Gauri A. Patwardhan

A role for ceramide in driving cancer cell resistance to doxorubicin

Advanced cancers acquire resistance to chemotherapy, and this results in treatment failure. The cellular mechanisms of chemotherapy resistance are not well understood. Here, for the first time, we show that ceramide contributes to cellular resistance to doxorubicin through up‐regulating the gene expression of glucosylceramide synthase (GCS). Ceramide, a cellular lipid messenger, modulates doxorubicin‐induced cell death. GCS catalyzes ceramide glycosylation, converting ceramide to glucosylceramide; this process hastens ceramide clearance and limits ceramide‐induced apoptosis. In the present study, we evaluated the role of the GCS gene in doxorubicin resistance using several paired wild‐type and drug‐resistant (doxorubicin‐selected) cancer cell lines, including breast, ovary, cervical, and colon. GCS was overexpressed in all drug‐resistant counterparts, and suppressing GCS overexpression using antisense oligonucleotide restored doxorubicin sensitivity. Characterizing the effect mechanism showed that doxorubicin exposure increased ceramide levels, enhanced GCS expression, and imparted cellular resistance. Exogenous C6‐ceramide and sphingomyelinase treatments mimicked the influence of doxorubicin on GCS, activating the GCS promoter and upregulating GCS gene expression. Fumonisin B1, an inhibitor of ceramide synthesis, significantly suppressed doxorubicin‐up‐regulated GCS expression. Promoter truncation, point mutation, gel‐shift, and protein‐DNA ELISA analysis showed that transcription factor Sp1 was essential for ceramide‐induced GCS up‐regulation. These data indicate that ceramide‐governed GCS gene expression drives cellular resistance to doxorubicin.—Liu, Y.‐Y., Yu, J. Y., Yin, D., Patwardhan, G. A., Gupta, V., Hirabayashi, Y., Holleran, W. M., Giuliano, A. E., Jazwinski, S. M., Gouaze‐Andersson, V., Consoli, D. P., Cabot, M. C. A role for ceramide in driving cancer cell resistance to doxorubicin. FASEB J. 22, 2541–2551 (2008)

Sphingolipids and expression regulation of genes in cancer.

Sphingolipids including glycosphingolipids have myriad effects on cell functions and affect cancer in aspects of tumorigenesis, metastasis and tumor response to treatments. Bioactive ones like ceramide, sphingosine 1-phosphate and globotriaosylceramide initiate and process cellular signaling to alter cell behaviors immediately responding to oncogenic stress or treatment challenges. Recent studies pinpoint that sphingolipid-mediated gene expression has long and profound impacts on cancer cells, and these play crucial roles in tumor progression and in treatment outcome. More than 10 sphingolipids and glycosphingolipids selectively mediate expressions of approximately 50 genes including c-myc, p21, c-fos, telomerase reverse transcriptase, caspase-9, Bcl-x, cyclooxygenase-2, matrix metalloproteinases, integrins, Oct-4, glucosylceramide synthase and multidrug-resistant gene 1. By diverse functions of these genes, sphingolipids enduringly affect cellular processes of mitosis, apoptosis, migration, stemness of cancer stem cells and cellular resistance to therapies. Mechanistic studies indicate that sphingolipids regulate particular gene expression by modulating phosphorylation and acetylation of proteins that serve as transcription factors (β-catenin, Sp1), repressor of transcription (histone H3), and regulators (SRp30a) in RNA splicing. Disclosing molecular mechanisms by which sphingolipids selectively regulate particular gene expression, instead of other relevant ones, requires understanding of the exact roles of individual lipid instead of a group, the signaling pathways that are implicated in and interaction with proteins or other lipids in details. These studies not only expand our knowledge of sphingolipids, but can also suggest novel targets for cancer treatments.

Suppression of Glucosylceramide Synthase Restores p53-Dependent Apoptosis in Mutant p53 Cancer Cells

Tumor suppressor p53 plays an essential role in protecting cells from malignant transformation by inducing cell-cycle arrest and apoptosis. Mutant p53 that is detected in more than 50% of cases of cancers loses its role in suppression of tumors but gains in oncogenic function. Strategies to convert mutant p53 into wild-type p53 have been suggested for cancer prevention and treatment, but they face a variety of challenges. Here, we report an alternative approach that involves suppression of glucosylceramide synthase (GCS), an enzyme that glycosylates ceramide and blunts its proapoptotic activity in cancer cells. Human ovarian cancer cells expressing mutant p53 displayed resistance to apoptosis induced by DNA damage. We found that GCS silencing sensitized these mutant p53 cells to doxorubicin but did not affect the sensitivity of cells with wild-type p53. GCS silencing increased the levels of phosphorylated p53 and p53-responsive genes, including p21(Waf1/Cip1), Bax, and Puma, consistent with a redirection of the mutant p53 cells to apoptosis. Reactivated p53-dependent apoptosis was similarly verified in p53-mutant tumors where GCS was silenced. Inhibition of ceramide synthase with fumonisin B1 prevented p53 reactivation induced by GCS silencing, whereas addition of exogenous C6-ceramide reactivated p53 function in p53-mutant cells. Our findings indicate that restoring active ceramide to cells can resuscitate wild-type p53 function in p53-mutant cells, offering preclinical support for a novel type of mechanism-based therapy in the many human cancers harboring p53 mutations.

Glucosylceramide synthase, a factor in modulating drug resistance, is overexpressed in metastatic breast carcinoma

Drug resistance causes treatment failure in approximately 50% of breast cancer patients with chemotherapy. Overexpression of glucosylceramide synthase (GCS) confers drug resistance in cancer cells, and suppression of GCS sensitizes cancers to chemotherapy in preclinical studies. Thus, GCS becomes a potential target to reverse drug resistance; however, little is known about GCS expression levels in normal tissues and whether GCS overexpression is associated with metastatic cancers. Herewith, we report our studies in GCS expression levels and breast cancer from patients. GCS levels were analyzed using cancer profiling arrays, breast cancer histo-arrays and quantitative RT-PCR in tumor tissues. We found that breast (18 exp. index) and other hormone-dependent organs (testis, cervix, ovary, prostate) displayed the lowest levels of GCS mRNA, whereas liver (52 exp. index) and other organs (kidney, bladder, stomach) displayed the highest levels of GCS. GCS mRNA levels were significantly elevated in tumors of breast, cervix, rectum and small intestine, as compared to each paired normal tissue. In mammary tissue, GCS overexpression was detected in breast cancers with metastasis, but not in benign fibroadenoma or primary tumors. GCS overexpression was coincident with HER2 expression (γ2=0.84) in ER-negative breast adenocarcinoma. In tumor specimens, GCS mRNA was elevated by 4-fold and significantly associated with stage III (5/7), lymph node-positive (7/8) and estrogen receptor-positive breast cancers (7/9). GCS expression was significantly and selectively elevated in breast cancer, in particular in metastatic disease. GCS overexpression was highly associated with ER-positive and HER2-positive breast cancer with metastasis. Although a small study, these data suggest that GCS may be a prognostic indicator and potential target for the treatment of chemotherapy-refractory breast cancer.

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.

Direct assessment of P-glycoprotein efflux to determine tumor response to chemotherapy.

Multidrug resistance is a major impediment to the success of cancer chemotherapy. The overproduced P-glycoprotein that extrudes anticancer drugs from cells, is the most common mechanism detected in multidrug-resistant cancers. Direct measurement of cellular efflux of tumors in vivo, rather than estimation of MDR1 mRNA and P-glycoprotein levels in samples stored or embedded, can functionally characterize the mechanism of drug resistance and determine the choice of anticancer drugs for cancer patients. Herewith, we introduce a new approach to directly determine P-glycoprotein efflux of tumors. Employing Flutax-2 (Oregon green-488 paclitaxel) and fluorescence spectrophotometry, this method has successfully measured cellular transportability including efflux and accumulation in diverse cancer cell lines, tumors and other tissues with high reproducibility. With this method, we have quantitatively determined cellular efflux that is correlated with P-glycoprotein levels and the reversal effects of agents in cell lines of breast, ovarian, cervical and colon cancers, and in tumor-bearing mice. It has sensitively detected these alterations of P-glycoprotein efflux in approximately 5mg tumor or other tissues with high confidence. This direct and quick functional assessment has a potential to determine drug resistance in different types of cancers after surgical resection. Further validation of this method in clinic settings for the diagnosis of drug resistance purpose is needed.

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.

Mixed Backbone Antisense Glucosylceramide Synthase Oligonucleotide (MBO-asGCS) loaded Solid Lipid Nanoparticles: In Vitro Characterization and Reversal of Multidrug Resistance in NCI/ADR-RES Cells

In this study, solid lipid nanoparticles (SLN) loaded with MBO-asGCS oligonucleotide were prepared, characterized and evaluated for cytotoxicity against NCI/ADR-RES human ovary cancer cells. Two types of cetyltrimethyl ammonium bromide (CTAB) stabilized SLN, with or without ceramide VI, were prepared by mixed homogenization/ultrasonication technique. Complexes were characterized for size, zeta-potential, and stability in biorelevant media and against DNaseI activity. Binding and release studies were further confirmed by gel electrophoresis. Cytotoxicity of the SLN against NCI/ADR-RES cells was evaluated by quantizing ATP. SLN with ceramide VI had lower particle size (74.6 nm) with improved stability in RPMI media when compared to reference SLN without ceramide VI (167.16 nm). Both SLN however had similar cytotoxicity profile with an optimum binding at CTAB to MBO-asGCS ratio of 6:1. Blank SLN, and free MBO-asGCS in the presence and absence of free doxorubicin had insignificant effect on the viability of NCI/ADR-RES cells. However, when cells were concurrently treated with MBO-asGCS loaded SLN and free doxorubicin, cell viability significantly decreased to approximately 12%. These results suggested that SLN enhanced internalization and uptake of MBO-asGCS oligonucleotide, which led to the downregulation of GCS and subsequently reversing the resistance of the cells to doxorubicin.

Ceramide modulates pre-mRNA splicing to restore the expression of wild-type tumor suppressor p53 in deletion-mutant cancer cells

Mutants of tumor suppressor p53 not only lose the activity in genome stabilizing and in tumor suppression, but also exhibit oncogenic function in cancer cells. Most efforts in restoring p53 biological activity focus on either altering mutant-protein conformation or introducing an exogenous p53 gene into cells to eliminate p53-mutant cancer cells. Being different from these, we report that ceramide can restore the expression of wild-type p53 and induce p53-dependent apoptosis in deletion-mutant cancer cells. We show that endogenous long-carbon chain ceramide species (C16- to C24-ceramides) and exogenous C6-ceramide, rather than other sphingolipids, restore wild-type mRNA (intact exon-5), phosphorylated protein (Ser15 in exon-5) of p53, and p53-responsive proteins, including p21 and Bax, in ovarian cancer cells, which predominantly express a deleted exon-5 of p53 mutant before treatments. Consequently, the restored p53 sensitizes these p53-mutant cancer cells to DNA damage-induced growth arrest and apoptosis. Furthermore, we elucidate that ceramide activates protein phosphatase-1, and then the dephosphorylated serine/arginine-rich splicing-factor 1 (SRSF1) is translocated to the nucleus, thus promoting pre-mRNA splicing preferentially to wild-type p53 expression. These findings disclose an unrecognized mechanism that pre-mRNA splicing dysfunction can result in p53 deletion-mutants. Ceramide through SRSF1 restores wild-type p53 expression versus deletion-mutant and leads cancer cells to apoptosis. This suggests that heterozygous deletion-mutants of p53 can be restored in posttranscriptional level by using epigenetic approaches.

Abstract 2073: A systematic investigation of the effect of scheduling of targeted combination therapies on response and dynamics of relapse in triple negative breast cancer cells

Cancer treatment typically involves administration of combination of targeted therapies, but initial response is often followed by disease relapse. The efficacy of a treatment regimen depends on the complex interplay between cancer growth dynamics, drug specificity and kinetics, treatment dose and its scheduling. In standard high-throughput drug screening assays, cells are treated with a single drug cocktail bolus and cell viability is assessed after 2-3 days, thus not considering treatment interactions and long-term effects. Recently we have reported a promising synergistic combination of crizotinib (ALK/MET inhibitor) and ABT-263 (BCL2/BCL-XL inhibitor) against triple negative breast cancer cells. To understand the effect of the sequence and combination doses of crizotnib and ABT-263, we designed a comprehensive experimental plan that involved a total of 567 treatment regimens by varying treatment duration with the first drug (1, 2, or 3 days), followed by drug withdrawal and recovery period (2, 5 or 10 days) and then by a second cycle of treatment and recovery periods over a 26-day period. Cell viability was assessed by the CellTiter-Glo luminescence assay. Interestingly, ABT-263 alone induced higher cytotoxicity than an equivalent dose of crizotinib, but the remaining viable cells recovered much faster after ABT-263 withdrawal than cells after crizotinib withdrawal. Furthermore, cells exposed to higher doses of ABT-263 eventually become less sensitive to crizotinib. Among sequential regimens, crizotinib followed by ABT-263 was significantly more effective than ABT-263 followed by crizotinib, and combinations that included lower doses of ABT-263 were most effective. Taken together, our results show a significant interaction between the two targeted therapies, and suggest that it may be possible to select treatment scheduling that can delay drug resistance and tumor relapse in vivo. Note: This abstract was not presented at the meeting. Citation Format: Gauri A. Patwardhan, Vikram B. Wali, Lajos Pusztai, Christos Hatzis. A systematic investigation of the effect of scheduling of targeted combination therapies on response and dynamics of relapse in triple negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2073. doi:10.1158/1538-7445.AM2017-2073