Activation of mitogen-activated protein kinase in estrogen receptor alpha-positive breast cancer cells in vitro induces an in vivo molecular phenotype of estrogen receptor alpha-negative human breast tumors

Breast cancer presents as either estrogen receptor alpha (ERalpha) positive or negative, with ERalpha+ tumors responding to antiestrogen therapy and having a better prognosis. By themselves, mRNA expression signatures of estrogen regulation in ERalpha+ breast cancer cells do not account for the vast molecular differences observed between ERalpha+ and ERalpha- cancers. In ERalpha- tumors, overexpression of epidermal growth factor receptor (EGFR) or c-erbB-2, leading to increased growth factor signaling, is observed such that mitogen-activated protein (MAP) kinase (MAPK) is significantly hyperactivated compared with ERalpha+ breast cancer. In ERalpha+/progesterone receptor-positive, estrogen-dependent MCF-7 breast cancer cells, we stably overexpressed EGFR or constitutively active erbB-2, Raf, or MAP/extracellular signal-regulated kinase kinase, resulting in cell lines exhibiting hyperactivation of MAPK, estrogen-independent growth, and the reversible down-regulation of ERalpha expression. By global mRNA profiling, we found a "MAPK signature" of approximately 400 genes consistently up-regulated or down-regulated in each of the MAPK+ cell lines. In several independent profile data sets of human breast tumors, the in vitro MAPK signature was able to accurately distinguish ER+ from ER- tumors. In addition, our in vitro mRNA profile data revealed distinct mRNA signatures specific to either erbB-2 or EGFR activation. A subset of breast tumor profiles was found to share extensive similarities with either the erbB-2-specific or the EGFR-specific signatures. Our results confirm that increased MAPK activation causes loss of ERalpha expression and suggest that hyperactivation of MAPK plays a role in the generation of the ERalpha- phenotype in breast cancer. These MAPK+ cell lines are excellent models for investigating the underlying mechanisms behind the ERalpha- phenotype.     

Long-term treatment with tamoxifen facilitates translocation of estrogen receptor alpha out of the nucleus and enhances its interaction with EGFR in MCF-7 breast cancer cells

The therapeutic benefit of tamoxifen in patients with hormone-dependent breast cancer is limited by acquired resistance to this drug. To investigate the biological alterations responsible for tamoxifen resistance, an in vitro model was established. After 6-month continuous exposure to tamoxifen (10(-7) mol/L), growth of MCF-7 breast cancer cells was no longer inhibited by this antiestrogen. Although there was no significant increase in the basal levels of activated mitogen-activated protein kinase (MAPK), tamoxifen-resistant (TAM-R) cells exhibited enhanced sensitivity to epidermal growth factor (EGF) and estradiol stimulated activation of MAPK. Tamoxifen elicited rapid phosphorylation of MAPK, in contrast to its antagonistic activity in control cells. Blockade of the EGF receptor (EGFR)/MAPK pathway caused more dramatic inhibition of growth of TAM-R cells than the control cells. An increased amount of estrogen receptor alpha (ERalpha) was coimmunoprecipitated with EGFR from TAM-R cells although the total levels of these receptors were not increased. Notably, ERalpha seemed to redistribute to extranuclear sites in TAM-R cells. Increased ERalpha immunoreactivity in the cytoplasm and plasma membrane of TAM-R cells was shown by fluorescent microscopy and by Western analysis of isolated cellular fractions. In TAM-R cells, an increased amount of c-Src was coprecipitated with EGFR or ERalpha. Blockade of c-Src activity resulted in redistribution of ERalpha back to the nucleus and in reduction of its interaction with EGFR. Prolonged blockade of c-Src activity restored sensitivity of TAM-R cells to tamoxifen. Our results suggest that enhanced nongenomic function of ERalpha via cooperation with the EGFR pathway is one of the mechanisms responsible for acquired tamoxifen resistance.     

Dual blockade of EGFR and ERK1/2 phosphorylation potentiates growth inhibition of breast cancer cells

One of the major targets for breast cancer therapy is the epidermal growth factor receptor (EGFR) and related receptors, which signal via different signal transduction pathways including the mitogen-activated protein kinase (MAPK) pathway. This study determined whether there is a correlation between EGFR/HER2 status and MAPK (ERK1/2) phosphorylation in breast cancer cells, and how this affects the response to an inhibitor of the receptors. Expression of EGFR, HER2 and phosphorylated ERK1/2 were measured by immunoblotting in a panel of breast cancer cell lines. Several lines expressed high levels of pERK1/2, with no obvious correlation with the level of EGFR/HER2. The EGFR tyrosine kinase inhibitor PKI166 inhibited growth and induced apoptosis in some cells with high levels of growth factor receptors (MDA-MB-468, SUM149, SKBR3), but was less effective in cells that also had high basal ERK1/2 activity (MDA-MB-231). The combination of an inhibitor of MAPK signalling (U0126) and PKI166 produced significantly more inhibition and apoptosis than either agent alone. This suggests that constitutive activation of the MAPK pathway may bypass inhibition of EGFR/HER2 tyrosine kinases, and lead to insensitivity to agents targeting the receptors. However, inhibiting both EGFR/HER2 and MAPK signalling can result in significant growth inhibition and apoptosis of EGFR-expressing breast cancer cells.     

Expression of c-erbB receptors, heregulin and oestrogen receptor in human breast cell lines

Members of the c-erbB family have been implicated in poor prognosis in breast cancer. Given the propensity for heterodimerisation within the erbB family, the pattern of co-expression of these receptors is likely to be as functionally important as aberrant expression of any given receptor alone. Therefore, the patterns of expression of the receptors, epidermal growth factor receptor (EGF-R), c-erbB-2, c-erbB-3, c-erbB-4, and one of the erbB ligands, heregulin (HRG), were examined in normal and malignant breast cell lines and compared with expression of oestrogen receptor (ER), a classical indicator of good prognosis. There was an inverse correlation between ER and EGF-R mRNA levels, as previously described, but no correlation between either of these receptors and c-erbB-2. c-erbB-3 expression was positively correlated with ER. In contrast, HRG expression was inversely related to ER. Expression of antisense-ER resulted in increased EGF-R mRNA, demonstrating a functional link between the expression of these 2 genes, however, there was no significant change in c-erbB-2 or c-erbB-3 mRNA, suggesting that ER is not directly involved in control of expression of these genes. A comparison of individual erbB receptors and HRG revealed that the majority of lines expressing increased levels of c-erbB-2 also expressed elevated levels of c-erbB-3 mRNA, and none of the cell lines that expressed both c-erbB-2 and either c-erbB-3 or c-erbB-4 expressed the ligand HRG. In summary, the levels of expression of c-erbB-1, -2, -3, and -4 varied in this series of breast cell lines, and the pattern of expression and the relationship of each growth factor receptor to the expression of ER was quite distinct. The lack of expression of HRG in cell lines that express receptors may be indicative of paracrine interactions between erbB ligands and their cognate receptors and may suggest that the ligand and receptors are expressed in different subtypes of breast epithelial cells from which the cell lines are derived.     

Inhibition of MAP kinase by sphingosine and its methylated derivative, N,N-dimethylsphingosine

Endogenous sphingolipid metabolites such as ceramides and sphingosines have been increasingly recognized as lipid mediators of cell growth, differentiation and apoptosis. We have previously studied the ability of sphingosine (Sph) and N,N-dimethylsphingosine (DMS) to induce apoptosis in a variety of solid tumor cell lines. Here we report that in tumor cell lines displaying high mitogen-activated protein kinase activity (MAPK), treatment with 5 mu M of these sphingolipids significantly inhibited MAPK activity within 2-5 min (p < 0.005-0.01 as compared to controls) and induced apoptosis within hours. In contrast, untransformed cells and those tumor cell lines with low MAPK activity showed no significant change in activity and no apoptosis. High concentrations of C2-ceramide (50-100 mM), which induced apoptosis in the solid tumor cells, did not show significant effect on MAPK activity. MAPK activity was not directly inhibited in vitro, but tyrosine phosphatase activity was increased 2-4 fold in solid tumor cells by Sph or DMS (p < 0.01-0.05), suggesting that a phosphatase may play an important role in sphingolipid-directed MAPK regulation. Sph/DMS-induced apoptosis, but not MAPK inhibition, was blocked by protease inhibitors, indicating that MAPK inhibition is an earlier step of Sph/DMS-induced apoptosis than proteolysis. Furthermore, in human breast carcinoma MDA468 cells and human epidermal carcinoma A431 cells, both of which overexpress the epidermal growth factor (EGF) receptor, 20-200 nM EGF inhibited MAPK (p < 0.005-0.01) and induced apoptosis. These observations suggest that inhibition of the MAPK cascade may be involved in apoptotic signaling by Sph/DMS in some solid tumor cells, or by EGF in some cancer cells which overexpress the EGF receptor. Finally, the PKC-specific inhibitor, calphostin C, under conditions in which PKC is completely suppressed, inhibited MAPK activity and induced apoptosis only weakly in these solid tumor cells, whereas the non-specific PKC inhibitor staurosporine induced both apoptosis and MAPK inhibition significantly, suggesting that MAPK inhibition and apoptosis by Sph/DMS occurs independently of PKC in these cell lines, although these pathways may act cooperatively in other cell types. This study provides insight into possible mechanisms involved in sphingolipid-induced apoptosis in solid cancer tumor cell lines.     

Targeting mixed lineage kinases in ER-positive breast cancer cells leads to G2/M cell cycle arrest and apoptosis

Estrogen receptor (ER)-positive tumors represent the most common type of breast cancer, and ER-targeted therapies such as antiestrogens and aromatase inhibitors have therefore been widely used in breast cancer treatment. While many patients have benefited from these therapies, both innate and acquired resistance continue to be causes of treatment failure. Novel targeted therapeutics that could be used alone or in combination with endocrine agents to treat resistant tumors or to prevent their development are therefore needed. In this report, we examined the effects of inhibiting mixed-lineage kinase (MLK) activity on ER-positive breast cancer cells and non-tumorigenic mammary epithelial cells. Inhibition of MLK activity with the pan-MLK inhibitor CEP-1347 blocked cell cycle progression in G2 and early M phase, and induced apoptosis in three ER-positive breast cancer cell lines, including one with acquired antiestrogen resistance. In contrast, it had no effect on the cell cycle or apoptosis in two non-tumorigenic mammary epithelial cell lines. CEP-1347 treatment did not decrease the level of active ERK or p38 in any of the cell lines tested. However, it resulted in decreased JNK and NF-κB activity in the breast cancer cell lines. A JNK inhibitor mimicked the effects of CEP-1347 in breast cancer cells, and overexpression of c-Jun rescued CEP-1347-induced Bax expression. These results indicate that proliferation and survival of ER-positive breast cancer cells are highly dependent on MLK activity, and suggest that MLK inhibitors may have therapeutic efficacy for ER-positive breast tumors, including ones that are resistant to current endocrine therapies.     

Activity of tamoxifen and its metabolites on endocrine-dependent and endocrine-independent breast cancer cells

To better understand the mechanism of action of antiestrogens, the growth-inhibitory effect of tamoxifen and its main metabolites N-desmethyltamoxifen and 4-hydroxytamoxifen, was studied in 6 breast cancer cell lines characterized by different steroid receptor contents. On the basis of the results, our cell lines could be classified into three groups: a first group, including 734B and ZR-75.1 cell lines, characterized by a clear endocrine-dependent behavior, in which cells were sensitive to antiestrogens although to different degrees; a second group, including MDA-MB 231 and BT20 cell lines, characterized by a clear endocrine-insensitive behavior, in which cells were affected only by the highest (10(-6) M) antiestrogen concentration; a third group, including MCF7 and T47D cell lines, characterized by a peculiar behavior. The T47D cell line displayed an increased growth rate after treatment with all three antiestrogens considered. Despite the positive receptor content in the MCF7 cell line, only 4-hydroxytamoxifen showed a clear antiestrogen dose-dependent effect, whereas tamoxifen decreased the cell growth rate only at lower concentrations (10(-8) and 10(-7) M). These results and the well-known heterogeneity of human breast tumors explain the failure of antiestrogen treatment in a certain percentage of patients with breast cancer with a positive estrogen receptor status.     

Cooperative effect of gefitinib and fumitremorgin c on cell growth and chemosensitivity in estrogen receptor alpha negative fulvestrant-resistant MCF-7 cells

The selective ER downregulator, fulvestrant, is currently approved as a second line endocrine therapy after onset of resistance to prior antiestrogen therapy in postmenopausal breast cancer patients. Resistance to antihormonal therapies is common and, therefore, we anticipate that fulvestrant-resistance will occur as well. The current study was undertaken to investigate the underlying molecular changes after fulvestrant-resistance and find new therapeutic targets and agents for fulvestrant-resistant breast cancer cells. We developed a unique fulvestrant-resistant cell line (MCF-7/F), derived from MCF-7 estrogen receptor alpha (ERalpha)-positive human breast cancer cells, by culturing them in 1 microM fulvestrant containing medium for approximately 18 months. MCF-7/F cells became irreversibly ERalpha negative as withdrawal of fulvestrant did not alter the ERalpha-negative phenotype, determined by real-time PCR, Western blot analysis, and ERE-luciferase transfection assays. MCF-7/F cells grew in a hormone-independent manner. Interestingly, MCF-7/F cells overexpressed both epidermal growth factor receptor (EGFR) and breast cancer resistant protein (BCRP). Gefitinib, a specific EGFR tyrosine kinase inhibitor, preferentially inhibited the growth of MCF-7/F cells relative to MCF-7 cells by inhibiting both MAPK44/42 and Akt phosphorylation. MCF-7/F cells became less sensitive to chemotherapeutic agents such as mitoxantrone. Moreover, fumitremorgin C, a specific BCRP inhibitor, significantly increased the efficacy of mitoxantrone in MCF-7/F cells. Gefitinib increased the inhibitory effect of mitoxantrone on cell growth. Similarly, fumitremorgin C increased the inhibitory effect of gefitinib on cell growth, suggesting that there is a bidirectional crosstalk between EGFR and BCRP. More importantly, these results provide a molecular basis for using gefitinib, BCRP inhibitors, and chemotherapeutic agents as combination therapy approaches in fulvestrant-resistant breast cancer.