MGMT Inhibition Restores ERα Functional Sensitivity to Antiestrogen Therapy

Antiestrogen therapy resistance remains a huge stumbling block in the treatment of breast cancer. We have found significant elevation of O⁶ methylguanine DNA methyl transferase (MGMT) expression in a small sample of consecutive patients who have failed tamoxifen treatment. Here, we show that tamoxifen resistance is accompanied by upregulation of MGMT. Further we show that administration of the MGMT inhibitor, O⁶-benzylguanine (BG), at nontoxic doses, leads to restoration of a favorable estrogen receptor alpha (ERα) phosphorylation phenotype (high p-ERα Ser167/low p-ERα Ser118), which has been reported to correlate with sensitivity to endocrine therapy and improved survival. We also show BG to be a dual inhibitor of MGMT and ERα. In tamoxifen-resistant breast cancer cells, BG alone or in combination with antiestrogen (tamoxifen [TAM]/ICI 182,780 [fulvestrant, Faslodex]) therapy enhances p53 upregulated modulator of apoptosis (PUMA) expression, cytochrome C release and poly (ADP-ribose) polymerase (PARP) cleavage, all indicative of apoptosis. In addition, BG increases the expression of p21^cip1/waf1. We also show that BG, alone or in combination therapy, curtails the growth of tamoxifen-resistant breast cancer in vitro and in vivo. In tamoxifen-resistant MCF7 breast cancer xenografts, BG alone or in combination treatment causes significant delay in tumor growth. Immunohistochemistry confirms that BG increases p21^cip1/waf1 and p-ERα Ser167 expression and inhibits MGMT, ERα, p-ERα Ser118 and ki-67 expression. Collectively, our results suggest that MGMT inhibition leads to growth inhibition of tamoxifen-resistant breast cancer in vitro and in vivo and resensitizes tamoxifen-resistant breast cancer cells to antiestrogen therapy. These findings suggest that MGMT inhibition may provide a novel therapeutic strategy for overcoming antiestrogen resistance.     

Silibinin prevents TPA-induced MMP-9 expression and VEGF secretion by inactivation of the Raf/MEK/ERK pathway in MCF-7 human breast cancer cells

Matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF) expression are pivotal steps in cancer metastasis. Herein, we investigated the effect of silibinin, a major constituent (flavanolignan) of the fruits of Silybum marianum, on 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced MMP-9 and VEGF expression in MCF-7 human breast cancer cells. The expression of MMP-9 and VEGF in response to TPA was increased, whereas TPA-induced MMP-9 and VEGF expression was decreased by silibinin. To investigate the regulatory mechanism of silibinin on TPA-induced MMP-9 and VEGF expression, we pretreated cells with various inhibitors, such as UO126 (MEK1/2 inhibitor), SP600125 (JNK inhibitor), and SB203580 (p38 inhibitor). Interestingly, TPA-induced MMP-9 expression was significantly inhibited by UO126, but not by SP600125 and SB203580. In addition, we pretreated cells with 100 μM silibinin prior to TPA treatment. TPA-induced MEK and ERK phosphorylation was significantly decreased by silibinin in MCF7 cells. TPA-induced VEGF expression was also suppressed by UO126. On the other hand, we found that adenoviral constitutive active-MEK (Ad-CA-MEK) significantly increased MMP-9 and VEGF expression. Taken together, we suggest that the inhibition of TPA-induced MMP-9 and VEGF expression by silibinin is mediated by the suppression of the Raf/MEK/ERK pathway in MCF-7 breast cancer cells.     

MGMT Inhibition Restores ERα Functional Sensitivity to Antiestrogen Therapy

Antiestrogen therapy resistance remains a huge stumbling block in the treatment of breast cancer. We have found significant elevation of O(6) methylguanine DNA methyl transferase (MGMT) expression in a small sample of consecutive patients who have failed tamoxifen treatment. Here, we show that tamoxifen resistance is accompanied by upregulation of MGMT. Further we show that administration of the MGMT inhibitor, O(6)-benzylguanine (BG), at nontoxic doses, leads to restoration of a favorable estrogen receptor alpha (ERα) phosphorylation phenotype (high p-ERα Ser167/low p-ERα Ser118), which has been reported to correlate with sensitivity to endocrine therapy and improved survival. We also show BG to be a dual inhibitor of MGMT and ERα. In tamoxifen-resistant breast cancer cells, BG alone or in combination with antiestrogen (tamoxifen [TAM]/ICI 182,780 [fulvestrant, Faslodex]) therapy enhances p53 upregulated modulator of apoptosis (PUMA) expression, cytochrome C release and poly (ADP-ribose) polymerase (PARP) cleavage, all indicative of apoptosis. In addition, BG increases the expression of p21(cip1/waf1). We also show that BG, alone or in combination therapy, curtails the growth of tamoxifen-resistant breast cancer in vitro and in vivo. In tamoxifen-resistant MCF7 breast cancer xenografts, BG alone or in combination treatment causes significant delay in tumor growth. Immunohistochemistry confirms that BG increases p21(cip1/waf1) and p-ERα Ser167 expression and inhibits MGMT, ERα, p-ERα Ser118 and ki-67 expression. Collectively, our results suggest that MGMT inhibition leads to growth inhibition of tamoxifen-resistant breast cancer in vitro and in vivo and resensitizes tamoxifen-resistant breast cancer cells to antiestrogen therapy. These findings suggest that MGMT inhibition may provide a novel therapeutic strategy for overcoming antiestrogen resistance.     

AIB1 is required for the acquisition of epithelial growth factor receptor-mediated tamoxifen resistance in breast cancer cells

Acquired resistance to tamoxifen has become a serious obstacle in breast cancer treatment. The underlying mechanism responsible for this condition has not been completely elucidated. In this study, a tamoxifen-resistant (Tam-R) MCF-7 breast cancer cell line was developed to mimic the occurrence of acquired tamoxifen resistance as seen in clinical practice. Increased expression levels of HER1, HER2 and the estrogen receptor (ER)-AIB1 complex were found in tamoxifen-resistant cells. EGF stimulation and gefitinib inhibition experiments further demonstrated that HER1/HER2 signaling and AIB1 were involved in the proliferation of cells that had acquired Tam resistance. However, when AIB1 was silenced with AIB1-siRNA in Tam-R cells, the cell growth stimulated by the HER1/HER2 signaling pathway was significantly reduced, and the cells were again found to be inhibited by tamoxifen. These results suggest that the AIB1 protein could be a limiting factor in the HER1/HER2-mediated hormone-independent growth of Tam-R cells. Thus, AIB1 may be a new therapeutic target, and the removal of AIB1 may decrease the crosstalk between ER and the HER1/HER2 pathway, resulting in the restoration of tamoxifen sensitivity in tamoxifen-resistant cells.     

Diverse and converging roles of ERK1/2 and ERK5 pathways on mesenchymal to epithelial transition in breast cancer

The epithelial to mesenchymal transition (EMT) is characterized by a loss of cell polarity, a decrease in the epithelial cell marker E-cadherin, and an increase in mesenchymal markers including the zinc-finger E-box binding homeobox (ZEB1). The EMT is also associated with an increase in cell migration and anchorage-independent growth. Induction of a reversal of the EMT, a mesenchymal to epithelial transition (MET), is an emerging strategy being explored to attenuate the metastatic potential of aggressive cancer types, such as triple-negative breast cancers (TNBCs) and tamoxifen-resistant (TAMR) ER-positive breast cancers, which have a mesenchymal phenotype. Patients with these aggressive cancers have poor prognoses, quick relapse, and resistance to most chemotherapeutic drugs. Overexpression of extracellular signal-regulated kinase (ERK) 1/2 and ERK5 is associated with poor patient survival in breast cancer. Moreover, TNBC and tamoxifen resistant cancers are unresponsive to most targeted clinical therapies and there is a dire need for alternative therapies.In the current study, we found that MAPK3, MAPK1, and MAPK7 gene expression correlated with EMT markers and poor overall survival in breast cancer patients using publicly available datasets. The effect of ERK1/2 and ERK5 pathway inhibition on MET was evaluated in MDA-MB-231, BT-549 TNBC cells, and tamoxifen-resistant MCF-7 breast cancer cells. Moreover, TU-BcX-4IC patient-derived primary TNBC cells were included to enhance the translational relevance of our study. We evaluated the effect of pharmacological inhibitors and lentivirus-induced activation or inhibition of the MEK1/2-ERK1/2 and MEK5-ERK5 pathways on cell morphology, E-cadherin, vimentin and ZEB1 expression. Additionally, the effects of pharmacological inhibition of trametinib and XMD8-92 on nuclear localization of ERK1/2 and ERK5, cell migration, proliferation, and spheroid formation were evaluated. Novel compounds that target the MEK1/2 and MEK5 pathways were used in combination with the AKT inhibitor ipatasertib to understand cell-specific responses to kinase inhibition. The results from this study will aid in the design of innovative therapeutic strategies that target cancer metastases.     

Cyclin G2 promotes cell cycle arrest in breast cancer cells responding to fulvestrant and metformin and correlates with patient survival

Definition of cell cycle control proteins that modify tumor cell resistance to estrogen (E2) signaling antagonists could inform clinical choice for estrogen receptor positive (ER+) breast cancer (BC) therapy. Cyclin G2 (CycG2) is upregulated during cell cycle arrest responses to cellular stresses and growth inhibitory signals and its gene, CCNG2, is directly repressed by E2-bound ER complexes. Our previous studies showed that blockade of HER2, PI3K and mTOR signaling upregulates CycG2 expression in HER2+ BC cells, and that CycG2 overexpression induces cell cycle arrest. Moreover, insulin and insulin-like growth factor-1 (IGF-1) receptor signaling strongly represses CycG2. Here we show that blockade of ER-signaling in MCF7 and T47D BC cell lines enhances the expression and nuclear localization of CycG2. Knockdown of CycG2 attenuated the cell cycle arrest response of E2-depleted and fulvestrant treated MCF7 cells. These muted responses were accompanied by sustained inhibitory phosphorylation of retinoblastoma (RB) protein, expression of cyclin D1, phospho-activation of ERK1/2 and MEK1/2 and expression of cRaf. Our work indicates that CycG2 can form complexes with CDK10, a CDK linked to modulation of RAF/MEK/MAPK signaling and tamoxifen resistance. We determined that metformin upregulates CycG2 and potentiates fulvestrant-induced CycG2 expression and cell cycle arrest. CycG2 knockdown blunts the enhanced anti-proliferative effect of metformin on fulvestrant treated cells. Meta-analysis of BC tumor microarrays indicates that CCNG2 expression is low in aggressive, poor-prognosis BC and that high CCNG2 expression correlates with longer periods of patient survival. Together these findings indicate that CycG2 contributes to signaling networks that limit BC.     

A role of SIPL1/SHARPIN in promoting resistance to hormone therapy in breast cancer

SIPL1 inhibits PTEN function and stimulates NF-κB signaling; both processes contribute to resistance to hormone therapy in estrogen receptor positive breast cancer (ER + BC). However, whether SIPL1 promotes tamoxifen resistance in BC remains unclear. We report here that SIPL1 enhances tamoxifen resistance in ER + BC. Overexpression of SIPL1 in MCF7 and TD47 cells conferred tamoxifen resistance. In MCF7 cell-derived tamoxifen resistant (TAM-R) cells, SIPL1 expression was upregulated and knockdown of SIPL1 in TAM-R cells re-sensitized the cells to tamoxifen. Furthermore, xenograft tumors produced by MCF7 SIPL1 cells but not by MCF7 empty vector cells resisted tamoxifen treatment. Collectively, we demonstrated a role of SIPL1 in promoting tamoxifen resistance in BC. Increases in AKT activation and NF-κB signaling were detected in both MCF7 SIPL1 and TAM-R cells; using specific inhibitors and unique SIPL1 mutants to inhibit either pathway significantly reduced tamoxifen resistance. A SIPL1 mutant defective in activating both pathways was incapable of conferring resistance to tamoxifen, showing that both pathways contributed to SIPL1-derived resistance to tamoxifen in ER + BCs. Using the Curtis dataset of breast cancer (n = 1980) within the cBioPortal database, we examined a correlation of SIPL1 expression with ER + BC and resistance to hormone therapy. SIPL1 upregulation strongly associates with reductions in overall survival in BC patients, particularly in patients with hormone naïve ER + BCs. Taken together, we provide data suggesting that SIPL1 contributes to promote resistance to tamoxifen in BC cells through both AKT and NF-κB actions.     

MTA1 is a novel regulator of autophagy that induces tamoxifen resistance in breast cancer cells

Tamoxifen is commonly used to treat patients with ESR/ER-positive breast cancer, but its therapeutic benefit is limited by the development of resistance. Recently, alterations in macroautophagy/autophagy function were demonstrated to be a potential mechanism for tamoxifen resistance. Although MTA1 (metastasis-associated 1) has been implicated in breast tumorigenesis and metastasis, its role in endocrine resistance has not been studied. Here, we report that the level of MTA1 expression was upregulated in the tamoxifen resistant breast cancer cell lines MCF7/TAMR and T47D/TR, and knockdown of MTA1 sensitized the cells to 4-hydroxytamoxifen (4OHT). Moreover, knockdown of MTA1 significantly decreased the enhanced autophagy flux in the tamoxifen resistant cell lines. To confirm the role of MTA1 in the development of tamoxifen resistance, we established a cell line, MCF7/MTA1, which stably expressed MTA1. Compared with parental MCF7, MCF7/MTA1 cells were more resistant to 4OHT-induced growth inhibition in vitro and in vivo, and showed increased autophagy flux and higher numbers of autophagosomes. Knockdown of ATG7 or cotreatment with hydroxychloroquine, an autophagy inhibitor, restored sensitivity to 4OHT in both the MCF7/MTA1 and tamoxifen resistant cells. In addition, AMP-activated protein kinase (AMPK) was activated, probably because of an increased AMP:ATP ratio and decreased expression of mitochondrial electron transport complex components. Finally, publicly available breast cancer patient datasets indicate that MTA1 levels correlate with poor prognosis and development of recurrence in patients with breast cancer treated with tamoxifen. Overall, our findings demonstrated that MTA1 induces AMPK activation and subsequent autophagy that could contribute to tamoxifen resistance in breast cancer.     

Disruption of the ER-α36-EGFR/HER2 Positive Regulatory Loops Restores Tamoxifen Sensitivity in Tamoxifen Resistance Breast Cancer Cells

Tamoxifen provided a successful treatment for ER-positive breast cancer for many years. However, most breast tumors develop tamoxifen resistance and are eventually refractory to tamoxifen therapy. The molecular mechanisms underlying development of tamoxifen resistance have not been well established. Recently, we reported that breast cancer cells with high levels of ER-α36, a variant of ER-α, were resistant to tamoxifen and knockdown of ER-α36 expression in tamoxifen resistant cells with the shRNA method restored tamoxifen sensitivity, indicating that gained ER-α36 expression is one of the underlying mechanisms of tamoxifen resistance. Here, we found that tamoxifen induced expression of ER-α36-EGFR/HER2 positive regulatory loops and tamoxifen resistant MCF7 cells (MCF7/TAM) expressed enhanced levels of the loops. Disruption of the ER-α36-EGFR/HER2 positive regulatory loops with the dual tyrosine kinase inhibitor Lapatinib or ER-α36 down-regulator Broussoflavonol B in tamoxifen resistant MCF7 cells restored tamoxifen sensitivity. In addition, we also found both Lapatinib and Broussoflavonol B increased the growth inhibitory activity of tamoxifen in tumorsphere cells derived from MCF7/TAM cells. Our results thus demonstrated that elevated expression of the ER-α36-EGFR/HER2 loops is one of the mechanisms by which ER-positive breast cancer cells escape tamoxifen therapy. Our results thus provided a rational to develop novel therapeutic approaches for tamoxifen resistant patients by targeting the ER-α36-EGFR/HER2 loops.     

Chemopreventive Activity of Ferulago angulate against Breast Tumor in Rats and the Apoptotic Effect of Polycerasoidin in MCF7 Cells: A Bioassay-Guided Approach

Ferulago angulata leaf hexane extract (FALHE) was found to be a potent inducer of MCF7 cell apoptosis. The aims of the present study were to investigate the in vivo chemopreventive effect of FALHE in rats, to identify the contributing anticancer compound in FALHE and to determine its potential mechanism of action against MCF7 cells. Thirty rats harboring LA7-induced breast tumors were divided into five groups: tumor control, low-dose FALHE, high-dose FALHE, treatment control (tamoxifen) and normal control. Breast tissues were then subjected to histopathological and immunohistochemical analyses. A bioassay-guided investigation on FALHE was performed to identify the cytotoxic compound and its mechanism of action through flow cytometry, real-time qPCR and western blotting analyses. An in vivo study showed that FALHE suppressed the expression of the tumor markers PCNA and Ki67. The tumor size was reduced from 2031 ± 281 mm³ to 432 ± 201 mm³ after FALHE treatment. FALHE administration induced apoptosis in breast tumor cells, and this was confirmed by high expression levels of Bax, p53 and caspase 3. Cell cycle arrest was suggested by the expression of p21 and p27. The in vitro experimental results resulted in the isolation of polycerasoidin as a bioactive ingredient of FALHE with an IC₅₀ value of 3.16 ± 0.31 μg/ml against MCF7 cells. Polycerasoidin induced mitochondrial-dependent apoptosis in breast cancer cells via caspase activation and changes in the mRNA and protein expression of Bax and Bcl-2. In addition, flow cytometric analysis demonstrated that the treated MCF7 cells were arrested at the G₁ phase, and this was associated with the up-regulation of p21 and p27 at both the mRNA and protein levels. The results of the present study reinforce further investigations scrutinizing the promising potential of the F. angulata chemical constituents as breast cancer chemopreventive agents.     

Involvement of peptidyl-prolyl isomerase Pin1 in the inhibitory effect of fluvastatin on endothelin-1-induced cardiomyocyte hypertrophy

Aims

Cardiac hypertrophy is elicited by endothelin (ET)-1 as well as other neurohumoral factors, hemodynamic overload, and oxidative stress; HMG-CoA reductase inhibitors (statins) were shown to inhibit cardiac hypertrophy partly via the anti-oxidative stress. One of their common intracellular pathways is the phosphorylation cascade of MEK signaling. Pin1 specifically isomerizes the phosphorylated protein with Ser/Thr-Pro bonds and regulates their activity through conformational changes. There is no report whether the Pin1 activation contributes to ET-1-induced cardiomyocyte hypertrophy and whether the Pin1 inactivation contributes to the inhibitory effect of statins. The aim of this study was to reveal these questions.

Main methods

We assessed neonatal rat cardiomyocyte hypertrophy using ET-1 and fluvastatin by the cell surface area, ANP mRNA expression, JNK and c-Jun phosphorylation, and [³H]-leucine incorporation.

Key findings

Fluvastatin inhibited ET-1-induced increase in the cell surface area, ANP expression, and [³H]-leucine incorporation; and it suppressed the signaling cascade from JNK to c-Jun. The phosphorylated Pin1 level, an inactive form, was decreased by ET-1; however, it reached basal level by fluvastatin. Furthermore, Pin1 overexpression clearly elicited cardiomyocyte hypertrophy, which was inhibited by fluvastatin.

Significance

This is the first report that ET-1-induced cardiomyocyte hypertrophy is mediated through the Pin1 activation and that the inhibitory effect of fluvastatin on cardiomyocyte hypertrophy would partly be attributed to the suppression of the Pin1 function. This study firstly suggests that Pin1 determines the size of hypertrophied cardiomyocyte by regulating the activity of phosphorylated molecules and that statins exert their pleiotropic effects partly via Pin1 inactivation.     

Expression of a phosphorylated p130Cas substrate domain attenuates the phosphatidylinositol 3‐kinase/Akt survival pathway in tamoxifen resistant breast cancer cells

Elevated expression of p130^Cas/BCAR1 (breast cancer anti estrogen resistance 1) in human breast tumors is a marker of poor prognosis and poor overall survival. Specifically, p130^Cas signaling has been associated with antiestrogen resistance, for which the mechanism is currently unknown. TAM‐R cells, which were established by long‐term exposure of estrogen (E₂)‐dependent MCF‐7 cells to tamoxifen, displayed elevated levels of total and activated p130^Cas. Here we have investigated the effects of p130^Cas inhibition on growth factor signaling in tamoxifen resistance. To inhibit p130^Cas, a phosphorylated substrate domain of p130^Cas, that acts as a dominant‐negative (DN) p130^Cas molecule by blocking signal transduction downstream of the p130^Cas substrate domain, as well as knockdown by siRNA was employed. Interference with p130^Cas signaling/expression induced morphological changes, which were consistent with a more epithelial‐like phenotype. The phenotypic reversion was accompanied by reduced migration, attenuation of the ERK and phosphatidylinositol 3‐kinase/Akt pathways, and induction of apoptosis. Apoptosis was accompanied by downregulation of the expression of the anti‐apoptotic protein Bcl‐2. Importantly, these changes re‐sensitized TAM‐R cells to tamoxifen treatment by inducing cell death. Therefore, our findings suggest that targeting the product of the BCAR1 gene by a peptide which mimics the phosphorylated substrate domain may provide a new molecular avenue for treatment of antiestrogen resistant breast cancers. J. Cell. Biochem. 107: 364–375, 2009. © 2009 Wiley‐Liss, Inc.     

Implication of protein tyrosine phosphatase 1B in MCF-7 cell proliferation and resistance to 4-OH tamoxifen

The protein tyrosine phosphatase 1B (PTP1B) and the T-cell protein tyrosine phosphatase (TC-PTP) were initially thought to be mainly anti-oncogenic. However, overexpression of PTP1B and TC-PTP has been observed in human tumors, and recent studies have demonstrated that PTP1B contributes to the appearance of breast tumors by modulating ERK pathway. In the present work, we observed that decreasing the expression of TC-PTP or PTP1B in MCF-7 cells using siRNA reduced cell proliferation without affecting cell death. This reduction in proliferation was associated with decreased ERK phosphorylation. Moreover, selection of tamoxifen-resistant MCF-7 cells, by long-term culture in presence of 4-OH tamoxifen, resulted in cells that display overexpression of PTP1B and TC-PTP, and concomitant increase in ERK and STAT3 phosphorylation. siRNA experiments showed that PTP1B, but not TC-PTP, is necessary for resistance to 4-OH tamoxifen. Therefore, our work indicates that PTP1B could be a relevant therapeutic target for treatment of tamoxifen-resistant breast cancers.