Methods and pharmaceutical compositions for preventing or reducing metastatic dissemination

Abstract

The present invention relates to methods and pharmaceutical compositions for preventing or reducing metastatic dissemination (i.e. reducing motility of cancer cells). In particular, the present invention relates to a method for preventing or reducing metastatic dissemination (i.e. reducing motility of cancer cells) in a subject suffering from a cancer comprising the steps consisting of i) determining the expression level of at least one biomarker selected from the group consisting of soluble CD95L and EMT promoting factors in a sample obtained from the subject, ii) comparing the expression level determined at step i) with a predetermined reference value and iii) administering the subject with a therapeutically effective amount of C16-ceramide or derivatives such as C16-sphingomyelin and C16-glycosphingolipids when the expression level determined at step i) is higher than the predetermined reference value.

Claims

1. A method for inhibiting or reducing metastatic dissemination in a subject suffering from a cancer comprising the steps of: i) measuring an expression level of at least one biomarker selected from the group consisting of: soluble CD95L and epithelial-to-mesenchymal transition (EMT) promoting factors in a sample obtained from the subject, ii) comparing the expression level measured at step i) with a reference expression level of the biomarker determined in an individual who has the same cancer but for whom metastatic dissemination did not occur, and iii) administering to the subject a therapeutically effective amount of at least one compound selected from the group consisting of C16-ceramide, C16-sphingomyelin and C16-glycosphingolipid when the expression level determined at step i) is higher than the reference biomarker expression level.

2. The method of claim 1 wherein the subject suffers from a cancer selected from the group consisting of breast cancer, colon cancer, lung cancer, prostate cancer, testicular cancer, brain cancer, skin cancer, rectal cancer, gastric cancer, esophageal cancer, sarcomas, tracheal cancer, head and neck cancer, pancreatic cancer, liver cancer, ovarian cancer, lymphoid cancer, cervical cancer, vulvar cancer, melanoma, mesothelioma, renal cancer, bladder cancer, thyroid cancer, bone cancers, carcinomas, sarcomas, and soft tissue cancers.

3. The method of claim 1 wherein the subject suffers from a triple negative breast cancer.

4. The method of claim 1 wherein the EMT-promoting factor is TGFbeta.

5. The method of claim 1 wherein the C16-ceramide, C16-sphingomyelin or C16-glycosphingolipid is incorporated into liposomal vesicles.

Description

FIGURES

(1) FIG. 1. Treatment of mesenchymal breast cancer cells with a non-toxic amount of exogenous C16:0 ceramide reduces plasma membrane fluidity and prevents basal and CD95-mediated cell migration. A. The indicated cells were pre-incubated with or without a non-cytotoxic dose (1 μM) of C16:0 ceramide (C16-Cer) for 60 min, and were then incubated with or without cl-CD95L (100 ng/mL) for 30 min. Membrane fluidity was assessed by EPR. Values are means±SEM of four independent experiments (*, p<0.05; ns, not significant). B. Indicated cells were pretreated with or without C16:0 ceramide (1 M) for 60 min and were then incubated with or without cl-CD95L (100 ng/mL) for 24 h. Cell migration was assessed by the Boyden chamber assay. Bars=70 μm. Giemsa-stained cells that migrated to the lower side of the membrane were lysed and absorbance at 560 nm was recorded. Values represent the means and SEM of three independent experiments (*p<0.05).

EXAMPLE

(2) Material & Methods

(3) Cell Lines

(4) The human cell lines derived from hematological cell lineage (Jurkat, CEM, H9, HL60, SKW6.4, K562, MOLT4, SR) were cultured in RPMI supplemented with 8% (v/v) heat-inactivated fetal calf serum (FCS) and 2 mM L-glutamine at 37° C. in a 5% CO2 incubator. All other cancer cell lines were cultured in DMEM supplemented with 8% (v/v) heat-inactivated FCS and 2 mM L-glutamine at 37° C. in a 5% CO2 incubator. The NCI-60 collection of human tumor cell lines came from Charles River Laboratories (Wilmington, Mass., USA). Other cells were purchased from American Type Culture Collection (LGC Standards, Molsheim, France). The human leukemic T-cell lines Jurkat and CEM, the lymphoma T-cell line H9, the promyelocytic cell line HL60, and the human B lymphoblastoid cell line SKW6.4 were cultured in RPMI supplemented with 8% (v/v) heat-inactivated fetal calf serum (FCS) and 2 mM L-glutamine at 37° C. in a 5% CO.sub.2 incubator. The human breast cancer cell lines hs578T, MDA-MB-231, MDA-MB-468, T47D, ZR-75-1, and MCF7, as well as the human kidney carcinoma cell line Caki-1, the human colorectal carcinoma cell line HCT116, and the human glioma cell line U251 were cultured in DMEM supplemented with 8% (v/v) heat-inactivated FCS and 2 mM L-glutamine at 37° C. in a 5% CO.sub.2 incubator. All cells were purchased from American Type Culture Collection (LGC Standards, Molsheim, France). The human mammary epithelial cell line MCF10A is spontaneously immortalized but not transformed. MCF10A cells infected with LXSN-K-RasV12 or an empty vector were kindly provided by Dr. B. H. Park (Baltimore, Md., USA).sup.16. MCF10A cells were grown in DMEM/F12 medium supplemented with 5% horse serum, EGF (20 ng/mL), insulin (10 μg/mL), cholera toxin (100 ng/mL), and hydrocortisone (0.5 μg/mL) at 37° C. in a 5% CO.sub.2 incubator. HMECs infected with a retrovirus carrying hTERT, Ras, and AgT were kindly provided by Dr R. A. Weinberg (Cambridge, Mass., USA).sup.4. HMECs immortalized by hTERT and Ras were designated HMEC-TR.

(5) In Vitro Motility Assay

(6) Boyden chambers contained membranes with a pore size of 8 μm (Millipore, Molsheim, France). After hydration of the membranes, mesenchymal cells (10.sup.5 cells per chamber) or epithelial cells (3×10.sup.5 cells per chamber) were added to the top chamber in low serum (1%)-containing medium. The bottom chamber was filled with low serum (1%)-containing medium in the presence or absence of cl-CD95L (100 ng/mL). Cells were cultured for 24 h at 37° C. To quantify migration, cells were mechanically removed from the top side of the membrane using a cotton-tipped swab, and migrating cells from the reverse side were fixed with methanol and stained with Giemsa. For each experiment, five representative pictures were taken for each insert, then cells were lyzed and absorbance at 560 nm correlated to the amount of Giemsa stain was measured.

(7) EPR

(8) Membrane fluidity was determined by a spin-labeling method using EPR, as previously described.sup.27. Briefly, plasma membranes of living cells were labeled by incubating cells for 15 min at 37° C. with 12-doxyl stearic acid (36 μg/mL), a fatty acid with a stable nitroxide radical ring at the C-12 position. Cells were then washed twice with PBS to remove excess 12-doxyl stearic acid and EPR spectra of the labeled samples were acquired at ambient temperature on a Bruker Elexsys EPR spectrometer operating at 3509.25 G center field, 20 mW microwave power, 9.86 GHz microwave frequency, 1.77 G modulation amplitude, and 100 kHz modulation frequency. Fluidity of the spin-labeled membranes was quantified by calculating the S parameter. An increase in the S parameter reflects a decrease in membrane fluidity, and vice versa.

(9) Immunoblotting

(10) Cells were lyzed for 30 min at 4° C. in lysis buffer (25 mM HEPES pH 7.4, 1% (v/v) Triton X-100, 150 mM NaCl, and 2 mM EGTA supplemented with a mixture of protease inhibitors). Protein concentration was determined by the bicinchoninic acid method (Pierce, Rockford, Ill., USA) according to the manufacturer's protocol. Proteins were resolved by 8, 10, or 12% SDS-PAGE and transferred to a nitrocellulose membrane (GE Healthcare, Buckinghamshire, UK). The membrane was blocked for 15 min with TBST (50 mM Tris, 160 mM NaCl, and 0.05% (v/v) Tween 20, pH 7.4) containing 5% (w/v) dried skimmed milk (TBSTM), and was then incubated overnight at 4° C. with the primary antibody diluted in TBSTM. The membrane was washed with TBST and was then incubated with peroxidase-labeled anti-mouse IgG1 or IgG2a (CliniSciences, Nanterre, France) for 45 min. Proteins were visualized using the enhanced chemiluminescence substrate kit (ECL RevelBlOt®, Ozyme, Saint Quentin en Yvelines, France).

(11) Results

EMT and Cl-CD95L-Induced Cell Migrations are Correlated with Increased Membrane Fluidity

(12) Using a transcriptomic analysis of 22 tumor cell lines of various histological origins [National Cancer Institute (NCI)] that respond differently to cytotoxic CD95L, we previously demonstrated that type I cells display a mesenchymal-like phenotype, whereas type II cells have an epithelial-like gene signature.sup.1. Cl-CD95L does not induce cell death but triggers cell motility.sup.34; therefore, we investigated whether EMT modifies the response of tumor cells to a CD95-driven pro-migratory cue. Using a Boyden chamber assay, cell motility was evaluated in various mesenchymal and epithelial-like breast cancer cells in the presence or absence of cl-CD95L. While mesenchymal-like cells migrated spontaneously across pore-containing membranes in the absence of cl-CD95L, epithelial cells did not. In addition, cl-CD95L treatment increased mesenchymal cell migration, but not epithelial cell migration. This suggests that CD95-mediated cell migration is blocked in the latter cells and/or promoted in the former cells.

(13) Increased membrane fluidity can promote the invasion/motility of tumor cells.sup.22, 30, 33, 39; therefore, we next evaluated the membrane lipid packing densities of a large set of tumor cell lines. To this end, electron paramagnetic resonance (EPR) was used to quantify the S parameter, a biophysical parameter that is inversely correlated with membrane fluidity.sup.2. The S parameter was higher in epithelial tumor cells than in mesenchymal tumor cells (0.496±0.0088, n=17 versus 0.467±0.0215, n=20, p≦0.001), indicating that EMT led to membrane fluidization. Confirming this, the S parameter was higher in epithelial breast cancer cells than in mesenchymal breast cancer cells than in epithelial breast cancer cells (0.476±0.0069 n=6 versus 0.464±0.0043, n=6, p=0.0043). In addition, the membrane fluidity of mesenchymal-like MDA-MB-231 breast tumor cells increased following cl-CD95L treatment. To determine the role of plasma membrane fluidity in cell migration, MDA-MB-231 cells were pretreated with non-cytotoxic doses of the membrane stabilizing agent ursodeoxycholic acid (UDCA).sup.17 or the cyclopropyl fatty acid ester A2C, a compound that fluidifies plasma membranes.sup.35. Pre-incubation of MDA-MB-231 cells with UDCA slightly decreased basal membrane fluidity, although not significantly; however, this treatment abrogated the CD95-mediated increase in plasma membrane fluidity. By contrast, A2C treatment significantly enhanced both basal and CD95-induced cell membrane fluidity. Importantly, CD95-mediated migration of MDA-MB-231 cells was completely abolished by UDCA treatment but stimulated by A2C treatment. Collectively, these data indicate that plasma membrane fluidity increases during EMT and that this biophysical parameter has a pivotal function in both EMT-driven and CD95-mediated cell motility.

(14) EMT Triggers Down-Regulation of CerS6.

(15) Cholesterol and SLs participate in plasma membrane fluidity and compaction; therefore, we next investigated whether enzymes involved in the biosynthesis of these lipids are up- or down-regulated during EMT. To this end, a transcriptomic meta-analysis was performed using data from 22 tumor cell lines. These cell lines were epithelium-like or mesenchymal-like.sup.25 and were previously categorized as type II and type I, respectively, based on their sensitivity to the CD95-mediated apoptotic signal.sup.1. Data were extracted from the NCBI Gene Expression Omnibus repository. The SAM tool (Significance Analysis of Microarrays) was used to identify 168 genes that were significantly up-regulated in type II cells and 542 genes that were significantly up-regulated in type I cells. Further analysis revealed that expression of CerS6, also known as LASS6, was significantly higher in epithelial-like (type II) cells than in mesenchymal-like (type I) cells. This in silico analysis was next validated experimentally at the mRNA. To rule out the possibility that this ceramide synthase was only associated with the way tumor cells respond to an apoptotic inducer, we next evaluated whether CerS6 was differentially expressed between NCI cell lines showing an epithelial or mesenchymal-like gene signature.sup.26. An in silico analysis performed on tumor cell lines classified as epithelial or mesenchymal-like cells according to their expression levels of E-cadherin (CDH1)(epithelial marker) and Vimentin (mesenchymal marker) confirmed that the level of CerS6 transcripts was reduced in mesenchymal-like cancer cells as compared to their counterparts exhibiting an epithelial-like gene signature. To confirm that CerS6 expression was down-regulated during EMT, we first evaluated the protein expression levels of two EMT markers, namely E-cadherin and Vimentin, in 47 NCI-60 tumor cell lines that have previously been reported to display an Epithelial or mesenchymal-like phenotype.sup.24We ranked these tumor cells based on their E-cadherin/Vimentin ratio into three groups: i) an epithelial group with high E-cadherin expression, ii) an undefined group that either expressed equally the two markers or did not express them, and iii) a mesenchymal group with high Vimentin expression. Minor differences were found when we compared our classification with the one from Peter's group.sup.24. Indeed, among the 47 tumor cell lines tested, only five of them (OSCAR-5, PC3, SW620, SR and IGROV-1) showed a difference of classification between the two studies. The protein expression levels of CerS6 were significantly increased in epithelial cells as compared to mesenchymal tumor cells. This suggests that CerS6 is a novel EMT-regulated gene, the expression of which in epithelial cells may alter the SL composition of plasma membranes.

(16) To confirm that CerS6 expression was regulated during EMT, the level of CerS6 expression was evaluated in various well-established EMT models. Primary human mammary epithelial cells (HMECs) were used that have undergone sequential retroviral infections to express the telomerase catalytic subunit (hTERT), the large-T and small-t antigens of SV40, and the oncogenic allele of H-Ras (H-Ras.sup.V12), and have thereby been converted to aggressive tumor cells.sup.4, 8. This stepwise EMT cellular model system, which gives rise to mammary cells exhibiting epithelial (E-cadherin) or mesenchymal-like (vimentin) gene signatures, confirmed that CerS6 expression was extinguished during EMT. To confirm this, CerS6 expression was evaluated in the immortal human mammary epithelial cell line MCF10A and its K-Ras.sup.V12-expressing counterpart. Expression of the mesenchymal marker N-cadherin (CDH-2) was higher and expression of the epithelial marker E-cadherin was lower in MCF10A-K-Ras.sup.V12 cells than in MCF10A cells, consistent with the former cells having undergone EMT.sup.19. Interestingly, EMT triggered by K-Ras.sup.V12 expression was accompanied by a decrease in the protein level of CerS6. Stimulation with TGF-β can trigger MCF10A and HMEC-TR breast epithelial cells to undergo EMT.sup.11. This inducible EMT model was used to establish that CerS6 expression is consistently reduced during EMT.

(17) Breast cancer is a heterogeneous pathology, with tumors being classified into various subtypes based on their genomic and clinical features.sup.31. While luminal A and B tumors express epithelial markers, such as E-cadherin, basal-like cancer cells have a mesenchymal-like phenotype and express markers such as vimentin and N-cadherin.sup.26. Importantly, prognosis is poorer in patients with basal breast tumors than in patients with luminal breast tumors, and EMT is associated with tumor aggressiveness and an increased risk of metastatic dissemination in these patients.sup.26. A meta-analysis of breast cancer patients using the bc-GenExMiner tool.sup.13 showed that CerS6 expression was lower in basal tumors than in luminal tumors. Overall, these data strongly support that CerS6 expression is down-regulated in tumor cells undergoing EMT.

(18) Overexpression of CerS6 in Mesenchymal Tumor Cells Decreases Membrane Fluidity and Inhibits Cell Motility.

(19) To characterize the cellular function of CerS6 during EMT, CerS6 was overexpressed in two mesenchymal breast cancer cells, MDA-MB-231 and MDA-MB-468. Transduction of these cells with a CerS6-encoding retroviral vector generated stable cell populations overexpressing CerS6. Strikingly, CerS6 overexpression did not alter the expression levels of the mesenchymal marker vimentin or the epithelial marker E-cadherin, suggesting that EMT controlled CerS6 expression but not vice versa. Lipidomic analysis using gas chromatography and mass spectrometry showed that CerS6 overexpression significantly increased the C16-ceramide contents of both cell lines. However, this increase did not alter the total ceramide content, indicating that a compensatory process served to ensure that the total ceramide content did not change in CerS6-overexpressing cells. Modulation of CerS6 expression will affect the acyl-CoA pool, which is required for the biosynthesis of glycerophospholipids, including phosphatidylcho line, phosphatidylinosito 1, phosphatidylserine and phosphatidylethanolamine; therefore, the levels of these lipids were evaluated in MDA-MB-231 cells, MDA-MB-468 cells, and their CerS6-overexpressing counterparts. CerS6 overexpression did not significantly affect the glycerophospholipid content. Ceramide can be metabolized to sphingomyelin, the major plasma membrane SL; therefore, sphingomyelin content was also monitored. Whereas CerS6 overexpression did not affect the total amount of sphingomyelin, the level of C16-sphingomyelin was significantly higher in CerS6-overexpressing cells than in control cells. These findings show that CerS6 overexpression leads to the accumulation of C16:0 ceramide, which is metabolized to more complex SLs such as C16:0 sphingomyelin. CerS6 overexpression slightly, but significantly, reduced basal membrane fluidity and prevented cl-CD95L-induced membrane fluidization. In addition, CerS6 overexpression in these mesenchymal-like breast cancer cells reduced basal and inhibited CD95-mediated cell motility. These findings indicate that ectopic CerS6 expression in mesenchymal-like tumor cells increases levels of C16:0 ceramide and its derivative C16:0 sphingomyelin, which in turn reduces membrane fluidity and inhibits cell motility.

(20) Down-Regulation of CerS6 in Epithelial Tumor Cells Enhances Membrane Fluidity and Stimulates Cell Migration.

(21) To confirm that CerS6 reduces membrane fluidity and cell motility, a pharmacological inhibitor was used to perturb CerS6 activity in epithelial-like breast cancer cells, and the effects of this on plasma membrane fluidity and cell migration were determined. Fumonisin B.sub.1 (FB1), a mycotoxin produced by Fusarium moniliforme, selectively inhibits CerS activity.sup.36. When the epithelial cell line MCF7 was treated with a non-cytotoxic concentration of FB1 for 48 hours, basal and CD95-induced membrane fluidity were slightly, but significantly, increased. In addition, FB1 treatment enhanced basal and CD95-mediated cell migration. To confirm these data, CerS6 expression was down-regulated in two epithelial breast cancer cells, MCF7 and T47D. These cells were transduced with lentiviruses encoding simultaneously GFP and shRNAs targeting different regions of CerS6 mRNA (shCerS6). All MCF7 cells and more than 80% of T47D cells were infected (GFP-positive). Consistent with the FACS analyses, CerS6 expression was efficiently silenced in MCF7 cells and to a lesser extent in T47D cells following transduction with lentiviruses containing either shCerS6. Furthermore, the reduction in CerS6 expression did not affect expression of EMT-related target genes, including E-cadherin and vimentin, confirming that CerS6 is not an EMT master regulator gene.

(22) Lipidomic analysis showed that down-regulation of CerS6 expression in MCF7 and T47D cells did not alter their total ceramide contents, whereas the level of C16:0 ceramide was significantly reduced. By contrast, down-regulation of CerS6 expression was associated with a slight increase in the level of C18:0, C20:0, C24:0, or C24:1 ceramide, according to the cell line. CerS6 knockdown increased basal and CD95-mediated plasma membrane fluidity and cell migration in these epithelial cancer cells. Of note, the pro-migratory effect of cl-CD95L was less pronounced in shCerS6-treated MCF7 cells than in shCerS6-treated T47D cells. This may be because CerS6 expression was down-regulated more in MCF7 cells than in T47D cells, which led to a dramatic increase in the basal motility of MCF7 cells that might partially mask CD95-induced cell motility. Overall, these findings demonstrate that down-regulation of CerS6 expression in epithelial-like tumor cells enhances cell migration without affecting EMT.

(23) C16-Ceramide, the Product of CerS6, Reduces Membrane Fluidity and Cell Migration.

(24) Finally, to show that CerS6 overexpression induced cell membrane compaction and thereby inhibited cell migration, we evaluated the impact of its main lipid product, C16-ceramide. To this end, two mesenchymal breast cancer cells were incubated for 24 hours with a non-cytotoxic concentration of C16:0 ceramide and membrane fluidity and cell motility were analyzed. While the addition of C16:0 ceramide slightly increased the basal S parameter in these cell lines (FIG. 1A), it completely inhibited the CD95-mediated increase in membrane fluidization (FIG. 1A). More importantly, basal and CD95-mediated migration of mesenchymal breast tumor cells was impaired when they were cultured in medium containing a non-cytotoxic concentration of C16-ceramide (FIG. 1B). These results indicate that exogenous C16-ceramide is adsorbed and retained in cell membranes, and confirm that an increased level of C16-ceramide, which is observed upon CerS6 overexpression, is instrumental in preventing CD95-mediated membrane fluidization and cell migration.

DISCUSSION

(25) EMT is a crucial process in embryonic development that can be partly phenocopied in carcinogenesis, giving rise to aggressive tumors with increased metastatic capacity. In the present study, we demonstrate that CerS6 expression is down-regulated during EMT, leading to increased plasma membrane fluidity, which in turn promotes cell migration.

(26) Cells undergoing EMT exhibit morphological changes and increased motility, which is linked to striking reductions in expression of epithelial markers, such as E-cadherin, and enhanced expression of mesenchymal markers, such as vimentin and N-cadherin. Using pharmacological and genetic approaches, we showed that the level of CerS6 expression does not affect EMT per se. This seems to disagree with data from the Hakomori group showing that pharmacological inhibition of glucosylceramide synthase or the addition of one of its products, gangliotetraosylceramide (Gg4), modulates cell motility and changes the expression ratio of EMT-related genes in various cellular models, including the epithelial breast cancer cell line MCF-7.sup.6. One explanation for this discrepancy is that the carbohydrate moiety complexity of Gg4 allows it to target other unidentified molecular targets and thereby exert additional effects on EMT compared to C16-ceramide and C16-sphingomyelin. In support of this hypothesis, the ganglioside GM2, whose expression is also decreased by inhibition of glucosylceramide synthase activity, does not reverse TGF-β-induced EMT of NMuMG cells, whereas Gg4 does.sup.6. In addition to the carbohydrate moiety, our findings provide insight into the pivotal roles played by the C16:0 fatty acid chain of ceramide and its derivatives in biological functions such as cell motility.

(27) Okazaki and colleagues recently showed that sphingomyelin impairs CXCL12-mediated cell migration.sup.3. The current study shows that CerS6 overexpression is associated with an increase in the C16:0 sphingomyelin content; therefore, we cannot exclude the possibility that sphingomyelin contributes to the phenotypes observed. However, given that CD95-mediated membrane fluidization and cell migration are inhibited by pre-incubation with exogenous C16-ceramide for a short period (60 minutes), C16-ceramide itself likely contributes to the modulation of membrane fluidity and cell motility during EMT.

(28) Glycosphingolipids (GSLs) modulate various cell signaling pathways by regulating the activities of tyrosine kinase receptors.sup.9, 21, and more specifically, epidermal growth factor (EGF) receptor.sup.9. Based on our recent findings showing that cl-CD95L induces a phosphoinositide 3-kinase (PI3K)-driven pro-migratory signaling pathway in mesenchymal breast tumor cells via recruitment of EGF receptor.sup.18, we investigated whether C16-ceramide can prevent this signaling pathway. Analysis of phosphorylation at serine 473 of Akt, a hallmark of PI3K activation, demonstrated that C16-ceramide did not affect the CD95-mediated PI3K signaling pathway. This indicates that C16-ceramide does not interfere with the initial steps of pro-migratory CD95-mediated cell signaling. The mechanistic link between CD95L and plasma membrane fluidization remains uncertain and requires further investigation.

(29) Exogenous short-chain ceramides such as C2-ceramide inhibit migration of lung cancer cells in response to nicotine via activation of protein phosphatase 2A.sup.38. This previous study suggested that C2-ceramide directly affects cell migration. However, the authors did not eliminate the possibility that C2-ceramide that accumulates in the lipid bilayers of pulmonary cells is hydrolyzed by acid ceramidase to form sphingosine that enters the salvage pathway to form new ceramides through the activities of CerS such as CerS6. In this regard, it would be interesting to investigate the activity of CerS6 in pulmonary epithelial cells exposed to nicotine.

(30) CerS6 expression was modulated in various cancer cell lines with epithelial- or mesenchymal-like gene signatures, in well-defined inducible EMT models, and also in a large panel of women affected by luminal or basal breast cancers, which show an epithelial- and mesenchymal-like gene signature, respectively.sup.26. Accordingly, we predict that in women with aggressive basal breast cancers in which CerS6 is not expressed, a slight increase in the level of C16-ceramide and its derivatives may help to reduce their elevated risk of metastatic dissemination.

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