Glucosylceramide synthase upregulates MDR1 expression in the regulation of cancer drug resistance through cSrc and β-catenin signaling

<p>Abstract</p> <p>Background</p> <p>Drug resistance is the outcome of multiple-gene interactions in cancer cells under stress of anticancer agents. <it>MDR1 </it>overexpression is most commonly detected in drug-resistant cancers and accompanied with other gene alterations including enhanced glucosylceramide synthase (GCS). <it>MDR1 </it>encodes for P-glycoprotein that extrudes anticancer drugs. Polymorphisms of <it>MDR1 </it>disrupt the effects of P-glycoprotein antagonists and limit the success of drug resistance reversal in clinical trials. GCS converts ceramide to glucosylceramide, reducing the impact of ceramide-induced apoptosis and increasing glycosphingolipid (GSL) synthesis. Understanding the molecular mechanisms underlying <it>MDR1 </it>overexpression and how it interacts with GCS may find effective approaches to reverse drug resistance.</p> <p>Results</p> <p><it>MDR1 </it>and <it>GCS </it>were coincidently overexpressed in drug-resistant breast, ovary, cervical and colon cancer cells; silencing <it>GCS </it>using a novel mixed-backbone oligonucleotide (MBO-asGCS) sensitized these four drug-resistant cell lines to doxorubicin. This sensitization was correlated with the decreased <it>MDR1 </it>expression and the increased doxorubicin accumulation. Doxorubicin treatment induced GCS and <it>MDR1 </it>expression in tumors, but MBO-asGCS treatment eliminated "in-vivo" growth of drug-resistant tumor (NCI/ADR-RES). MBO-asGCS suppressed the expression of <it>MDR1 </it>with GCS and sensitized NCI/ADR-RES tumor to doxorubicin. The expression of P-glycoprotein and the function of its drug efflux of tumors were decreased by 4 and 8 times after MBO-asGCS treatment, even though this treatment did not have a significant effect on P-glycoprotein in normal small intestine. GCS transient transfection induced <it>MDR1 </it>overexpression and increased P-glycoprotein efflux in dose-dependent fashion in OVCAR-8 cancer cells. GSL profiling, silencing of globotriaosylceramide synthase and assessment of signaling pathway indicated that GCS transfection significantly increased globo series GSLs (globotriaosylceramide Gb3, globotetraosylceramide Gb4) on GSL-enriched microdomain (GEM), activated cSrc kinase, decreased β-catenin phosphorylation, and increased nuclear β-catenin. These consequently increased <it>MDR1 </it>promoter activation and its expression. Conversely, MBO-asGCS treatments decreased globo series GSLs (Gb3, Gb4), cSrc kinase and nuclear β-catenin, and suppressed <it>MDR-1 </it>expression in dose-dependent pattern.</p> <p>Conclusion</p> <p>This study demonstrates, for the first time, that GCS upregulates <it>MDR1 </it>expression modulating drug resistance of cancer. GSLs, in particular globo series GSLs mediate gene expression of <it>MDR1 </it>through cSrc and β-catenin signaling pathway.</p

A New Mixed-Backbone Oligonucleotide against Glucosylceramide Synthase Sensitizes Multidrug-Resistant Tumors to Apoptosis

Enhanced ceramide glycosylation catalyzed by glucosylceramide synthase (GCS) limits therapeutic efficiencies of antineoplastic agents including doxorubicin in drug-resistant cancer cells. Aimed to determine the role of GCS in tumor response to chemotherapy, a new mixed-backbone oligonucleotide (MBO-asGCS) with higher stability and efficiency has been generated to silence human GCS gene. MBO-asGCS was taken up efficiently in both drug-sensitive and drug-resistant cells, but it selectively suppressed GCS overexpression, and sensitized drug-resistant cells. MBO-asGCS increased doxorubicin sensitivity by 83-fold in human NCI/ADR-RES, and 43-fold in murine EMT6/AR1 breast cancer cells, respectively. In tumor-bearing mice, MBO-asGCS treatment dramatically inhibited the growth of multidrug-resistant NCI/ADR-RE tumors, decreasing tumor volume to 37%, as compared with scrambled control. Furthermore, MBO-asGCS sensitized multidrug-resistant tumors to chemotherapy, increasing doxorubicin efficiency greater than 2-fold. The sensitization effects of MBO-asGCS relied on the decreases of gene expression and enzyme activity of GCS, and on the increases of C18-ceramide and of caspase-executed apoptosis. MBO-asGCS was accumulation in tumor xenografts was greater in other tissues, excepting liver and kidneys; but MBO-asGCS did not exert significant toxic effects on liver and kidneys. This study, for the first time in vivo, has demonstrated that GCS is a promising therapeutic target for cancer drug resistance, and MBO-asGCS has the potential to be developed as an antineoplastic agent

Identification and validation of a novel biologics target in triple negative breast cancer

The goal of this study was to identify a novel target for antibody-drug conjugate (ADC) development in triple negative breast cancer (TNBC), which has limited treatment options, using gene expression datasets and in vitro siRNA/CRISPR and in vivo functional assays. We analyzed 4467 breast cancers and identified GABRP as top expressed gene in TNBC with low expression in most normal tissues. GABRP protein was localized to cell membrane with broad range of receptors/cell (815–53,714) and expressed by nearly half of breast cancers tissues. GABRP gene knockdown inhibited TNBC cell growth and colony formation in vitro and growth of MDA-MB-468 xenografts in nude mice. Commercially available anti-GABRP antibody (5–100 μg/ml) or de novo generated Fabs (20 μg/ml) inhibited TNBC cell growth in vitro. The same antibody conjugated to mertansine (DM1) also showed significant anticancer activity at nanomolar concentrations. Our results indicate that GABRP is a potential novel therapeutic target for ADC development

Glucosylceramide synthase upregulates MDR1 expression in the regulation of cancer drug resistance through cSrc and beta-catenin signaling

Abstract Background Drug resistance is the outcome of multiple-gene interactions in cancer cells under stress of anticancer agents. MDR1 overexpression is most commonly detected in drug-resistant cancers and accompanied with other gene alterations including enhanced glucosylceramide synthase (GCS). MDR1 encodes for P-glycoprotein that extrudes anticancer drugs. Polymorphisms of MDR1 disrupt the effects of P-glycoprotein antagonists and limit the success of drug resistance reversal in clinical trials. GCS converts ceramide to glucosylceramide, reducing the impact of ceramide-induced apoptosis and increasing glycosphingolipid (GSL) synthesis. Understanding the molecular mechanisms underlying MDR1 overexpression and how it interacts with GCS may find effective approaches to reverse drug resistance. Results MDR1 and GCS were coincidently overexpressed in drug-resistant breast, ovary, cervical and colon cancer cells; silencing GCS using a novel mixed-backbone oligonucleotide (MBO-asGCS) sensitized these four drug-resistant cell lines to doxorubicin. This sensitization was correlated with the decreased MDR1 expression and the increased doxorubicin accumulation. Doxorubicin treatment induced GCS and MDR1 expression in tumors, but MBO-asGCS treatment eliminated "in-vivo" growth of drug-resistant tumor (NCI/ADR-RES). MBO-asGCS suppressed the expression of MDR1 with GCS and sensitized NCI/ADR-RES tumor to doxorubicin. The expression of P-glycoprotein and the function of its drug efflux of tumors were decreased by 4 and 8 times after MBO-asGCS treatment, even though this treatment did not have a significant effect on P-glycoprotein in normal small intestine. GCS transient transfection induced MDR1 overexpression and increased P-glycoprotein efflux in dose-dependent fashion in OVCAR-8 cancer cells. GSL profiling, silencing of globotriaosylceramide synthase and assessment of signaling pathway indicated that GCS transfection significantly increased globo series GSLs (globotriaosylceramide Gb3, globotetraosylceramide Gb4) on GSL-enriched microdomain (GEM), activated cSrc kinase, decreased β-catenin phosphorylation, and increased nuclear β-catenin. These consequently increased MDR1 promoter activation and its expression. Conversely, MBO-asGCS treatments decreased globo series GSLs (Gb3, Gb4), cSrc kinase and nuclear β-catenin, and suppressed MDR-1 expression in dose-dependent pattern. Conclusion This study demonstrates, for the first time, that GCS upregulates MDR1 expression modulating drug resistance of cancer. GSLs, in particular globo series GSLs mediate gene expression of MDR1 through cSrc and β-catenin signaling pathway

Multi-Omics Investigation of Innate Navitoclax Resistance in Triple-Negative Breast Cancer Cells

Cancer cells employ various defense mechanisms against drug-induced cell death. Investigating multi-omics landscapes of cancer cells before and after treatment can reveal resistance mechanisms and inform new therapeutic strategies. We assessed the effects of navitoclax, a BCL2 family inhibitor, on the transcriptome, methylome, chromatin structure, and copy number variations of MDA-MB-231 triple-negative breast cancer (TNBC) cells. Cells were sampled before treatment, at 72 h of exposure, and after 10-day drug-free recovery from treatment. We observed transient alterations in the expression of stress response genes that were accompanied by corresponding changes in chromatin accessibility. Most of these changes returned to baseline after the recovery period. We also detected lasting alterations in methylation states and genome structure that suggest permanent changes in cell population composition. Using single-cell analyses, we identified 2350 genes significantly upregulated in navitoclax-resistant cells and derived an 18-gene navitoclax resistance signature. We assessed the navitoclax-response-predictive function of this signature in four additional TNBC cell lines in vitro and in silico in 619 cell lines treated with 251 different drugs. We observed a drug-specific predictive value in both experiments, suggesting that this signature could help guiding clinical biomarker studies involving navitoclax

Direct quantitative determination of ceramide glycosylation in vivo: a new approach to evaluate cellular enzyme activity of glucosylceramide synthase

Glucosylceramide synthase (GCS or GlcT-1), converting ceramide to glucosylceramide, is a key enzyme for the synthesis of glycosphingolipids. Due to its diverse roles in physiology and diseases, GCS may be a disease marker and drug target. Current assays for enzymes including GCS are based on reactions conducted in a test tube using enzyme preparations. Measurement of enzyme activity in laboratory-made conditions cannot directly evaluate the role of GCS in cells. Here, we introduce a new approach to determine GCS cellular activity using fluorescent NBD C6-ceramide in vivo. Cellular GCS transfers UDP-glucose to NBD C6-ceramide and produces NBD C6-glucosylceramide. C6-glucosylceramide is then separated from C6-ceramide by thin-layer chromatography and both are then quantitated by spectrophotometer. This cell-based method is able to quantitate glucosylceramide in pmol range, produced by approximately 50,000 cells or 1.0 mg tissue. This method has been used successfully to evaluate the degrees of GCS enzyme in cells and in tumors subjected to gene manipulation and chemical inhibition. These data indicate that this cell-based fluorescent method is direct, reproducible, and simple for assessing ceramide glycosylation. It is applicable to validate GCS activity in drug-resistant cancers and in other disorders

MBO-asGCS suppresses tumor growth in athymic nude mice.

<p>Athymic nude mice (<i>Foxn1^nu/Foxn1⁺</i>, 10 per group) bearing drug-resistant tumors (NCI/ADR-RE cells) were treated with either MBOs (1 mg/kg every 3 days, intratumoral injection) alone or MBOs combined with doxorubicin (Dox, 2 mg/kg once a week, intraperitoneal injection) for 38 days. Treatments were started once tumors were visible (2 mm diameter, day 0). a. Tumor growth. *, p<0.001 compared with treatments in the presence of saline or MBO-SC. **, p<0.001 compared with treatments in the presence of doxorubicin or doxorubicin combined with MBO-SC. b. Body weight of mice after treatments. c. Tumors after treatments. Tumors were photographed when mice were sacrificed at the end of treatment regimens.</p

MBO-asGCS increases doxorubicin sensitivity in drug-resistant cancer cells.

<p>Cells (4,000/well) were plated in 96-well plates and pretreated with MBO-asGCS (50 nM). After 24 hr growth, cells were shifted to 5% FBS medium containing increasing concentrations of agents and grown for additional 72 hr. Cell viability was measured using the CellTiter-Glo luminescent cell viability assay. a. Cell viability after C₆-ceramide treatment. ADR-RE, NCI/ADR-RE cells; *, p<0.01 compared with vehicle treatment. b. EC₅₀ values for C₆-ceramide. *, p<0.001 compared with vehicle treatments. c. Cell viability after doxorubicin treatment. d. EC₅₀ values for doxorubicin. *, p<0.001 compared with vehicle treatment.</p