Modeling the estrogen receptor to growth factor receptor signaling switch in human breast cancer cells

Breast cancer cells develop resistance to endocrine therapies by shifting between estrogen receptor (ER)-regulated and growth factor receptor (GFR)-regulated survival signaling pathways. To study this switch, we propose a mathematical model of crosstalk between these pathways. The model explains why MCF7 sub-clones transfected with HER2 or EGFR show three GFR-distribution patterns, and why the bimodal distribution pattern can be reversibly modulated by estrogen. The model illustrates how transient overexpression of ER activates GFR signaling and promotes estrogen-independent growth. Understanding this survival-signaling switch can help in the design of future therapies to overcome resistance in breast cancer.     

Sphingosine kinase type 1 promotes estrogen-dependent tumorigenesis of breast cancer MCF-7 cells

The sphingolipid metabolite, sphingosine-1-phosphate (S1P), formed by phosphorylation of sphingosine, has been implicated in cell growth, suppression of apoptosis, and angiogenesis. In this study, we have examined the contribution of intracellular S1P to tumorigenesis of breast adenocarcinoma MCF-7 cells. Enforced expression of sphingosine kinase type 1 (SPHK1) increased S1P levels and blocked MCF-7 cell death induced by anti-cancer drugs, sphingosine, and TNF-alpha. SPHK1 also conferred a growth advantage, as determined by proliferation and growth in soft agar, which was estrogen dependent. While both ERK and Akt have been implicated in MCF-7 cell growth, SPHK1 stimulated ERK1/2 but had no effect on Akt. Surprisingly, parental growth of MCF-7 cells was only weakly stimulated by S1P or dihydro-S1P, ligands for the S1P receptors which usually mediate growth effects. When injected into mammary fat pads of ovariectomized nude mice implanted with estrogen pellets, MCF-7/SPHK1 cells formed more and larger tumors than vector transfectants with higher microvessel density in their periphery. Collectively, our results suggest that SPHK1 may play an important role in breast cancer progression by regulating tumor cell growth and survival.     

Membrane initiated estrogen signaling in breast cancer

Recent research has focused on effects of the estrogen receptor acting at the level of the cell membrane in breast cancer. In this review we describe 17beta-estradiol (E2)-initiated membrane signaling pathways involving the activation of several kinases that contribute to the regulation of cell proliferation and prevention of apoptosis. Although classical concepts had assigned priority to the nuclear actions of estrogen receptor, recent studies document the additional importance of estrogen receptor residing in or near the plasma membrane. A small fraction of estrogen receptor is associated with the cell membrane and mediates the rapid effects of E2. Unlike classical growth factor receptors, such as insulin-like growth factor 1 receptor (IGF1R) and epidermal growth factor receptor (EGFR), estrogen receptor has no transmembrane and kinase domains and is known to initiate E2 rapid signals by forming a protein complex with many signaling molecules. The formation of the protein complex is a critical step, leading to the activation of the MAPK1/3 (also known as MAP kinase) and AKT1 (also known as Akt) pathways. A full understanding of the mechanisms underlying these relationships, with the ultimate aim of abrogating specific steps, should lead to more-targeted strategies for treatment of hormone dependent-breast cancer.     

Sphingosine kinase 1 is required for migration, proliferation and survival of MCF-7 human breast cancer cells

Sphingosine-1-phosphate (S1P) is a potent lysolipid involved in a variety of biological responses important for cancer progression. Therefore, we investigated the role of sphingosine kinase type 1 (SphK1), the enzyme that makes S1P, in the motility, growth, and chemoresistance of MCF-7 breast cancer cells. Epidermal growth factor (EGF), an important growth factor for breast cancer progression, activated and translocated SphK1 to plasma membrane. SphK1 was required for EGF-directed motility. Downregulation of SphK1 in MCF-7 cells reduced EGF- and serum-stimulated growth and enhanced sensitivity to doxorubicin, a potent chemotherapeutic agent. These results suggest that SphK1 may be critical for growth, metastasis and chemoresistance of human breast cancers.     

Epigenetic regulation of estrogen signaling in breast cancer

Estrogen signaling is mediated by ERα and ERβ in hormone dependent, breast cancer (BC). Over the last decade the implication of epigenetic pathways in BC tumorigenesis has emerged: cancer-related epigenetic modifications are implicated in both gene expression regulation, and chromosomal instability. In this review, the epigenetic-mediated estrogen signaling, controlling both ER level and ER-targeted gene expression in BC, are discussed: (1) ER silencing is frequently observed in BC and is often associated with epigenetic regulations while chemical epigenetic modulators restore ER expression and increase response to treatment;(2) ER-targeted gene expression is tightly regulated by co-recruitment of ER and both coactivators/corepressors including HATs, HDACs, HMTs, Dnmts and Polycomb proteins.     

Androgens inhibit estrogen action in MCF-7 human breast cancer cells

We have observed that the estrogen-dependent augmentation of cytoplasmic progesterone receptor (PR_c) content in MCF-7 human breast cancer cells is completely inhibited in the presence of testosterone (T) or dihydrotestosterone (DHT)¹. This effect is neither a result of altered PR_c affinity for test ligand nor a result of the direct interaction of either androgen with PR_c. Furthermore, the antiestrogenic activity of DHT is blocked in the presence of the antiandrogen, cyproterone, indicating that it is mediated through the androgen receptor. Further investigations of this cell line may provide important insights into the effects of sex steroids upon the hormonal regulation of a variety of healthy and malignant human tissues.     

Protein kinase C subspecies in estrogen receptor-positive and -negative human breast cancer cell lines

Estrogen receptor-positive (MCF7) and -negative (BT20) human breast cancer cell lines, which are frequently used for studies on cancer chemotherapy with triphenylethylene (TPE) anti-estrogens, express at least three protein kinase C subspecies. Two of them are identified as type II PKC having the beta-sequence and type III PKC having the alpha-sequence. The other one shows typical characteristics of PKC which responds to Ca2+, phosphatidylserine and diacylglycerol, but shows kinetic properties subtly different from the previously known PKC subspecies. Immunoblot analysis has shown that this enzyme does not correspond to any of the well defined subspecies with known sequence structures. All of these PKC subspecies are similarly susceptible to the TPE antiestrogens.     

Alternate estrogen receptors promote invasion of inflammatory breast cancer cells via non-genomic signaling

Although Inflammatory Breast Cancer (IBC) is a rare and an aggressive type of locally advanced breast cancer with a generally worst prognosis, little work has been done in identifying the status of non-genomic signaling in the invasiveness of IBC. The present study was performed to explore the status of non-genomic signaling as affected by various estrogenic and anti-estrogenic agents in IBC cell lines SUM149 and SUM190. We have identified the presence of estrogen receptor α (ERα) variant, ERα36 in SUM149 and SUM190 cells. This variant as well as ERβ was present in a substantial concentration in IBC cells. The treatment with estradiol (E2), anti-estrogenic agents 4-hydroxytamoxifen and ICI 182780, ERβ specific ligand DPN and GPR30 agonist G1 led to a rapid activation of p-ERK1/2, suggesting the involvement of ERα36, ERβ and GPR30 in the non-genomic signaling pathway in these cells. We also found a substantial increase in the cell migration and invasiveness of SUM149 cells upon the treatment with these ligands. Both basal and ligand-induced migration and invasiveness of SUM149 cells were drastically reduced in the presence of MEK inhibitor U0126, implicating that the phosphorylation of ERK1/2 by MEK is involved in the observed motility and invasiveness of IBC cells. We also provide evidence for the upregulation of p-ERK1/2 through immunostaining in IBC patient samples. These findings suggest a role of non-genomic signaling through the activation of p-ERK1/2 in the hormonal dependence of IBC by a combination of estrogen receptors. These findings only explain the failure of traditional anti-estrogen therapies in ER-positive IBC which induces the non-genomic signaling, but also opens newer avenues for design of modified therapies targeting these estrogen receptors.     

Ubiquitination and downregulation of ErbB2 and estrogen receptor-alpha by kinase inhibitor MP-412 in human breast cancer cells

ErbB2 has been proven to be an important target for breast cancer therapy. MP-412 is a dual ErbB2 and epidermal growth factor receptor tyrosine kinase inhibitor belonging to an irreversible-type anilinoquinazoline derivative. We demonstrate herein that along with the kinase inhibition, MP-412 has the ability to induce ubiquitination, internalization, and degradation of ErbB2 in several human breast cancer cell lines at concentrations relatively higher than those required for kinase inhibition. Another irreversible inhibitor, CI-1033, showed similar activity, while the reversible compounds were ineffective, suggesting a crucial role of covalent bonding functionality in these effects. In MCF7 cells, MP-412 depleted not only ErbB2 but also estrogen receptor (ER)-α, and to some extent, affected Raf-1, while MP-412 activated Hsp70 expression. Moreover, we observed that MP-412 increased immunocomplexing of Hsp70 with ErbB2 and ER-α, with simultaneous induction of ubiquitination of these client proteins. Furthermore, in combination with proteasome inhibitor, MP-412 resulted in the noticeable accumulation of ErbB2 and ER-α in the detergent insoluble fraction of cell lysates. These results suggest that MP-412 acts as an inhibitor of Hsp90 function, whereas MP-412 did not bind directly to ATP-binding site of Hsp90, unlike geldanamycin. We also found that new protein synthesis was involved in the activity of MP-412 on Hsp90 modulation. Since downregulation of ErbB2 and ER-α by accelerating the ubiquitin-proteolysis system will become an attractive approach for breast cancer therapy, we expect MP-412 to be a lead compound for the drug design and the development of such agents.     

Mesothelin promotes anchorage-independent growth and prevents anoikis via extracellular signal-regulated kinase signaling pathway in human breast cancer cells

Mesothelin (MSLN) is a glycoprotein that is overexpressed in various tumors. MSLN is present on the cell surface and is also released into body fluids or culture supernatants from MSLN-positive tumor cells. Despite intensive study of MSLN as a diagnostic marker or target for immunotherapy, its biological function is largely unknown. In the present study, we examined the effects of ectopic expression of MSLN in human breast cancer cell lines (MCF-7, T47D, and MDA-MB-231). We found that overexpression of MSLN promoted anchorage-independent growth in soft agar. In addition, MDA-MB-231 cells expressing high levels of MSLN exhibited resistance to anoikis (a type of apoptosis induced by detachment from substratum), as indicated by decreased DNA fragmentation and down-regulation of the proapoptotic protein Bim. Incubating MSLN-expressing MDA-MB-231 cells in the presence of U0126, an inhibitor of mitogen-activated protein/extracellular-signal-regulated kinase kinase, induced accumulation of Bim and restored susceptibility to anoikis. Western blot analysis also revealed that overexpression of MSLN resulted in sustained activation of extracellular signal-regulated kinase 1/2 and suppression of Bim. The present results constitute novel evidence that MSLN enables cells to survive under anchorage-independent conditions by suppressing Bim induction via the extracellular signal-regulated kinase signaling pathway.     

The R-Ras GTPase mediates cross talk between estrogen and insulin signaling in breast cancer cells

The signaling cascades activated by insulin and IGF-1 contribute to the control of multiple cellular functions, including glucose metabolism and cell proliferation. In most cases these effects are mediated, at least in part, by insulin receptor substrates (IRS), one of which is insulin receptor substrate 1 (IRS-1). R-Ras is a member of the Ras family of GTPases and is involved in a variety of biological processes, including integrin activation, cell migration, and control of cell proliferation. Here we demonstrate that both R-Ras and BCAR3, a regulator of R-Ras activity that has been implicated in breast cancer, regulate the level of IRS-1 protein in estrogen-dependent MCF-7 and ZR75 breast cancer cells. In particular, expression of a constitutively activated R-Ras mutant, R-Ras38V, or of BCAR3 accelerates the degradation of IRS-1, leading to the impairment of signaling through insulin but not epidermal growth factor receptors. Moreover, knockdown of endogenous R-Ras levels in MCF-7 cells inhibits IRS-1 degradation induced by estrogen signaling blockade but not by long-term insulin treatment. Consistent with these results, both R-Ras38V expression and estrogen signaling blockade lead to the degradation of IRS-1, at least in part, through calpain activity. These findings show that R-Ras activity mediates inhibition of insulin signaling associated with suppression of estrogen action, implicating this GTPase in a growth-inhibitory mechanism associated with antiestrogen treatment of breast cancer.