ER alpha negative breast cancer cells restore response to endocrine therapy by combination treatment with both HDAC inhibitor and DNMT inhibitor.
OBJECTIVE
Estrogen receptor alpha (ER alpha) mediates the growth stimulation of estrogen in breast cancer cells and is a useful predictive factor for response to endocrine therapy. It is reported that ER alpha was induced in ER alpha negative breast cancer cells by both DNA methyltransferase-1 (DNMT1) inhibitor 5-aza-2'-deoxycytidine (AZA) and histone deacetylase (HDAC) inhibitor trichostatin A (TSA). However, whether the breast cancer cells with induced ER alpha restore response to endocrine therapy requires to be further researched.


METHODS
Reverse transcriptase-polymerase chain reaction (RT-PCR) method was used to explore the change in the mRNA of ER alpha, PR and pS2 in the ER alpha negative breast cancer cells MDA-MB-435 treated with two chemicals (AZA + TSA). Water-soluble tetrazolium salt-8 (WST-8) method was used to study the proliferation rate of the breast cancer cells. Flow cytometer (FCW) was used to analyze the distribution of cell cycle of these breast cancer cells. Some xenograft models in nude mice were used to further study the results we found in vitro.


RESULTS
In this study we observed that the mRNA of ER alpha, PR and pS2 in the ER alpha negative breast cancer cells MDA-MB-435 was re-expressed by treatment with AZA + TSA. The proliferation assay analysis showed AZA + TSA suppressed the proliferation of MDA-MB-435 cells, which were further suppressed by addition of 4-OH Tamoxifen (4-OHT). On the contrary, the proliferation of cells treated with 4-OHT alone showed no difference compared with the vehicle control. Cell cycle analysis showed AZA + TSA treated cells showed S phase arrest, which was partially attenuated by addition of estradiol (E2); furthermore, the effect of E2 on stimulation of cell cycle could be reversed by 4-OHT in the treated cells with induced ER alpha. In vivo experiment xenograft volume of MDA-MB-435 cells treated with AZA + TSA was smaller than that of the control (P < 0.01), and the xenograft of AZA + TSA treated cells was further suppressed by ovariectomy (P < 0.01).


CONCLUSIONS
Our data indicate that DNMT1 inhibitor AZA and HDAC inhibitor TSA play important roles in restoring sensitivity of the ER alpha negative breast cancer cells to endocrine therapy in vitro and in vivo.
Fan
Jiang
J
Department of Breast Surgery, Cancer Hospital/Cancer Institute, Fudan University, 399 Ling-Ling Road, 200032, Shanghai, People.s Republic of China. drjiangfan@gmail.com
Yin
Wen-Jin
WJ
Lu
Jin-Song
JS
Wang
Lei
L
Wu
Jiong
J
Wu
Feng-Ying
FY
Di
Gen-Hong
GH
Shen
Zhen-Zhou
ZZ
Shao
Zhi-Min
ZM
D000818:Animals; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D001943:Breast Neoplasms / Q000378:metabolism*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000037:antagonists & inhibitors*; D000077209:Decitabine; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004958:Estradiol / Q000494:pharmacology; D004965:Estrogen Antagonists / Q000494:pharmacology; D047628:Estrogen Receptor alpha / Q000187:drug effects / Q000378:metabolism*; D004967:Estrogens / Q000494:pharmacology; D005260:Female; D005434:Flow Cytometry; D056572:Histone Deacetylase Inhibitors; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology*; D051379:Mice; D012333:RNA, Messenger / Q000032:analysis; D011980:Receptors, Progesterone / Q000187:drug effects; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013629:Tamoxifen / Q000031:analogs & derivatives / Q000494:pharmacology; D000071163:Trefoil Factor-1; D025521:Tumor Suppressor Proteins / Q000187:drug effects; D023041:Xenograft Model Antitumor Assays
D004791:Enzyme Inhibitors; D004965:Estrogen Antagonists; D047628:Estrogen Receptor alpha; D004967:Estrogens; D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; D012333:RNA, Messenger; D011980:Receptors, Progesterone; C051838:TFF1 protein, human; D000071163:Trefoil Factor-1; D025521:Tumor Suppressor Proteins; D013629:Tamoxifen; C016601:afimoxifene; C012589:trichostatin A; D004958:Estradiol; D000077209:Decitabine; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; C000619380:DNMT1 protein, human; C000619381:Dnmt1 protein, mouse; D001374:Azacitidine