Sarmila Majumder

We explored the role of microRNAs (miRNAs) in acquiring resistance to tamoxifen, a drug successfully used to treat women with estrogen receptor-positive breast cancer. miRNA microarray analysis of MCF-7 cell lines that are either sensitive (parental) or resistant (4-hydroxytamoxifen-resistant (OHTR)) to tamoxifen showed significant (>1.8-fold) up-regulation of eight miRNAs and marked down-regulation (>50%) of seven miRNAs in OHTR cells compared with parental MCF-7 cells. Increased expression of three of the most promising up-regulated (miR-221, miR-222, and miR-181) and down-regulated (miR-21, miR-342, and miR-489) miRNAs was validated by real-time reverse transcription-PCR. The expression of miR-221 and miR-222 was also significantly (2-fold) elevated in HER2/neu-positive primary human breast cancer tissues that are known to be resistant to endocrine therapy compared with HER2/neu-negative tissue samples. Ectopic expression of miR-221/222 rendered the parental MCF-7 cells resistant to tamoxifen. The protein level of the cell cycle inhibitor p27Kip1, a known target of miR-221/222, was reduced by 50% in OHTR cells and by 28–50% in miR-221/222-overexpressing MCF-7 cells. Furthermore, overexpression of p27Kip1 in the resistant OHTR cells caused enhanced cell death when exposed to tamoxifen. This is the first study demonstrating a relationship between miR-221/222 expression and HER2/neu overexpression in primary breast tumors that are generally resistant to tamoxifen therapy. This finding also provides the rationale for the application of altered expression of specific miRNAs as a predictive tamoxifen-resistant breast cancer marker. We explored the role of microRNAs (miRNAs) in acquiring resistance to tamoxifen, a drug successfully used to treat women with estrogen receptor-positive breast cancer. miRNA microarray analysis of MCF-7 cell lines that are either sensitive (parental) or resistant (4-hydroxytamoxifen-resistant (OHTR)) to tamoxifen showed significant (>1.8-fold) up-regulation of eight miRNAs and marked down-regulation (>50%) of seven miRNAs in OHTR cells compared with parental MCF-7 cells. Increased expression of three of the most promising up-regulated (miR-221, miR-222, and miR-181) and down-regulated (miR-21, miR-342, and miR-489) miRNAs was validated by real-time reverse transcription-PCR. The expression of miR-221 and miR-222 was also significantly (2-fold) elevated in HER2/neu-positive primary human breast cancer tissues that are known to be resistant to endocrine therapy compared with HER2/neu-negative tissue samples. Ectopic expression of miR-221/222 rendered the parental MCF-7 cells resistant to tamoxifen. The protein level of the cell cycle inhibitor p27Kip1, a known target of miR-221/222, was reduced by 50% in OHTR cells and by 28–50% in miR-221/222-overexpressing MCF-7 cells. Furthermore, overexpression of p27Kip1 in the resistant OHTR cells caused enhanced cell death when exposed to tamoxifen. This is the first study demonstrating a relationship between miR-221/222 expression and HER2/neu overexpression in primary breast tumors that are generally resistant to tamoxifen therapy. This finding also provides the rationale for the application of altered expression of specific miRNAs as a predictive tamoxifen-resistant breast cancer marker. Breast cancer is the most common malignancy in women, accounting for 31% of all female cancers. An estimated 178,480 new cases of invasive breast cancer was diagnosed in the United States in 2007, and 40,460 women will die of this cancer. Over two-thirds of breast cancers exhibit high concentrations of estrogen receptor, which contribute to tumor growth and progression. Blocking the steroid hormone pathway with tamoxifen and/or oophorectomy has been shown to be effective in this patient population. The Early Breast Cancer Trialists' Collaborative Group overview demonstrated a significant improvement in 15-year survival with the addition of adjuvant tamoxifen for 5 years following surgery (1Early Breast Cancer Trialists' Collaborative GroupLancet. 2005; 365: 1687-1717Abstract Full Text Full Text PDF PubMed Scopus (6347) Google Scholar). Furthermore, tamoxifen can also reduce the incidence of contralateral breast cancer and has been approved as a prophylactic agent to prevent breast cancer. Despite this accomplishment in the management of women with potentially endocrine-responsive breast cancers, a significant proportion of women will to either or resistance to of growth by tamoxifen is to tamoxifen This to of the protein and which cell and be by the growth inhibitor that growth is in tamoxifen resistance Cancer PubMed Scopus Google Scholar). The are by that tumors exhibit when with tamoxifen PubMed Scopus Google Scholar). of the for tamoxifen resistance 2005; PubMed Scopus Google to the of a expression that can resistant tumors and of as the of microRNAs (miRNAs) used reverse used reverse in all in and expression of miRNAs in primary human cancers has been used for tumor and PubMed Scopus Google Scholar). expression of target by of the target and in cases by of the PubMed Scopus Google Scholar). study with and breast tissue altered expression of miRNAs that the of the tumor Cancer 2005; PubMed Scopus Google Scholar). This study to miRNA expression in drug used a cell to the miRNA expression of a tamoxifen-resistant cell that was validated in primary human breast cancers. We a target protein of the miRNAs in tamoxifen-resistant breast cancer and role in tamoxifen and MCF-7 cells and cells and as Cancer PubMed Scopus Google Scholar). all MCF-7 and OHTR cells in growth with and in the of for cells and in human breast the miRNA microarray was the Cancer for for miRNA was primary human breast cancer tissue with a and the cell lines with a reverse and to real-time a a in miRNA expression was to as in and with 5 tamoxifen in was a was by Cancer PubMed Scopus Google and cell cycle analysis for was by and was to be miRNA with in Breast Cancer a first to miRNAs in tamoxifen-resistant breast compared the miRNA of the OHTR and MCF-7 breast cancer cell lines by microarray cell lines a MCF-7 Cancer PubMed Scopus Google and in the miRNA expression is to tamoxifen the microarray the expression of miRNA was to the of all the and compared between the cell a of miRNAs in the OHTR and MCF-7 cells. of significantly miRNAs is in miRNA miR-222, and significantly in OHTR cells compared with MCF-7 cells. seven miR-342, and in OHTR cells the of in the expression in OHTR validated expression of three up-regulated miRNAs (miR-221, miR-222, and miR-181) and three down-regulated miRNAs miR-342, and by real-time of the miRNA expression to and in the miR-221 and miR-222 in OHTR cells compared with MCF-7 cells the miR-342, and expression in OHTR cells reduced by and compared with MCF-7 cells and miR-222 in HER2/neu-positive Breast was in miRNA expression in a cell also in primary breast cancer samples. this a of primary human breast cancer that either HER2/neu-positive or expression and HER2/neu in breast cancer are with of endocrine therapy Cancer PubMed Scopus Google used HER2/neu expression as a for tamoxifen and to expression significant in miR-221 and miR-222 expression in the HER2/neu-positive tumor compared with the HER2/neu-negative The the patient the the cell of tamoxifen also miR-221 and miR-222 expression in breast cancer cell lines with a expression of to patient in miR-221 and miR-222 expression was in cells compared with MCF-7 cells of miR-221 and miR-222 in MCF-7 to expression of miR-221 and miR-222 in primary 2005; PubMed Scopus Google PubMed Scopus Google and cancer PubMed Scopus Google Scholar). the of miRNAs also significantly elevated in HER2/neu-positive breast explored role in acquiring resistance to endocrine therapy. miR-221 and miR-222 in MCF-7 cells and miR-221/222-overexpressing cells We used the and a cells for Increased expression of miR-221/222 in the and was by real-time MCF-7 cells with the and miR-221/222-overexpressing cells with 5 tamoxifen, and cell was the The of the miR-221/222-overexpressing and in the of tamoxifen was significantly that of the demonstrating that miR-221/222 can resistance to tamoxifen in the breast cancer cell expression of miR-221/222 in MCF-7 cells in tamoxifen expression of miR-221 and miR-222 was MCF-7 cells by real-time and to is shown the the of MCF-7 and the MCF-7 cell with 5 tamoxifen for in the of the drug was the The of cells was as The are the of and MCF-7 cells with and tamoxifen for The cells a cell the and MCF-7 cells by and with and that and The was with to protein The in was of and and was to The are of the and MCF-7 cells with tamoxifen for The cells and with and cell cycle was by in a The of cells in and cells is in the three tamoxifen is known to in breast cancer cells Full Text Full Text PDF PubMed Scopus Google explored the role of miR-221/222 in this miR-221/222-overexpressing MCF-7 cells with concentrations of the drug and cell a The cells of in the of tamoxifen that with tamoxifen the and showed of when with tamoxifen the was significantly in cells compared with cells can be as the in of tamoxifen in breast cancer with a of tamoxifen was to be Cancer PubMed Scopus Google Scholar). the of the and cells to and by and in cells with concentrations of tamoxifen the in The of in the miR-221/222-overexpressing was also reduced by to the cells. is that the level of miR-221/222 expression in the was compared with that in and a of miR-221/222 in MCF-7 cells was expression of miR-221/222 the of in cell lines by of the cells tamoxifen of and of the miR-221/222-overexpressing the This of demonstrated that the miR-221/222 a role in tamoxifen resistance to MCF-7 a of miR-221/222, the cell cycle inhibitor p27Kip1 is a known target of miR-221/222 PubMed Scopus Google Scholar). shown that of p27Kip1 in the by with of breast cancer in PubMed Scopus Google Scholar). this that the of miR-221/222 in tamoxifen resistance is by the of p27Kip1 protein in breast cancer. analysis 50% in the level of p27Kip1 in MCF-7 cells compared with OHTR cells Furthermore, the level of p27Kip1 was reduced by and in and the compared with the cells analysis showed that the level of p27Kip1 was also reduced in cell lines that miR-221/222 p27Kip1 the level of and a target of miR-221/222, tamoxifen to MCF-7 cells. cell MCF-7 OHTR and and MCF-7 cells to and with The was with and p27Kip1 expression was to MCF-7 OHTR and and MCF-7 cells by real-time with specific for p27Kip1 and cell the OHTR cell was to analysis with and with OHTR cells with and expression with 5 tamoxifen for was the The of cells was as The are the of and OHTR cells with and tamoxifen for The cells a The in and cell the and OHTR cells to and with and The was with to protein The in was of and and is in the The are of three the expression of p27Kip1 in OHTR cells can the OHTR cells to tamoxifen. this a p27Kip1 expression OHTR cells and a for The expression of p27Kip1 was by analysis with The of OHTR cells and the and cell in the of tamoxifen was by the significant of cell was when p27Kip1 was in OHTR cells the of p27Kip1 in cell in the and OHTR cells in the of tamoxifen. The of cells was as a was in cells this and in cell was in the cells in the the was significantly in the cells in the cells all concentrations of tamoxifen that the of high p27Kip1 a role in the endocrine therapy of breast is the adjuvant therapy in women with estrogen receptor-positive breast cancer. of tumors are resistant to tamoxifen, is in the of acquiring resistance to this altered expression of miRNAs in primary human cancers has been used for tumor and the of miRNAs in of drug tamoxifen has been demonstrated the role of in resistance in cancer by Cancer PubMed Scopus Google Scholar). with showed that of and to Full Text Full Text PDF PubMed Scopus Google Scholar). The study showed significantly expression of eight miRNAs and down-regulation of seven miRNAs in a tamoxifen-resistant breast cancer cell compared with a cell finding of was the expression of miR-221 and miR-222 in HER2/neu-positive compared with HER2/neu-negative primary human breast tumors that are resistant to endocrine therapy. expression of miR-221 and miR-222 as a of primary 2005; PubMed Scopus Google PubMed Scopus Google and cancer PubMed Scopus Google Scholar). for the first expression of miR-221/222 as a of tamoxifen resistance in breast as by the cell cycle inhibitor p27Kip1, cell PubMed Scopus Google PubMed Scopus Google Scholar). expression of miRNAs to by Cancer PubMed Scopus Google Scholar). The study has a relationship between tamoxifen resistance and reduced of p27Kip1 by miR-221/222 the of this cell cycle inhibitor in the elevated expression of this cell cycle inhibitor has been with in cell cancer Full Text Full Text PDF PubMed Scopus Google Scholar). in and in OHTR cells that is in the death of cells. The cell death also by PubMed Scopus Google Scholar). the of in the cells and that the to and be in this the tumor of p27Kip1 is by by or of p27Kip1 the of of and to growth in PubMed Scopus Google Scholar). be of to study in the of the level of p27Kip1 in tamoxifen-resistant growth in the of this miRNAs that are in tamoxifen-resistant cell of miRNAs as of tamoxifen-resistant of target of miRNAs will of tamoxifen resistance and of new Breast cancer is the most common malignancy in women, accounting for 31% of all female cancers. An estimated 178,480 new cases of invasive breast cancer was diagnosed in the United States in 2007, and 40,460 women will die of this cancer. Over two-thirds of breast cancers exhibit high concentrations of estrogen receptor, which contribute to tumor growth and progression. Blocking the steroid hormone pathway with tamoxifen and/or oophorectomy has been shown to be effective in this patient population. The Early Breast Cancer Trialists' Collaborative Group overview demonstrated a significant improvement in 15-year survival with the addition of adjuvant tamoxifen for 5 years following surgery (1Early Breast Cancer Trialists' Collaborative GroupLancet. 2005; 365: 1687-1717Abstract Full Text Full Text PDF PubMed Scopus (6347) Google Scholar). Furthermore, tamoxifen can also reduce the incidence of contralateral breast cancer and has been approved as a prophylactic agent to prevent breast cancer. Despite this accomplishment in the management of women with potentially endocrine-responsive breast cancers, a significant proportion of women will to either or resistance to tamoxifen. of growth by tamoxifen is to tamoxifen This to of the protein and which cell and be by the growth inhibitor that growth is in tamoxifen resistance Cancer PubMed Scopus Google Scholar). The are by that tumors exhibit when with tamoxifen PubMed Scopus Google Scholar). of the for tamoxifen resistance 2005; PubMed Scopus Google to the of a expression that can resistant tumors and of as the of microRNAs (miRNAs) used reverse used reverse in all in and expression of miRNAs in primary human cancers has been used for tumor and PubMed Scopus Google Scholar). expression of target by of the target and in cases by of the PubMed Scopus Google Scholar). study with and breast tissue altered expression of miRNAs that the of the tumor Cancer 2005; PubMed Scopus Google Scholar). This study to miRNA expression in drug used a cell to the miRNA expression of a tamoxifen-resistant cell that was validated in primary human breast cancers. We a target protein of the miRNAs in tamoxifen-resistant breast cancer and role in tamoxifen and MCF-7 cells and cells and as Cancer PubMed Scopus Google Scholar). all MCF-7 and OHTR cells in growth with and in the of for cells and in human breast the miRNA microarray was the Cancer for for miRNA was primary human breast cancer tissue with a and the cell lines with a reverse and to real-time a a in miRNA expression was to as in and with 5 tamoxifen in was a was by Cancer PubMed Scopus Google and cell cycle analysis for was by and was to be and MCF-7 cells and cells and as Cancer PubMed Scopus Google Scholar). all MCF-7 and OHTR cells in growth with and in the of for cells and in human breast the miRNA miRNA microarray was the Cancer for for miRNA was primary human breast cancer tissue with a and the cell lines with a reverse and to real-time a a in miRNA expression was to as Scholar). in and with 5 tamoxifen in was a was by Cancer PubMed Scopus Google and cell cycle analysis for was by and was to be miRNA with in Breast Cancer a first to miRNAs in tamoxifen-resistant breast compared the miRNA of the OHTR and MCF-7 breast cancer cell lines by microarray cell lines a MCF-7 Cancer PubMed Scopus Google and in the miRNA expression is to tamoxifen the microarray the expression of miRNA was to the of all the and compared between the cell a of miRNAs in the OHTR and MCF-7 cells. of significantly miRNAs is in miRNA miR-222, and significantly in OHTR cells compared with MCF-7 cells. seven miR-342, and in OHTR cells the of in the expression in OHTR validated expression of three up-regulated miRNAs (miR-221, miR-222, and miR-181) and three down-regulated miRNAs miR-342, and by real-time of the miRNA expression to and in the miR-221 and miR-222 in OHTR cells compared with MCF-7 cells the miR-342, and expression in OHTR cells reduced by and compared with MCF-7 cells and miR-222 in HER2/neu-positive Breast was in miRNA expression in a cell also in primary breast cancer samples. this a of primary human breast cancer that either HER2/neu-positive or expression and HER2/neu in breast cancer are with of endocrine therapy Cancer PubMed Scopus Google used HER2/neu expression as a for tamoxifen and to expression significant in miR-221 and miR-222 expression in the HER2/neu-positive tumor compared with the HER2/neu-negative The the patient the the cell of tamoxifen also miR-221 and miR-222 expression in breast cancer cell lines with a expression of to patient in miR-221 and miR-222 expression was in cells compared with MCF-7 cells of miR-221 and miR-222 in MCF-7 to expression of miR-221 and miR-222 in primary 2005; PubMed Scopus Google PubMed Scopus Google and cancer PubMed Scopus Google Scholar). the of miRNAs also significantly elevated in HER2/neu-positive breast explored role in acquiring resistance to endocrine therapy. miR-221 and miR-222 in MCF-7 cells and miR-221/222-overexpressing cells We used the and a cells for Increased expression of miR-221/222 in the and was by real-time MCF-7 cells with the and miR-221/222-overexpressing cells with 5 tamoxifen, and cell was the The of the miR-221/222-overexpressing and in the of tamoxifen was significantly that of the demonstrating that miR-221/222 can resistance to tamoxifen in the breast cancer cell tamoxifen is known to in breast cancer cells Full Text Full Text PDF PubMed Scopus Google explored the role of miR-221/222 in this miR-221/222-overexpressing MCF-7 cells with concentrations of the drug and cell a The cells of in the of tamoxifen that with tamoxifen the and showed of when with tamoxifen the was significantly in cells compared with cells can be as the in of tamoxifen in breast cancer with a of tamoxifen was to be Cancer PubMed Scopus Google Scholar). the of the and cells to and by and in cells with concentrations of tamoxifen the in The of in the miR-221/222-overexpressing was also reduced by to the cells. is that the level of miR-221/222 expression in the was compared with that in and a of miR-221/222 in MCF-7 cells was expression of miR-221/222 the of in cell lines by of the cells tamoxifen of and of the miR-221/222-overexpressing the This of demonstrated that the miR-221/222 a role in tamoxifen resistance to MCF-7 a of miR-221/222, the cell cycle inhibitor p27Kip1 is a known target of miR-221/222 PubMed Scopus Google Scholar). shown that of p27Kip1 in the by with of breast cancer in PubMed Scopus Google Scholar). this that the of miR-221/222 in tamoxifen resistance is by the of p27Kip1 protein in breast cancer. analysis 50% in the level of p27Kip1 in MCF-7 cells compared with OHTR cells Furthermore, the level of p27Kip1 was reduced by and in and the compared with the cells analysis showed that the level of p27Kip1 was also reduced in cell lines that miR-221/222 p27Kip1 the level of and a target of miR-221/222, tamoxifen to MCF-7 cells. cell MCF-7 OHTR and and MCF-7 cells to and with The was with and p27Kip1 expression was to MCF-7 OHTR and and MCF-7 cells by real-time with specific for p27Kip1 and cell the OHTR cell was to analysis with and with OHTR cells with and expression with 5 tamoxifen for was the The of cells was as The are the of and OHTR cells with and tamoxifen for The cells a The in and cell the and OHTR cells to and with and The was with to protein The in was of and and is in the The are of three the expression of p27Kip1 in OHTR cells can the OHTR cells to tamoxifen. this a p27Kip1 expression OHTR cells and a for The expression of p27Kip1 was by analysis with The of OHTR cells and the and cell in the of tamoxifen was by the significant of cell was when p27Kip1 was in OHTR cells the of p27Kip1 in cell in the and OHTR cells in the of tamoxifen. The of cells was as a was in cells this and in cell was in the cells in the the was significantly in the cells in the cells all concentrations of tamoxifen that the of high p27Kip1 a role in the endocrine therapy of breast cancer. miRNA with in Breast Cancer a first to miRNAs in tamoxifen-resistant breast compared the miRNA of the OHTR and MCF-7 breast cancer cell lines by microarray cell lines a MCF-7 Cancer PubMed Scopus Google and in the miRNA expression is to tamoxifen the microarray the expression of miRNA was to the of all the and compared between the cell a of miRNAs in the OHTR and MCF-7 cells. of significantly miRNAs is in miRNA miR-222, and significantly in OHTR cells compared with MCF-7 cells. seven miR-342, and in OHTR cells the of in the expression in OHTR validated expression of three up-regulated miRNAs (miR-221, miR-222, and miR-181) and three down-regulated miRNAs miR-342, and by real-time of the miRNA expression to and in the miR-221 and miR-222 in OHTR cells compared with MCF-7 cells the miR-342, and expression in OHTR cells reduced by and compared with MCF-7 cells miR-221 and miR-222 in HER2/neu-positive Breast was in miRNA expression in a cell also in primary breast cancer samples. this a of primary human breast cancer that either HER2/neu-positive or expression and HER2/neu in breast cancer are with of endocrine therapy Cancer PubMed Scopus Google used HER2/neu expression as a for tamoxifen and to expression significant in miR-221 and miR-222 expression in the HER2/neu-positive tumor compared with the HER2/neu-negative The the patient the the cell of tamoxifen We also miR-221 and miR-222 expression in breast cancer cell lines with a expression of to patient in miR-221 and miR-222 expression was in cells compared with MCF-7 cells of miR-221 and miR-222 in MCF-7 to expression of miR-221 and miR-222 in primary 2005; PubMed Scopus Google PubMed Scopus Google and cancer PubMed Scopus Google Scholar). the of miRNAs also significantly elevated in HER2/neu-positive breast explored role in acquiring resistance to endocrine therapy. miR-221 and miR-222 in MCF-7 cells and miR-221/222-overexpressing cells We used the and a cells for Increased expression of miR-221/222 in the and was by real-time MCF-7 cells with the and miR-221/222-overexpressing cells with 5 tamoxifen, and cell was the The of the miR-221/222-overexpressing and in the of tamoxifen was significantly that of the demonstrating that miR-221/222 can resistance to tamoxifen in the breast cancer cell tamoxifen is known to in breast cancer cells Full Text Full Text PDF PubMed Scopus Google explored the role of miR-221/222 in this miR-221/222-overexpressing MCF-7 cells with concentrations of the drug and cell a The cells of in the of tamoxifen that with tamoxifen the and showed of when with tamoxifen the was significantly in cells compared with cells can be as the in of tamoxifen in breast cancer with a of tamoxifen was to be Cancer PubMed Scopus Google Scholar). the of the and cells to and by and in cells with concentrations of tamoxifen the in The of in the miR-221/222-overexpressing was also reduced by to the cells. is that the level of miR-221/222 expression in the was compared with that in and a of miR-221/222 in MCF-7 cells was expression of miR-221/222 We the of in cell lines by of the cells tamoxifen of and of the miR-221/222-overexpressing the This of demonstrated that the miR-221/222 a role in tamoxifen resistance to MCF-7 cells. p27Kip1 a of miR-221/222, the cell cycle inhibitor p27Kip1 is a known target of miR-221/222 PubMed Scopus Google Scholar). shown that of p27Kip1 in the by with of breast cancer in PubMed Scopus Google Scholar). this that the of miR-221/222 in tamoxifen resistance is by the of p27Kip1 protein in breast cancer. analysis 50% in the level of p27Kip1 in MCF-7 cells compared with OHTR cells Furthermore, the level of p27Kip1 was reduced by and in and the compared with the cells analysis showed that the level of p27Kip1 was also reduced in cell lines that miR-221/222 p27Kip1 the level of and We the expression of p27Kip1 in OHTR cells can the OHTR cells to tamoxifen. this a p27Kip1 expression OHTR cells and a for The expression of p27Kip1 was by analysis with The of OHTR cells and the and cell in the of tamoxifen was by the significant of cell was when p27Kip1 was in OHTR cells the of p27Kip1 in cell in the and OHTR cells in the of tamoxifen. The of cells was as a was in cells this and in cell was in the cells in the the was significantly in the cells in the cells all concentrations of tamoxifen that the of high p27Kip1 a role in the endocrine therapy of breast cancer. is the adjuvant therapy in women with estrogen receptor-positive breast cancer. of tumors are resistant to tamoxifen, is in the of acquiring resistance to this altered expression of miRNAs in primary human cancers has been used for tumor and the of miRNAs in of drug tamoxifen has been demonstrated the role of in resistance in cancer by Cancer PubMed Scopus Google Scholar). with showed that of and to Full Text Full Text PDF PubMed Scopus Google Scholar). The study showed significantly expression of eight miRNAs and down-regulation of seven miRNAs in a tamoxifen-resistant breast cancer cell compared with a cell finding of was the expression of miR-221 and miR-222 in HER2/neu-positive compared with HER2/neu-negative primary human breast tumors that are resistant to endocrine therapy. expression of miR-221 and miR-222 as a of primary 2005; PubMed Scopus Google PubMed Scopus Google and cancer PubMed Scopus Google Scholar). for the first expression of miR-221/222 as a of tamoxifen resistance in breast as by the cell cycle inhibitor p27Kip1, cell PubMed Scopus Google PubMed Scopus Google Scholar). expression of miRNAs to by Cancer PubMed Scopus Google Scholar). The study has a relationship between tamoxifen resistance and reduced of p27Kip1 by miR-221/222 the of this cell cycle inhibitor in the elevated expression of this cell cycle inhibitor has been with in cell cancer Full Text Full Text PDF PubMed Scopus Google Scholar). in and in OHTR cells that is in the death of cells. The cell death also by PubMed Scopus Google Scholar). the of in the cells and that the to and be in this the tumor of p27Kip1 is by by or of p27Kip1 the of of and to growth in PubMed Scopus Google Scholar). be of to study in the of the level of p27Kip1 in tamoxifen-resistant growth in the of this miRNAs that are in tamoxifen-resistant cell of miRNAs as of tamoxifen-resistant of target of miRNAs will of tamoxifen resistance and of new is the adjuvant therapy in women with estrogen receptor-positive breast cancer. of tumors are resistant to tamoxifen, is in the of acquiring resistance to this altered expression of miRNAs in primary human cancers has been used for tumor and the of miRNAs in of drug tamoxifen has been demonstrated the role of in resistance in cancer by Cancer PubMed Scopus Google Scholar). with showed that of and to Full Text Full Text PDF PubMed Scopus Google Scholar). The study showed significantly expression of eight miRNAs and down-regulation of seven miRNAs in a tamoxifen-resistant breast cancer cell compared with a cell finding of was the expression of miR-221 and miR-222 in HER2/neu-positive compared with HER2/neu-negative primary human breast tumors that are resistant to endocrine therapy. expression of miR-221 and miR-222 as a of primary 2005; PubMed Scopus Google PubMed Scopus Google and cancer PubMed Scopus Google Scholar). for the first expression of miR-221/222 as a of tamoxifen resistance in breast cancer. miR-221/222 as by the cell cycle inhibitor p27Kip1, cell PubMed Scopus Google PubMed Scopus Google Scholar). expression of miRNAs to by Cancer PubMed Scopus Google Scholar). The study has a relationship between tamoxifen resistance and reduced of p27Kip1 by miR-221/222 the of this cell cycle inhibitor in the elevated expression of this cell cycle inhibitor has been with in cell cancer Full Text Full Text PDF PubMed Scopus Google Scholar). in and in OHTR cells that is in the death of cells. The cell death also by PubMed Scopus Google Scholar). the of in the cells and that the to and be in this the tumor of p27Kip1 is by by or of p27Kip1 the of of and to growth in PubMed Scopus Google Scholar). be of to study in the of the level of p27Kip1 in tamoxifen-resistant growth in the of this We miRNAs that are in tamoxifen-resistant cell of miRNAs as of tamoxifen-resistant of target of miRNAs will of tamoxifen resistance and of new We for the OHTR and MCF-7 cells and and for the miR-221/222 and breast cancer with with

MicroRNAs (miR) are a class of small ( approximately 21 nucleotide) noncoding RNAs that, in general, negatively regulate gene expression. Some miRs harboring CGIs undergo methylation-mediated silencing, a characteristic of many tumor suppressor genes. To identify such miRs in liver cancer, the miRNA expression profile was analyzed in hepatocellular carcinoma (HCC) cell lines treated with 5-azacytidine (DNA hypomethylating agent) and/or trichostatin A (histone deacetylase inhibitor). The results showed that these epigenetic drugs differentially regulate expression of a few miRs, particularly miR-1-1, in HCC cells. The CGI spanning exon 1 and intron 1 of miR-1-1 was methylated in HCC cell lines and in primary human HCCs but not in matching liver tissues. The miR-1-1 gene was hypomethylated and activated in DNMT1-/- HCT 116 cells but not in DNMT3B null cells, indicating a key role for DNMT1 in its methylation. miR-1 expression was also markedly reduced in primary human hepatocellular carcinomas compared with matching normal liver tissues. Ectopic expression of miR-1 in HCC cells inhibited cell growth and reduced replication potential and clonogenic survival. The expression of FoxP1 and MET harboring three and two miR-1 cognate sites, respectively, in their respective 3'-untranslated regions, was markedly reduced by ectopic miR-1. Up-regulation of several miR-1 targets including FoxP1, MET, and HDAC4 in primary human HCCs and down-regulation of their expression in 5-AzaC-treated HCC cells suggest their role in hepatocarcinogenesis. The inhibition of cell cycle progression and induction of apoptosis after re-expression of miR-1 are some of the mechanisms by which DNA hypomethylating agents suppress hepatocarcinoma cell growth.

5-Azacytidine- and 5-aza-deoxycytidine (5-aza-CdR)-mediated reactivation of tumor suppressor genes silenced by promoter methylation has provided an alternate approach in cancer therapy. Despite the importance of epigenetic therapy, the mechanism of action of DNA-hypomethylating agents in vivo has not been completely elucidated. Here we report that among three functional DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B), the maintenance methyltransferase, DNMT1, was rapidly degraded by the proteasomal pathway upon treatment of cells with these drugs. The 5-aza-CdR-induced degradation, which occurs in the nucleus, could be blocked by proteasomal inhibitors and required a functional ubiquitin-activating enzyme. The drug-induced degradation occurred even in the absence of DNA replication. Treatment of cells with other nucleoside analogs modified at C-5, 5-fluorodeoxyuridine and 5-fluorocytidine, did not induce the degradation of DNMT1. Mutation of cysteine at the catalytic site of Dnmt1 (involved in the formation of a covalent intermediate with cytidine in DNA) to serine (CS) did not impede 5-aza-CdR-induced degradation. Neither the wild type nor the catalytic site mutant of Dnmt3a or Dnmt3b was sensitive to 5-aza-CdR-mediated degradation. These results indicate that covalent bond formation between the enzyme and 5-aza-CdR-incorporated DNA is not essential for enzyme degradation. Mutation of the conserved KEN box, a targeting signal for proteasomal degradation, to AAA increased the basal level of Dnmt1 and blocked its degradation by 5-aza-CdR. Deletion of the catalytic domain increased the expression of Dnmt1 but did not confer resistance to 5-aza-CdR-induced degradation. Both the nuclear localization signal and the bromo-adjacent homology domain were essential for nuclear localization and for the 5-aza-CdR-mediated degradation of Dnmt1. Polyubiquitination of Dnmt1 in vivo and its stabilization upon treatment of cells with a proteasomal inhibitor indicate that the level of Dnmt1 is controlled by ubiquitin-dependent proteasomal degradation. Overexpression of the substrate recognition component, Cdh1 but not Cdc20, of APC (anaphase-promoting complex)/cyclosome ubiquitin ligase reduced the level of Dnmt1 in both untreated and 5-aza-CdR-treated cells. In contrast, the depletion of Cdh1 with small interfering RNA increased the basal level of DNMT1 that blocked 5-aza-CdR-induced degradation. Dnmt1 interacted with Cdh1 and colocalized in the nucleus at discrete foci. Both Dnmt1 and Cdh1 were phosphorylated in vivo, but only Cdh1 was significantly dephosphorylated upon 5-aza-CdR treatment, suggesting its involvement in initiating the proteasomal degradation of DNMT1. These results demonstrate a unique mechanism for the selective degradation of DNMT1, the maintenance DNA methyltransferase, by well-known DNA-hypomethylating agents.

Micro-RNAs are ∼21–25-nucleotide-long noncoding RNAs that regulate gene expression primarily at the post-transcriptional level in animals. Here, we report that micro-RNA-1 (miR-1), abundant in the cardiac and smooth muscles, is expressed in the lung and is down-regulated in human primary lung cancer tissues and cell lines. In situ hybridization demonstrated localization in in lung expression in the lung cancer in lung cancer the in lung cancer in and and in expression and in lung the and that in cell in in the and and the lung Micro-RNAs are ∼21–25-nucleotide-long noncoding RNAs that regulate gene expression primarily at the post-transcriptional level in animals. Here, we report that micro-RNA-1 (miR-1), abundant in the cardiac and smooth muscles, is expressed in the lung and is down-regulated in human primary lung cancer tissues and cell lines. 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