Increased Expression of P-Glycoprotein and Doxorubicin Chemoresistance of Metastatic Breast Cancer Is Regulated by miR-298

Abstract

MicroRNAs (miRNAs) are short, noncoding RNA molecules that regulate the expression of a number of genes involved in cancer; therefore, they offer great diagnostic and therapeutic targets. We have developed doxorubicin-resistant and -sensitive metastatic human breast cancer cell lines (MDA-MB-231) to study the chemoresistant mechanisms regulated by miRNAs. We found that doxorubicin localized exclusively to the cytoplasm and was unable to reach the nuclei of resistant tumor cells because of the increased nuclear expression of MDR1/P-glycoprotein (P-gp). An miRNA array between doxorubicin-sensitive and -resistant breast cancer cells showed that reduced expression of miR-298 in doxorubicin-resistant human breast cancer cells was associated with increased expression of P-gp. In a transient transfection experiment, miR-298 directly bound to the MDR1 3′ untranslated region and regulated the expression of firefly luciferase reporter in a dose-dependent manner. Overexpression of miR-298 down-regulated P-gp expression, increasing nuclear accumulation of doxorubicin and cytotoxicity in doxorubicin-resistant breast cancer cells. Furthermore, down-regulation of miR-298 increased P-gp expression and induced doxorubicin resistance in sensitive breast cancer cells. In summary, these results suggest that miR-298 directly modulates P-gp expression and is associated with the chemoresistant mechanisms of metastatic human breast cancer. Therefore, miR-298 has diagnostic and therapeutic potential for predicting doxorubicin chemoresistance in human breast cancer. MicroRNAs (miRNAs) are short, noncoding RNA molecules that regulate the expression of a number of genes involved in cancer; therefore, they offer great diagnostic and therapeutic targets. We have developed doxorubicin-resistant and -sensitive metastatic human breast cancer cell lines (MDA-MB-231) to study the chemoresistant mechanisms regulated by miRNAs. We found that doxorubicin localized exclusively to the cytoplasm and was unable to reach the nuclei of resistant tumor cells because of the increased nuclear expression of MDR1/P-glycoprotein (P-gp). An miRNA array between doxorubicin-sensitive and -resistant breast cancer cells showed that reduced expression of miR-298 in doxorubicin-resistant human breast cancer cells was associated with increased expression of P-gp. In a transient transfection experiment, miR-298 directly bound to the MDR1 3′ untranslated region and regulated the expression of firefly luciferase reporter in a dose-dependent manner. Overexpression of miR-298 down-regulated P-gp expression, increasing nuclear accumulation of doxorubicin and cytotoxicity in doxorubicin-resistant breast cancer cells. Furthermore, down-regulation of miR-298 increased P-gp expression and induced doxorubicin resistance in sensitive breast cancer cells. In summary, these results suggest that miR-298 directly modulates P-gp expression and is associated with the chemoresistant mechanisms of metastatic human breast cancer. Therefore, miR-298 has diagnostic and therapeutic potential for predicting doxorubicin chemoresistance in human breast cancer. A number of chemotherapy regimens have been used to treat metastatic breast cancer in humans. The success of treating breast cancer by chemotherapy is hampered by the development of multidrug resistance (MDR) of cancer cells.1Szakacs G. Paterson J.K. Ludwig J.A. Booth-Genthe C. Gottesman M.M. Targeting multidrug resistance in cancer.Nat Rev Drug Discov. 2006; 5: 219-234Crossref PubMed Scopus (2829) Google Scholar, 2Lehnert M. Chemotherapy resistance in breast cancer.Anticancer Res. 1998; 18: 2225-2226PubMed Google Scholar, 3Gottesman M.M. Fojo T. Bates S.E. Multidrug resistance in cancer: role of ATP-dependent transporters.Nature Rev Cancer. 2002; 2: 48-58Crossref PubMed Scopus (4443) Google Scholar MDR of cancer cell occurs because of the overexpression of one or more of the ATP binding cassette (ABC) transporters.4Leonard G.D. Fojo T. Bates S.E. The role of ABC transporters in clinical medicine.Oncologist. 2003; 8: 411-424Crossref PubMed Scopus (645) Google Scholar, 5Fojo T. Multiple paths to a drug resistance phenotype: mutations, translations, deletions and amplification of coding genes or promoter regions, epigenetics changes and microRNAs.Drug Resist Update. 2007; 10: 59-67Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar There are three well-characterized transporters, ABCB1 (MDR-1/P-gp), ABCC1 (MRP-1), and ABCG2 (BCRP), associated with the chemoresistance of breast cancer.6Dean M. Hamon Y. Chimini G. The human ATP-binding cassette (ABC) transporter superfamily.J Lipid Res. 2001; 42: 1007-1017Abstract Full Text Full Text PDF PubMed Google Scholar, 7Xu Dong Ye Dongjiu Fisher Michael Juliano R.L. Selective inhibition of P-glycoprotein expression in multidrug-resistance tumor cells by a designed transcriptional tegulator.J Pharmacol Exp Ther. 2002; 302: 963-971Crossref PubMed Scopus (48) Google Scholar, 8Wu Hao Hait William Yang Jin-ming Small interfering RNA-induced suppression of MDR1 (P-glycoprotein) restores sensitivity to multidrug-resistant cancer cells.Cancer Res. 2003; 63: 1515-1519PubMed Google Scholar, 9Faneyte I.F. Kristel P.M. Maliepaard M. Scheffer G.L. Scheper R.J. Schellens J.H. van de Vijver M.J. Expression of the breast cancer resistance protein (BCRP) in breast cancer.Clin Cancer Res. 2002; 8: 1068-1074PubMed Google Scholar, 10Bailey-Dell K.J. Hassel B. Doyle L.A. Ross D.D. Promoter characterization and genomic organization of the human breast cancer resistance protein (ATP-binding cassette transporter G2) gene.Biochim Biophys Acta. 2001; 1520: 234-241Crossref PubMed Scopus (198) Google Scholar The P-glycoprotein (P-gp) overexpression in breast cancer cells has been found to be strongly associated with chemoresistant mechanisms of a variety of drugs.11Abolhoda A. Wilson A.E. Ross H. Danenberg P.V. Burt M. Scotto K.W. Rapid activation of MDR1 gene expression in human metastatic sarcoma after in vivo exposure to doxorubicin.Clin Cancer Res. 1999; 5: 3352-3356PubMed Google Scholar, 12Chaudhary P.M. Roninson I.B. Induction of multidrug resistance in human cancer cells by transient exposure to different chemotherapy drugs.J Natl Cancer Inst. 1993; 85: 632-639Crossref PubMed Scopus (360) Google Scholar, 13Chin K.V. Chauhan S.S. Pastan I. Gottesman M.M. Regulation of MDR RNA levels in response to cytotoxic drugs in rodent cells.Cell Growth Differ. 1990; 1: 361-365PubMed Google Scholar P-gp is a 170-kDa transmembrane glycoprotein that acts as an energy-dependent efflux transporter that enhances drug efflux from the nucleus or prevents entry of drugs to the nucleus, thereby decreasing cytotoxicity of anticancer drugs.12Chaudhary P.M. Roninson I.B. Induction of multidrug resistance in human cancer cells by transient exposure to different chemotherapy drugs.J Natl Cancer Inst. 1993; 85: 632-639Crossref PubMed Scopus (360) Google Scholar, 13Chin K.V. Chauhan S.S. Pastan I. Gottesman M.M. Regulation of MDR RNA levels in response to cytotoxic drugs in rodent cells.Cell Growth Differ. 1990; 1: 361-365PubMed Google Scholar, 14Chin K.V. Tanaka S. Darlington G. Pastan I. Gottesman M.M. Heat shock and arsenite increase expression of the multidrug resistance (MDR1) gene in human renal carcinoma cells.J Biol Chem. 1990; 265: 221-226Abstract Full Text PDF PubMed Google Scholar A number of mechanisms have been proposed to explain the transcriptional activation of the P-gp gene (ABCB1) in cancer cells.15Hu Z. Jin S. Scotto K.W. Transcriptional activation of the MDR1 gene by UV irradiation Role of NF-Y and SP-1.J Biol Chem. 2000; 275: 2979-2985Crossref PubMed Scopus (134) Google Scholar, 16Uchiumi T. Kohno K. Tanimura H. Matsuo K. Sato S. Uchida Y. Kuwano M. Enhanced expression of the human multidrug resistance 1 gene in response to UV light irradiation.Cell Growth Differ. 1993; 4: 147-157PubMed Google Scholar, 17Yang J.M. Vassil A.D. Hait W.N. Activation of phospholipase C induces the expression of the multidrug resistance (MDR1) gene through Raf-MAPK pathway.Mol Pharmacol. 2001; 60: 674-680Crossref PubMed Scopus (148) Google Scholar, 18Jin W. Scotto K.W. Hait W.N. Yang J.M. Involvement of CtBP1 in the transcriptional activation of the MDR1 gene in human multidrug resistant cancer cells.Biochem Pharmacol. 2007; 74: 851-859Crossref PubMed Scopus (47) Google Scholar Studies indicate that increased resistance of chemotherapeutic agents is also associated with epigenetic alterations, including DNA methylation and histone modifications.19Glasspool R.M. Teodoridis J.M. Brown R. Epigenetics as a mechanism driving polygenic clinical drug resistance.Br J Cancer. 2006; 94: 1087-1092Crossref PubMed Scopus (191) Google Scholar, 20Iwasa Y. Nowak M.A. Michor F. Evolution of resistance during clonal expansion.Genetics. 2006; 172: 2557-2566Crossref PubMed Scopus (171) Google Scholar, 21Matarazzo M.R. Boyle S. D'Esposito M. Bickmore W.A. Chromosome territory reorganization in human disease with altered DNA methylation.Proc Natl Acad Sci U S A. 2007; 104: 16546-16551Crossref PubMed Scopus (56) Google Scholar The precise cellular and molecular mechanisms underlying the increased MDR1 gene expression is not clear. New evidence indicates that changes in gene expression associated with cell proliferation, apoptosis, signaling, and chemotherapy response are regulated by altered expression of cellular microRNAs (miRNAs). miRNAs are small non–protein-coding RNAs that regulate gene expression through base pairing with target mRNAs, resulting in translation inhibition or mRNA cleavage.22Lai E.C. MicroRNAs are complementary to 3′ that 2002; PubMed Scopus Google Scholar miRNAs are through a of that are in the nucleus miRNAs are by RNA The are to to miRNA by an and H. microRNAs target of genes with 3′ Natl Acad Sci U S A. PubMed Scopus Google Scholar, A. microRNAs to gene expression and have a Full Text Full Text PDF PubMed Scopus Google Scholar, M. for of by Res. PubMed Scopus Google Scholar are to the cytoplasm by and by to a miRNA Y. M. S. genes are by RNA PubMed Scopus Google Scholar, of microRNAs by the PubMed Scopus Google Scholar, G. T. B. R. the of PubMed Scopus Google Scholar, R. Y. the nuclear of and 2003; PubMed Scopus Google Scholar, S. A. of PubMed Scopus Google Scholar The is the miRNA and is the RNA-induced the is of microRNAs by the PubMed Scopus Google Scholar the role of miRNAs in the of MDR of breast cancer developed doxorubicin-sensitive and -resistant metastatic human breast cancer cells We showed that expression of P-gp to the nuclear of doxorubicin and the doxorubicin chemoresistance of study the role of miRNA in the doxorubicin chemoresistance a miRNA array between the doxorubicin-sensitive and -resistant metastatic breast cancer cells. We found and down-regulation of miRNAs in the doxorubicin-resistant human breast cancer cells with the sensitive cells. We have that miR-298 is down-regulated in the doxorubicin-resistant cells with the doxorubicin-sensitive cells. the miRNA found that human miR-298 to the 3′ untranslated region of the human P-gp the role of development of resistance to the chemotherapeutic drug is study the evidence to suggest that the of miR-298 because of expression of is associated with an increased expression of P-gp and to the doxorubicin resistance in breast cancer cells. have an in the of cancer cell resistance to a variety of chemotherapeutic drugs used in the of breast cancer. The and human breast cancer cell lines from cell lines in with and and in a with and was from A of doxorubicin was in cells in in the of that in the of doxorubicin as cancer cells. The cells been for in the with doxorubicin to that they resistant to the of a cells increased cytotoxicity the cytotoxicity in the as doxorubicin-sensitive cells. cells from the of of and in with and 1 after doxorubicin was by the The a was reduced to by the The of by cells indicates the of cell The of tumor cells was by the of by the The or cells a of in with and to was and with different and of of cells with of of with of in for the tumor cells in and with 1 of for of was in a The of cell was by with cells as and cells in with the the was and with of of cells with and with The cells in in and for The cells with and for The cells in a in the for The cell was with the of cells by a The of tumor cells in the and of the cell was with the the cells in a and with of protein in the was by protein of protein from was in by and a The was with in with for 1 The was three and with the to P-gp or the in the was three with and for 1 with the or that was with a of The bound with the of and the with the of The and cells the The with and with 1 of for The with a for 1 by a for a for the the with and miRNA was from and cells with the of the miRNA to the the of miRNA expression in these used the miRNA array a of the expression of and miRNA molecules to from the cells with and from the cells with the miRNA in and to the array in a a with the of and A of was to and from The and in to miRNAs to be they a in expression Expression miRNA in the doxorubicin-sensitive and -resistant breast cancer cells was by with the of a miRNA RNA was from and cells with the of the RNA RNA with of RNA was for and RNA in a a in from the of the RNAs to a with the of with UV and for The was a with and with of for The was with of with of miRNA and The was to an and with of and with of for with the was with 1 of the of for the was and the was three with of for The was with of with of A and for was by the with the of a The was with and to miRNAs. The 3′ of P-gp gene (ABCB1) to to was by and different with of miR-298 and binding by and with the of the and The DNA with in the miR-298 binding was by of 1 and The was with the of the and and as a The was from with the of and was to the and was to the The DNA was with and and the of the firefly luciferase reporter gene in the of The of the was by DNA and was with of The amplification for for for for 1 by for the DNA with in the binding was by of and the was with the of and the as a The was with the of the and with the of the as a was to the and was to the that the DNA be with the of the The amplification are the as The of the was by DNA and is with of The or cells in and with of the firefly luciferase with the miRNA by or miRNA by with the of to the transfection for cells with a and the of luciferase was with the firefly luciferase reporter to the The by protein of cell The of transfection to breast cancer cells was by with the of RNA accumulation was by a A. K. S. K. R. S. expression of P-glycoprotein is associated with doxorubicin chemoresistance in metastatic breast cancer J Full Text Full Text PDF PubMed Scopus Google Scholar cells or cells with the miRNAs or miR-298 with the of cells with doxorubicin cells with with and a The of doxorubicin the and breast cancer cells was by A in the cytotoxicity between the and breast cancer cell was The doxorubicin a of to a in the to was with the of the cells The cellular cytotoxicity of doxorubicin with the of the cell lines was also by for and a increase in cytotoxicity The of doxorubicin to cell and between the and cell lines was by results indicate that doxorubicin to in the of cells and that of cells in the S of cell in was in the of the in the breast cancer have that doxorubicin induced cell because of DNA in of the breast cancer A. K. S. K. R. S. expression of P-glycoprotein is associated with doxorubicin chemoresistance in metastatic breast cancer J Full Text Full Text PDF PubMed Scopus Google Scholar in the resistant breast cancer cell cells the mechanism of doxorubicin the cellular of doxorubicin between the and cells was by and cells with doxorubicin the doxorubicin was by The results indicate that a in the because of doxorubicin between the and cells indicates that doxorubicin is to the cell The results indicate that of the doxorubicin localized in the nucleus of cells. In the doxorubicin is unable to reach the nucleus and exclusively in the cytoplasm expression of different drug efflux with the nuclear of doxorubicin in the expression of and between the and cells was by the the expression of and was also by We a nuclear and expression of P-gp in cells expression in cells. cell lines showed expression of and by and the role of increased expression of P-gp in to the expression of P-gp in the cells increase the nuclear of doxorubicin and We used a MDR efflux R. P-glycoprotein (ABCB1) modulates sensitivity of a multidrug resistant cell to PubMed Scopus Google Scholar The cells in with a of for the cells with doxorubicin an increasing and and results indicate that with doxorubicin of cells after The cytotoxicity was to increased nuclear of doxorubicin a We also used a P-gp to cytotoxicity in cells. is as a that H. MDR1 expression by and Pharmacol. PubMed Scopus Google Scholar showed that of cells with a of after the expression of P-gp of cells with by doxorubicin the cytotoxicity by results indicate that P-gp expression is for nuclear of doxorubicin and and nuclear of doxorubicin in the cells. and cells in a with doxorubicin for and for cellular and of and cells the of doxorubicin There is a in the in the cells with the cells. the and cells after doxorubicin The that doxorubicin localized in the nucleus of cells. The that of doxorubicin was in the cytoplasm of cells. The the of The are the with nuclear expression of P-gp in doxorubicin-resistant breast cancer cell and inhibition of expression cellular the expression of three different drug efflux and in and cells with the of and and nuclear of P-gp expression in the cells. The expression of P-gp in cells was The expression of and in cells was the expression of these in and cells with the of and to was used as the cytotoxicity of cells with for and with an increasing of doxorubicin for and cytotoxicity was by nuclear of doxorubicin cells in with for and with doxorubicin for was a The the nuclear of doxorubicin in the cells The the nuclear of doxorubicin in the cells in the of with the P-gp expression in cells by in a to that with the doxorubicin cytotoxicity of cells. are as and the miRNAs are an of cancer cell and chemotherapy an miRNA between and cells. We found a and down-regulation of miRNAs in cells with cells. the miRNAs expression between these cell lines A and The P-gp is the target of miR-298 and showed down-regulated expression in cells. was used to the of miR-298 expression between and cells. The results of indicate that miR-298 and are in cells with cells We also found that the of miR-298 was in cells because the miRNA miRNA as as indicate that and regulate the expression of P-gp by binding to the 3′ of the MDR1 Wilson M. A. C. The and Res. PubMed Scopus Google Scholar, Y. Involvement of in resistance of the breast cancer cells to chemotherapeutic drug Cancer Ther. PubMed Scopus Google Scholar, H. H. Yang J.M. Role of and in the of MDR1/P-glycoprotein expression in human cancer cells.Biochem Pharmacol. PubMed Scopus Google Scholar We not of these miRNAs levels in cells by the of in the miRNA in the to the levels of the miRNA and by is the that the of miRNA in the of microRNAs by the PubMed Scopus Google Scholar and to a in a of the of the the is to the the by G. T. B. R. the of PubMed Scopus Google Scholar The of in cells was protein was associated with small RNAs that miRNA or a of G. T. B. R. the of PubMed Scopus Google Scholar showed a down-regulation of levels in different expression of and in and cells was The of explain the potential for the expression of miRNAs in the cells. the role of miRNAs in the P-gp used a of the 3′ of MDR1 that found a binding for Wilson M. A. C. The and Res. PubMed Scopus Google Scholar of miR-298 MDR1 mRNA to and to MDR1 is by miR-298 and 3′ of MDR1 with miR-298 and was the for 3′ for miRNA target with a of miRNA or was and used as a in and the of a transient found that miR-298 the firefly luciferase expression the with the miR-298 binding in a The luciferase expression of the miR-298 binding not after transfection and with the that showed a dose-dependent in the luciferase expression of the by transient the transfection and the that the not luciferase expression of miRNA transfection the expression of firefly luciferase in cells. miR-298 the expression of firefly luciferase in a dose-dependent manner. The miR-298 not regulate the expression of firefly luciferase of or with of miR-298 binding also the expression of dose-dependent not the and with of are as of of the and found that cells a expression of with cells. We used miRNA to that miR-298 the expression levels of P-gp in cells. cells with miR-298 by and after the of P-gp expression in the and of miRNA was by indicate that miR-298 and or in the expression of P-gp. We P-gp by miRNA also increase with miR-298 cells and the with and cell was by after the nuclear was by the expression of transfection of the miRNA the nuclear of doxorubicin in cells The results that with miR-298 and or in increased nuclear of The of transfection to breast cancer cells with the of was to be with RNA the potential between miR-298 and P-gp expression in to the doxorubicin chemoresistance be by the expression of P-gp in cells by the expression of miR-298 by transient transfection with miR-298 cells with the of P-gp was by and luciferase We found that miR-298 by to a dose-dependent increase in the of luciferase in cells not in the was to down-regulation of miR-298 the doxorubicin cytotoxicity of cells. cells with miR-298 by for and cells with an increasing of doxorubicin for was by The expression of P-gp in cells with or transfection with miR-298 was by cells with miR-298 showed nuclear of doxorubicin with the cells that transfection with miR-298 in cells has resistant to doxorubicin because of increased P-gp The role of miR-298 in P-gp expression was with human breast cancer cell resistant to was to the doxorubicin cytotoxicity between doxorubicin-sensitive and doxorubicin-resistant breast cancer cells. a in the cytotoxicity between the and the breast cancer cell The doxorubicin a of to a in the was with the cells. The expression of P-gp between the and cell lines was by We a expression of P-gp in cells expression in cells. that the miR-298 also P-gp luciferase cells with with or miR-298 binding and miR-298 luciferase was with the protein of cells. We that the miR-298 the firefly luciferase expression from in a in the luciferase expression was with the of with of the miR-298 The role of of P-gp luciferase expression in cells was in a transient transfection with We that also luciferase expression by a to a The luciferase expression of of miR-298 not inhibition and to the cells The results of are to that with the cells. miRNAs are noncoding RNAs that regulate expression of the target genes in a cells of development cellular response to chemotherapy are evidence to indicate that they are in the expression of genes involved in chemoresistant mechanisms in cancer not be for chemoresistance and potential Cancer Ther. PubMed Scopus Google Scholar the chemoresistant mechanisms of human breast have developed chemoresistant human breast cancer (MDA-MB-231) cells. We used doxorubicin as a drug that was as We showed that the increased expression of P-gp in cells is associated with nuclear of In that doxorubicin was localized in the cytoplasm in doxorubicin was localized to the nucleus in the cells. We that doxorubicin chemoresistance of breast cancer is to expression of miRNA was by miRNA array between and cells. The miRNA that miRNAs are and down-regulated in cells with cells. the increased drug efflux because of the expression of P-gp is the mechanism involved in the doxorubicin found the down-regulated expression of miRNAs that to the P-gp We have miR-298 expression is associated with increased P-gp results that the miR-298 expression is altered in cells by of the expression of of the miR-298 in the the role of miR-298 in the expression of that transfection of miR-298 cells down-regulated expression of P-gp by as as luciferase We also found that transfection of miR-298 cells drug and nuclear of doxorubicin and cytotoxicity by The nuclear of doxorubicin in the cells is with cells. of these the role of miR-298 in the expression of P-gp. these suggest that the expression of miRNAs drug resistance and cytotoxicity to The role of miRNAs in chemotherapy resistant mechanisms is are including that study the of P-gp by Y. Involvement of in resistance of the breast cancer cells to chemotherapeutic drug Cancer Ther. PubMed Scopus Google Scholar, H. H. Yang J.M. Role of and in the of MDR1/P-glycoprotein expression in human cancer cells.Biochem Pharmacol. PubMed Scopus Google Scholar the of the are miRNAs to be associated with chemoresistant H. H. Yang J.M. Role of and in the of MDR1/P-glycoprotein expression in human cancer cells.Biochem Pharmacol. PubMed Scopus Google Scholar that increased expression of and to the MDR and modulates the expression of P-glycoprotein in human cancer cell showed that transfection of cells with of or the expression, that these miRNAs regulate expression of P-gp. In Y. Involvement of in resistance of the breast cancer cells to chemotherapeutic drug Cancer Ther. PubMed Scopus Google Scholar found that expression of is associated with increased expression of P-gp and doxorubicin chemoresistance of the breast cancer cell There are also that multidrug-resistant and are also regulated by Z. H. Y. Y. K. Y. S. H. Involvement of in chemotherapy resistance of breast cancer through expression of multidrug protein Pharmacol. PubMed Scopus Google Scholar, the expression of breast cancer resistant protein in human cancer Pharmacol. PubMed Scopus Google Scholar In the role of and in the expression of P-gp in cells. these miRNAs miR-298 expression was by We not miRNAs by The has showed results in the expression of P-gp and increased of doxorubicin and in results also evidence to indicate that the altered expression of is altered in the doxorubicin-resistant cell to of results are also by a study that altered expression of miRNA are associated with a of human breast as the cell K.J. R. M.A. K. R.L. of the miRNA and is associated with of breast J Cancer. Full Text Full Text PDF PubMed Scopus Google Scholar The mechanism underlying the of altered expression of miR-298 and P-gp to be in the In summary, the results of study suggest that miR-298 is a potential for chemoresistance of human breast cancer. We that of miR-298 the expression of P-gp and chemotherapy response of metastatic human breast cancer. We for the