the expression of Cdc25A, cyclins D1/A and cyclin-dependent kinases (CDK2/4), and upregulating the expression of cyclin-dependent kinase (CDK) inhibitors (p21Cip1 and p27Kip1), leading to decreased phosphorylation of Rb. SKLB188 also induced caspase-dependent apoptosis of HNSCC cells by downregulating the expression of Mcl-1 and survivin. Molecular docking revealed that SKLB188 could bind to the kinase domain of EGFR through hydrogen bonds and hydrophobic interactions. In vitro kinase assay showed that SKLB188 inhibited the activity of a recombinant human EGFR very potently (IC50=5 nM). Western blot analysis demonstrated that SKLB188 inhibited the phosphorylation of EGFR and its downstream targets, extracellular signal-regulated protein kinases 1 and 2 (Erk1/2) and Akt in the cells. In addition, SKLB188 dose-dependently inhibited FaDu xenograft growth in nude mice, and concurrently inhibited the phosphorylation of Erk1/2 and Akt in the tumours. CONCLUSIONS: SKLB188 potently inhibits the growth of HNSCC cells in vitro and in vivo by targeting EGFR signalling. The results provide a basis for further clinical investigation of SKLB188 as a targeted therapy for HNSCC. Our findings may open a new avenue for development of novel EGFR inhibitors for treatment of HNSCC and other cancers.

Ciclopirox inhibits cancer cell proliferation by suppression of Cdc25A.

Ciclopirox olamine (CPX), an off-patent fungicide, has recently been identified as a novel anticancer agent. However, the molecular mechanism underlying its anticancer action remains to be elucidated. Here we show that CPX inhibits cell proliferation in part by downregulating the protein level of Cdc25A in tumor cells. Our studies revealed that CPX did not significantly reduce Cdc25A mRNA level or Cdc25A protein synthesis, but remarkably promoted Cdc25A protein degradation. This resulted in inhibition of G1-cyclin dependent kinases (CDKs), as evidenced by increased inhibitory phosphorylation of G1-CDKs. Since Cdc25A degradation is tightly related to its phosphorylation status, we further examined whether CPX alters Cdc25A phosphorylation. The results showed that CPX treatment increased the phosphorylation of Cdc25A (S76 and S82), but only Cdc25A-S82A mutant was resistant to CPX-induced degradation. Furthermore, ectopic expression of Cdc25A-S82A partially conferred resistance to CPX inhibition of cell proliferation. Therefore, our findings indicate that CPX inhibits cell proliferation at least in part by promoting Cdc25A degradation.

Ciclopirox olamine inhibits mTORC1 signaling by activation of AMPK.

Ciclopirox olamine (CPX), an off-patent antifungal agent, has recently been identified as a potential anticancer agent. The mammalian target of rapamycin (mTOR) is a central controller of cell growth, proliferation and survival. Little is known about whether and how CPX executes its anticancer action by inhibiting mTOR. Here we show that CPX inhibited the phosphorylation of p70 S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E binding protein 1 (4E-BP1), two downstream effector molecules of mTOR complex 1 (mTORC1), in a spectrum of human tumor cells, indicating that CPX inhibits mTORC1 signaling. Using rhabdomyosarcoma cells as an experimental model, we found that expression of constitutively active mTOR (E2419K) conferred resistance to CPX inhibition of cell proliferation, suggesting that CPX inhibition of mTORC1 contributed to its anticancer effect. In line with this, treatment with CPX inhibited tumor growth and concurrently suppressed mTORC1 signaling in RD xenografts. Mechanistically, CPX inhibition of mTORC1 was neither via inhibition of IGF-I receptor or phosphoinositide 3-kinase (PI3K), nor by activation of phosphatase and tensin homolog (PTEN). Instead, CPX inhibition of mTORC1 was attributed to activation of AMP-activated protein kinase (AMPK)-tuberous sclerosis complexes (TSC)/raptor pathways. This is supported by the findings that CPX activated AMPK; inhibition of AMPK with Compound C or ectopic expression of dominant negative AMPKα partially prevented CPX from inhibiting mTORC1; silencing TSC2 attenuated CPX inhibition of mTORC1; and CPX also increased AMPK-mediated phosphorylation of raptor (S792). Therefore, the results indicate that CPX exerts the anticancer effect by activating AMPK, resulting in inhibition of mTORC1 signaling.

Fusarochromanone-induced reactive oxygen species results in activation of JNK cascade and cell death by inhibiting protein phosphatases 2A and 5.

Recent studies have shown that fusarochromanone (FC101), a mycotoxin, is cytotoxic in a variety of cell lines. However, the molecular mechanism underlying its cytotoxicity remains elusive. Here we found that FC101 induced cell death in COS7 and HEK293 cells in part by activating JNK pathway. This is evidenced by the findings that inhibition of JNK with SP600125 or expression of dominant negative c-Jun partially prevented FC101-induced cell death. Furthermore, we observed that FC101-activated JNK pathway was attributed to induction of reactive oxygen species (ROS). Pretreatment with N-acetyl-L-cysteine (NAC), a ROS scavenger and antioxidant, suppressed FC101-induced activation of JNK and cell death. Moreover, we noticed that FC101 inhibited the serine/threonine protein phosphatases 2A (PP2A) and 5 (PP5) in the cells, which was abrogated by NAC. Overexpression of PP2A or PP5 partially prevented FC101-induced activation of JNK and cell death. The results indicate that FC101-induced ROS inhibits PP2A and PP5, leading to activation of JNK pathway and consequently resulting in cell death.

Maduramicin inhibits proliferation and induces apoptosis in myoblast cells.

Maduramicin, a polyether ionophore antibiotic derived from the bacterium Actinomadura yumaensis, is currently used as a feed additive against coccidiosis in poultry worldwide. It has been clinically observed that maduramicin can cause skeletal muscle and heart cell damage, resulting in skeletal muscle degeneration, heart failure, and even death in animals and humans, if improperly used. However, the mechanism of its toxic action in myoblasts is not well understood. Using mouse myoblasts (C2C12) and human rhabdomyosarcoma (RD and Rh30) cells as an experimental model for myoblasts, here we found that maduramicin inhibited cell proliferation and induced cell death in a concentration-dependent manner. Further studies revealed that maduramicin induced accumulation of the cells at G0/G1 phase of the cell cycle, and induced apoptosis in the cells. Concurrently, maduramicin downregulated protein expression of cyclin D1, cyclin-dependent kinases (CDK4 and CDK6), and CDC25A, and upregulated expression of the CDK inhibitors (p21Cip1 and p27Kip1), resulting in decreased phosphorylation of Rb. Maduramicin also induced expression of BAK, BAD, DR4, TRADD and TRAIL, leading to activation of caspases 8, 9 and 3 as well as cleavage of poly ADP ribose polymerase (PARP). Taken together, our results suggest that maduramicin executes its toxicity in myoblasts at least by inhibiting cell proliferation and inducing apoptotic cell death.

Fusarochromanone induces G1 cell cycle arrest and apoptosis in COS7 and HEK293 cells.

Fusarochromanone (FC101), a mycotoxin produced by the fungus Fusarium equiseti, is frequently observed in the contaminated grains and feedstuffs, which is toxic to animals and humans. However, the underlying molecular mechanism remains to be defined. In this study, we found that FC101 inhibited cell proliferation and induced cell death in COS7 and HEK293 cells in a concentration-dependent manner. Flow cytometric analysis showed that FC101 induced G1 cell cycle arrest and apoptosis in the cells. Concurrently, FC101 downregulated protein expression of cyclin D1, cyclin-dependent kinases (CDK4 and CDK6), and Cdc25A, and upregulated expression of the CDK inhibitors (p21Cip1 and p27Kip1), resulting in hypophosphorylation of Rb. FC101 also inhibited protein expression of Bcl-2, Bcl-xL, Mcl-1 and survivin, and induced expression of BAD, leading to activation of caspase 3 and cleavage of PARP, indicating caspase-dependent apoptosis. However, Z-VAD-FMK, a pan-caspase inhibitor, only partially prevented FC101-induced cell death, implying that FC101 may induce cell death through both caspase-dependent and -independent mechanisms. Our results support the notion that FC101 executes its toxicity at least by inhibiting cell proliferation and inducing cell death.

Ciclopirox induces autophagy through reactive oxygen species-mediated activation of JNK signaling pathway.

Ciclopirox olamine (CPX), a fungicide, has been demonstrated as a potential anticancer agent. However, the underlying anticancer mechanism is not well understood. Here, we found that CPX induced autophagy in human rhabdomyosarcoma (Rh30 and RD) cells. It appeared that CPX-induced autophagy was attributed to induction of reactive oxygen species (ROS), as N-acetyl-L-cysteine (NAC), a ROS scavenger and antioxidant, prevented this process. Furthermore, we observed that CPX induced activation of mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK) and p38 MAPK, which was also blocked by NAC. However, only inhibition of JNK (with SP600125) or expression of dominant negative c-Jun partially prevented CPX-induced autophagy, indicating that ROS-mediated activation of JNK signaling pathway contributed to CPX-induced autophagy. Of interest, inhibition of autophagy by chloroquine (CQ) enhanced CPX-induced cell death, indicating that CPX-induced autophagy plays a pro-survival role in human rhabdomyosarcoma cells. Our finding suggests that the combination with autophagy inhibitors may be a novel strategy in potentiating the anticancer activity of CPX for treatment of rhabdomyosarcoma.

Dihydroartemisinin inhibits the mammalian target of rapamycin-mediated signaling pathways in tumor cells.

Dihydroartemisinin (DHA), an antimalarial drug, has previously unrecognized anticancer activity, and is in clinical trials as a new anticancer agent for skin, lung, colon and breast cancer treatment. However, the anticancer mechanism is not well understood. Here, we show that DHA inhibited proliferation and induced apoptosis in rhabdomyosarcoma (Rh30 and RD) cells, and concurrently inhibited the signaling pathways mediated by the mammalian target of rapamycin (mTOR), a central controller for cell proliferation and survival, at concentrations (<3 µM) that are pharmacologically achievable. Of interest, in contrast to the effects of conventional mTOR inhibitors (rapalogs), DHA potently inhibited mTORC1-mediated phosphorylation of p70 S6 kinase 1 and eukaryotic initiation factor 4E binding protein 1 but did not obviously affect mTORC2-mediated phosphorylation of Akt. The results suggest that DHA may represent a novel class of mTORC1 inhibitor and may execute its anticancer activity primarily by blocking mTORC1-mediated signaling pathways in the tumor cells.