Induction of tamoxifen resistance in breast cancer cells by ELF electromagnetic fields

The incidence of breast cancer in western societies has been rising ever since the Second World War. Besides the exposure to a multitude of new chemical compounds, electromagnetic field exposure has been linked to breast cancer through a radiation-mediated anti-melatonin pathway. We investigated, whether low-frequency electromagnetic field exposure interferes with the anti-estrogenic activity of tamoxifen. Two different clones of the breast cancer cell line MCF-7 were exposed to highly homogeneous 50Hz electromagnetic fields and IC(50) values were calculated from dose-response curves of tamoxifen at various field intensities. An intensity-dependent shift of tamoxifen dose-response curves to higher concentrations with a maximal response at 1.2muT was observed. Hypothetically, electromagnetic field exposure could contribute to tamoxifen resistance observed in breast cancer after long-term treatment.     

Down-regulation of thiamine transporter THTR2 gene expression in breast cancer and its association with resistance to apoptosis

The recent molecular identification of two thiamine transporters, SLC19A2 (THTR1) and SLC19A3 (THTR2), has provided the opportunity to study thiamine transporter gene expression in human malignancies. We compared RNA levels of both THTR1 and THTR2 in a panel of human breast tumors and corresponding normal tissues. THTR2 RNA levels were down-regulated in breast cancer to 14% of the level found in corresponding normal tissues, while THTR1 levels were unchanged. Both thiamine transport genes were cloned and expressed in a breast cancer cell line to examine the impact of reconstituted thiamine transport gene expression on drug and radiation sensitivity and on resistance to apoptosis. THTR2-transfected breast cancer cells showed a 2.5-fold increase in specific THTR2 activity and a 3-fold increase in cytotoxicity against a bromoacetyl ester derivative of thiamine. Surprisingly, these cells also showed a 3-fold increase in sensitivity to doxorubicin and an increase in sensitivity to ionizing radiation, but no change in sensitivity to methotrexate or paclitaxel. TUNEL assays demonstrate an increase in apoptosis in THTR2-transfected cells exposed to doxorubicin and radiation, and Western blot analysis suggests that apoptosis associated with these cytotoxic stresses is mediated at least in part by a caspase-3-dependent pathway. Therefore, thiamine transporter THTR2 gene expression is down-regulated in breast cancer, which may contribute to resistance to apoptosis in these tumors.     

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.     

Identification of eight genes that are potentially involved in tamoxifen sensitivity in breast cancer cells

Although the antiestrogen agent tamoxifen has long been used to treat women with hormone receptor positive invasive breast carcinoma, the mechanisms of its action and acquired resistance to tamoxifen during treatment are largely unknown. A number of studies have revealed that over-activation of some signaling pathways can cause tamoxifen resistance; however, very little information is available regarding the genes whose loss-of-function alternation contribute to tamoxifen resistance. Here we used a forward genetic approach in vitro to generate tamoxifen resistant cells from the tamoxifen sensitive breast cancer cell line ZR-75-1, and further identified the disrupted gene in different tamoxifen resistant clones. Retinol binding protein 7, DNA polymerase-transactivated protein 3, γ-glutamyltransferase-like activity 1,slit-robo RhoGTPase-activating protein, tetraspan NET-4, HSPC194, amiloride-sensitive epithelial sodium channel gene,and Notch2, were the eight mutated genes identified in different tamoxifen resistant clones, suggesting their requirement for tamoxifen sensitivity in ZR-75-1 cells. Since the functions of these genes are not related to each other, it suggests that multiple pathways can influence tamoxifen sensitivity in breast cancer ceils.     

Characterization of BCAR4, a novel oncogene causing endocrine resistance in human breast cancer cells

Resistance to the antiestrogen tamoxifen remains a major problem in the management of estrogen receptor-positive breast cancer. Knowledge on the resistance mechanisms is needed to develop more effective therapies. Breast cancer antiestrogen resistance 4 (BCAR4) was identified in a functional screen for genes involved in tamoxifen resistance. BCAR4 is expressed in 27% of primary breast tumors. In patients treated with tamoxifen for metastized disease high BCAR4 mRNA levels are associated with reduced clinical benefit and progression-free survival. Regarding tumor aggressiveness high BCAR4 mRNA levels are associated with a shorter metastasis free survival and overall survival. In the present study, we investigated the role of BCAR4 in endocrine resistance. Forced expression of BCAR4 in human ZR-75-1 and MCF7 breast cancer cells resulted in cell proliferation in the absence of estrogen and in the presence of various antiestrogens. Inhibition of estrogen receptor 1 (ESR1) expression with small interfering RNA (siRNA), implied that the BCAR4-induced mechanism of resistance is independent of ESR1. Highly conserved BCAR4 homologues of rhesus monkey, green monkey, and the less conserved common marmoset gene induced tamoxifen-resistant cell proliferation, in contrast to the distant BCAR4 homologues of bovine and rabbit. Injection of BCAR4-expressing ZR-75-1 cells into nude mice resulted in rapidly growing tumors. In silico analysis showed that BCAR4 mRNA is highly expressed in human placenta and oocyte, and absent in other normal tissues. In conclusion, BCAR4 is a strong transforming gene causing estrogen-independent growth and antiestrogen resistance, and induces tumor formation in vivo. Due to its restricted expression, BCAR4 may be a good target for treating antiestrogen-resistant breast cancer.     

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.     

Activation function-1 domain of estrogen receptor regulates the agonistic and antagonistic actions of tamoxifen

The antiestrogen tamoxifen has been widely used for decades as selective estrogen receptor (ER) modulator for ERalpha-positive breast tumors. Tamoxifen significantly reduces tumor recurrence by binding to the activation function-2 (AF-2) domain of the ER. Acquired resistance to tamoxifen in breast cancer patients is a serious therapeutic problem. Antiestrogen-resistant breast cancer often shows increased expression of the epidermal growth factor receptor (EGFR) family members, EGFR and ErbB2. In this report we now show that overexpression of EGFR or activated AKT-2 in MCF-7 cells leads to phosphorylation of Ser167 in the AF-1 domain of ERalpha, enhanced ER-amplified in breast cancer 1 (ER:AIB1) interaction in the presence of tamoxifen, and resistance to tamoxifen. In contrast, transfection of activated MAPK kinase, an immediate upstream activator of MAPK (ERK 1 and 2) into MCF-7 cells leads to phosphorylation of Ser118 in the AF-1 domain of ERalpha, inhibition of ER-amplified in breast cancer 1 (ER:AIB1) interaction in the presence of Tam, and maintenance of sensitivity to tamoxifen. Inhibition of AKT by short inhibitory RNA blocked Ser167 phosphorylation in ER and restored tamoxifen sensitivity. However, maximum sensitivity to tamoxifen was observed when both AKT and MAPK were inhibited. Taken together, these data demonstrate that different phosphorylation sites in the AF-1 domain of ERalpha regulate the agonistic and antagonistic actions of tamoxifen in human breast cancer cells.     

Elevated CRB3 expression suppresses breast cancer stemness by inhibiting β‐catenin signalling to restore tamoxifen sensitivity

Tamoxifen is a first‐line drug for hormone therapy (HT) in oestrogen receptor‐positive breast cancer patients. However, 20% to 30% of those patients are resistant to tamoxifen treatment. Cancer stem cells (CSCs) have been implicated as one of the mechanisms responsible for tamoxifen resistance. Our previous study indicated that decreased expression of the CRB3 gene confers stem cell characteristics to breast cancer cells. In the current investigation, we found that most of the breast cancer patient tissues resistant to tamoxifen were negative for CRB3 protein and positive for β‐catenin protein, in contrast to their matched primary tumours by immunohistochemical analysis. Furthermore, expression of CRB3 mRNA and protein was low, while expression of β‐catenin mRNA and protein was high in tamoxifen resistance cells (LCC2 and T47D TamR) contrast to their corresponding cell lines MCF7 and T47D. Similarly, CRB3 overexpression markedly restored the tamoxifen sensitivity of TamR cells by the MTT viability assay. Finally, we found that CRB3 suppressed the stemness of TamR cells by inhibiting β‐catenin signalling, which may in turn lead to a decrease in the breast cancer cell population. Furthermore, these findings indicate that CRB3 is an important regulator for breast cancer stemness, which is associated with tamoxifen resistance.     

Downregulation of CXXC Finger Protein 4 Leads to a Tamoxifen-resistant Phenotype in Breast Cancer Cells Through Activation of the Wnt/β-catenin Pathway

Tamoxifen is a successful endocrine therapy drug for estrogen receptor–positive (ER+) breast cancer. However, resistance to tamoxifen compromises the efficacy of endocrine treatment. In the present study, we identified potential tamoxifen resistance–related gene markers and investigated their mechanistic details. First, we established two ER + breast cancer cell lines resistant to tamoxifen, named MCF-7/TMR and BT474/TMR. Gene expression profiling showed that CXXC finger protein 4 (CXXC4) expression is lower in MCF-7/TMR cells than in MCF-7 cells. Furthermore, CXXC4 mRNA and protein expression are lower in the resistant cell lines than in the corresponding parental cell lines. We also investigated the correlation between CXXC4 and endocrine resistance in ER + breast cancer cells. CXXC4 knockdown accelerates cell proliferation in vitro and in vivo and renders breast cancer cells insensitive to tamoxifen, whereas CXXC4 overexpression inhibits cancer cell growth and increases tamoxifen sensitivity of resistant cells. In addition, we demonstrated that CXXC4 inhibits Wnt/β-catenin signaling in cancer cells by modulating the phosphorylation of GSK-3β, influencing the integrity of the β-catenin degradation complex. Silencing the CXXC4 gene upregulates expression of cyclinD1 and c-myc (the downstream targets of Wnt signaling) and promotes cell cycle progression. Conversely, ectopic expression of CXXC4 downregulates the expression of these proteins and arrests the cell cycle in the G0/G1 phase. Finally, the small-molecule inhibitor XAV939 suppresses Wnt signaling and sensitizes resistant cells to tamoxifen. These results indicate that components of Wnt pathway that are early in response to tamoxifen could be involved as an intrinsic factor of the transition to endocrine resistance, and inhibition of Wnt signaling may be an effective therapeutic strategy to overcome tamoxifen resistance.     

Elevated expression of CUEDC2 protein confers endocrine resistance in breast cancer

Endocrine resistance is a major obstacle to hormonal therapy for breast cancers. Although reduced expression of estrogen receptor-α (ER-α) is a known contributing factor to endocrine resistance, the mechanism of ER-α downregulation in endocrine resistance is still not fully understood. Here we report that CUE domain-containing protein-2 (CUEDC2), a ubiquitin-binding motif-containing protein, is a key factor in endocrine resistance in breast cancer. We show that CUEDC2 modulates ER-α protein stability through the ubiquitin-proteasome pathway. Through the study of specimens from a large cohort of subjects with breast cancer, we found a strong inverse correlation between CUEDC2 and ER-α protein expression. Notably, subjects with tumors that highly expressed CUEDC2 had poor responsiveness to tamoxifen treatment and high potential for relapse. We further show that ectopic CUEDC2 expression impaired the responsiveness of breast cancer cells to tamoxifen. Therefore, our findings suggest that CUEDC2 is a crucial determinant of resistance to endocrine therapies in breast cancer.     

Novel function of transcription factor Nrf2 as an inhibitor of RON tyrosine kinase receptor-mediated cancer cell invasion

Recepteur d' origine nantais (RON), a tyrosine kinase receptor, is aberrantly expressed in human tumors and promotes cancer cell invasion. RON receptor activation is also associated with resistance to tamoxifen treatment in breast cancer cells. Nrf2 is a positive regulator of cytoprotective genes. Using chromatin immunoprecipitation (ChIP) and site-directed mutagenesis studies of the RON promoter, we identified Nrf2 as a negative regulator of RON gene expression. High Nrf2 and low RON expression was observed in normal mammary tissue whereas high RON and low or undetectable expression of Nrf2 was observed in breast tumors. The Nrf2 inducer sulforaphane (SFN) as well as ectopic Nrf2 expression or knock-down of the Nrf2 negative regulator keap1, which stabilizes Nrf2, inhibited RON expression and invasion of carcinoma cells. Consequently, our studies identified a novel functional role for Nrf2 as a "repressor" and RON kinase as a molecular target of SFN, which mediates the anti-tumor effects of SFN. These results are not limited to breast cancer cells since the Nrf2 inducer SFN stabilized Nrf2 and inhibited RON expression in carcinoma cells from various tumor types.