Single-Cell FISH Analysis Reveals Distinct Shifts in PKM Isoform Populations during Drug Resistance Acquisition.

The Warburg effect, i.e., the utilization of glycolysis under aerobic conditions, is recognized as a survival advantage of cancer cells. However, how the glycolytic activity is affected during drug resistance acquisition has not been explored at single-cell resolution. Because the relative ratio of the splicing isoform of pyruvate kinase M (PKM), PKM2/PKM1, can be used to estimate glycolytic activity, we utilized a single-molecule fluorescence in situ hybridization (SM-FISH) method to simultaneously quantify the mRNA levels of PKM1 and PKM2. Treatment of HCT116 cells with gefitinib (GE) resulted in two distinct populations of cells. However, as cells developed GE resistance, the GE-sensitive population with reduced PKM2 expression disappeared, and GE-resistant cells (Res) demonstrated enhanced PKM1 expression and a tightly regulated PKM2/PKM1 ratio. Our data suggest that maintaining an appropriate PKM2 level is important for cell survival upon GE treatment, whereas increased PKM1 expression becomes crucial in GE Res. This approach demonstrates the importance of single-cell-based analysis for our understanding of cancer cell metabolic responses to drugs, which could aid in the design of treatment strategies for drug-resistant cancers.

Probing Physical Properties of the Cellular Membrane in Senescent Cells by Fluorescence Imaging.

Cellular senescence is the irreversible cell cycle arrest in response to various types of stress. Although the plasma membrane and its composition are significantly affected by cellular senescence, detailed studies on the physical properties of the plasma membrane have shown inconclusive results. In this study, we utilized both ensemble and single-molecule fluorescence imaging to investigate how membrane properties, such as fluidity, hydrophobicity, and ganglioside GM1 level are affected by cellular senescence. The diffusion coefficient of lipid probes, as well as the type of diffusion determined by an exponent α, which is the slope of the log-log plot of mean squared displacement as a function of time lag, were analyzed. We found that the number of molecules with a lower diffusion coefficient increased as cells became senescent. The changes in the population with a lower diffusion coefficient, observed after methyl-ß-cyclodextrin treatment, and the increase in ceramide levels, detected using a ceramide-specific antibody, suggest that ceramide-rich lipid rafts were enhanced in senescent cells. Our results emphasize the importance of membrane properties in cellular senescence and might serve as a base for in-depth studies to determine how such domains facilitate the signaling pathway specific to cellular senescence.

Regulation of mRNA export through API5 and nuclear FGF2 interaction.

API5 (APoptosis Inhibitor 5) and nuclear FGF2 (Fibroblast Growth Factor 2) are upregulated in various human cancers and are correlated with poor prognosis. Although their physical interaction has been identified, the function related to the resulting complex is unknown. Here, we determined the crystal structure of the API5-FGF2 complex and identified critical residues driving the protein interaction. These findings provided a structural basis for the nuclear localization of the FGF2 isoform lacking a canonical nuclear localization signal and identified a cryptic nuclear localization sequence in FGF2. The interaction between API5 and FGF2 was important for mRNA nuclear export through both the TREX and eIF4E/LRPPRC mRNA export complexes, thus regulating the export of bulk mRNA and specific mRNAs containing eIF4E sensitivity elements, such as c-MYC and cyclin D1. These data show the newly identified molecular function of API5 and nuclear FGF2, and provide a clue to understanding the dynamic regulation of mRNA export.

Farnesyl diphosphate synthase is important for the maintenance of glioblastoma stemness.

Glioblastoma is a highly malignant tumor that easily acquires resistance to treatment. The stem-cell-like character (stemness) has been thought to be closely associated with the treatment resistance of glioblastoma cells. In this study, we determined that farnesyl diphosphate synthase (FDPS), a key enzyme in isoprenoid biosynthesis, plays an important role in maintaining glioblastoma stemness. A comparison of the mRNA expression in patient-derived glioblastoma sphere cells, which maintain stemness, and their differentiated counterparts, which lose stemness, via RNA sequencing showed that most of the altered genes were networked in the cholesterol biosynthesis pathway. We screened Federal Drug Administration (FDA)-approved drugs targeting specific enzymes in the cholesterol biosynthesis pathway for their ability to inhibit glioblastoma sphere formation. Inhibitors of FDPS, such as alendronate and zoledronate, significantly reduced the formation of glioblastoma spheres, and alendronate was effective at a lower molar concentration than zoledronate. Knockdown of FDPS using short hairpin RNA also completely inhibited the formation of secondary spheres. FDPS mRNA in patients with glioblastoma was associated with malignancy in three independent microarray data sets. RNA sequencing showed that alendronate treatment reduced the embryonic stem cell signature and activated development- and necrosis-related pathways in glioblastoma spheres. These results suggest that FDPS is important for the maintenance of glioblastoma stemness and that alendronate, a drug widely used to treat osteoporosis, can be repositioned to treat glioblastoma.

Metformin induces degradation of cyclin D1 via AMPK/GSK3ß axis in ovarian cancer.

Metformin, which is widely used as an anti-diabetic drug, reduces cancer related morbidity and mortality. However, the role of metformin in cancer is not fully understood. Here, we first describe that the anti-cancer effect of metformin is mediated by cyclin D1 deregulation via AMPK/GSK3ß axis in ovarian cancer cells. Metformin promoted cytotoxic effects only in the cancer cells irrespective of the p53 status and not in the normal primary-cultured cells. Metformin induced the G1 cell cycle arrest, in parallel with a decrease in the protein expressions of cyclin D1 without affecting its transcriptional levels. Using a proteasomal inhibitor, we could address that metformin-induced decrease in cyclin D1 through the ubiquitin/proteasome process. Cyclin D1 degradation by metformin requires the activation of GSK3ß, as determined based on the treatment with GSK3ß inhibitors. The activation of GSK3ß correlated with the inhibitory phosphorylation by Akt as well as p70S6K through AMPK activation in response to metformin. These findings suggested that the anticancer effects of metformin was induced due to cyclin D1 degradation via AMPK/GSK3ß signaling axis that involved the ubiquitin/proteasome pathway specifically in ovarian cancer cells. © 2016 Wiley Periodicals, Inc.

Malignant ascites enhances migratory and invasive properties of ovarian cancer cells with membrane bound IL-6R in vitro.

Transcoelomic route is the most common and the earliest route of metastasis, causing the ascites formation in advanced epithelial ovarian cancer (EOC). We demonstrated that interleukin 6 (IL-6) is enriched in the malignant ascites from patients with ovarian cancer, which enhanced invasive properties of EOC cells. Interestingly, the expression of IL-6R on cell membrane of EOC cells correlated with ascites-induced invasion. Selective knockdown of IL-6R or inhibition with IL-6 neutralizing antibody, suppressed the stimulatory effects of ascites on EOC invasion. Moreover, the ascites treatment induced the phosphorylation of JAK2-STAT3 and use of selective inhibitors of JAK2 and STAT3, blocked the expression of epithelial-mesenchymal transition related proteins in parallel with the suppression of EOC invasion. Thus, IL-6/IL-6R mediated JAK2-STAT3 signaling pathway could be a promising therapeutic target for anticancer therapy in ovarian cancer patients with ascites.

Autophagy and protein kinase RNA-like endoplasmic reticulum kinase (PERK)/eukaryotic initiation factor 2 alpha kinase (eIF2α) pathway protect ovarian cancer cells from metformin-induced apoptosis.

Metformin, an oral biguanide for the treatment of type II diabetes, has been shown to have anticancer effects in ovarian cancer. Energy starvation induced by metformin causes endoplasmic reticulum stress-mediated unfolded protein response (UPR) and autophagy. UPR and autophagy act as a survival or death mechanism in cells. In this study, we observed that metformin-induced apoptosis was relieved by autophagy and the PERK/eIF2α pathway in ovarian cancer cells, but not in peripheral blood mononuclear cells (PBMC) or 'normal' ovarian surface epithelial cells (OSE). Increased PARP cleavage and increased LC3B-II with ATG5-ATG12 complex suggested the induction of apoptosis and autophagy, respectively, in metformin-treated ovarian cancer cells. Accumulation of acidic vacuoles in the cytoplasm and downregulation of p62 further supported late-stage autophagy. Interestingly, metformin induced interdependent activation between autophagy and the UPR, especially the PERK/eIF2α pathway. Inhibition of autophagy-induced PERK inhibition, and vice versa, were demonstrated using small molecular inhibitors (PERK inhibitor I, GSK2606414; autophagy inhibitor, 3-MA, and BafA1). Moreover, autophagy and PERK activation protected ovarian cancer cells against metformin-induced apoptosis. Metformin treatment in the presence of inhibitors of PERK and autophagy, however, had no cytotoxic effects on OSE or PBMC. In conclusion, these results suggest that inhibition of autophagy and PERK can enhance the selective anticancer effects of metformin on ovarian cancer cells. © 2015 Wiley Periodicals, Inc.

Resveratrol triggers ER stress-mediated apoptosis by disrupting N-linked glycosylation of proteins in ovarian cancer cells.

Malignant tumors have a high glucose demand and alter cellular metabolism to survive. Herein, focusing on the utility of glucose metabolism as a therapeutic target, we found that resveratrol induced endoplasmic reticulum (ER) stress-mediated apoptosis by interrupting protein glycosylation in a cancer-specific manner. Our results indicated that resveratrol suppressed the hexosamine biosynthetic pathway and interrupted protein glycosylation through GSK3ß activation. Application of either biochemical intermediates of the hexosamine pathway or small molecular inhibitors of GSK3ß reversed the effects of resveratrol on the disruption of protein glycosylation. Additionally, an ER UDPase, ectonucleoside triphosphate diphosphohydrolase 5 (ENTPD5), modulated protein glycosylation by Akt attenuation in response to resveratrol. By inhibition or overexpression of Akt functions, we confirmed that the glycosylation activities were dependent on ENTPD5 expression and regulated by the action of Akt in ovarian cancer cells. Resveratrol-mediated disruption of protein glycosylation induced cellular apoptosis as indicated by the up-regulation of GADD153, followed by the activation of ER-stress sensors (PERK and ATF6α). Thus, our results provide novel insight into cancer cell metabolism and protein glycosylation as a therapeutic target for cancers.

Cancer-specific interruption of glucose metabolism by resveratrol is mediated through inhibition of Akt/GLUT1 axis in ovarian cancer cells.

The metabolic phenotype of cancer is considered an ideal target for anticancer therapy. In ovarian cancer, glucose transporter 1 (GLUT1) is overexpressed and positron emission tomography (PET) using [18(F)] fluorodeoxyglucose (FDG), as a metabolic tumor parameter, has been found to be an effective diagnostic tool. In this study, we have characterized the selective cytotoxicity of resveratrol (RSV) in ovarian cancer cells through glucose metabolism regulation via GLUT1 modulation. We have demonstrated that, in contrast to primary normal ovarian epithelial cells, RSV selectively inhibited glucose uptake and induced apoptosis irrespective of p53 status in vitro. RSV had no affect on GLUT1 mRNA and protein expressions but interrupted intracellular GLUT1 trafficking to the plasma membrane. Suppressed plasma membrane GLUT1 localization in ovarian cancer was found to be associated with the inhibition of Akt activity by RSV, as confirmed by the action of the Akt inhibitors (LY294002 and Akt inhibitor IV), as well as overexpression of a constitutive active form of Akt. Taken together, these findings suggested that RSV induced apoptosis in ovarian cancer cells by impairing glucose uptake, a process involving Akt-regulated plasma membrane GLUT1 trafficking.