Selective turnover of the essential fatty acid ester components of estradiol-17 beta lipoidal derivatives formed by human mammary cancer cells in culture

The properties of fatty acyl coenzyme A: estradiol-17 beta acyl transferase in microsomes derived from pooled human mammary cancer tissue have been examined. A pH optimum of 5.5 was found and addition of long-chained fatty acyl CoAs increased estradiol-17 beta (E2) 17-monoacyl ester synthesis; the apparent Km for E2 being 8 microM when oleoyl CoA, linolenoyl CoA or palmitoyl CoA were employed. Testosterone, dehydroepiandrosterone, and 5-androsterone-3 beta, 17 beta-diol acted as competitive inhibitors with Ki values of 36, 36 and 46 microM, respectively. The composition of E2 fatty acyl esters (E2-L) formed by incubation of [3H]E2 with human mammary cancer tissue and human mammary cancer cell lines has been determined by HPLC. Although the composition of E2-L in estrogen receptor negative cell lines (MDA-MB-231 and MDA-MB-330) was generally similar to that found for MCF-7 cells (estrogen receptor positive) and pooled human mammary cancer tissue, the former cell lines contained a 3-fold higher relative concentration of E2-17 beta stearate. MCF-7 cells were exposed to 30 nM [3H]E2 and the composition of the isolated [3H]E2-L fraction studied at various time intervals. At 0.5 h, the intracellular concentration of E2-L was 1.8 +/- 0.4 (SEM) pmol/mg DNA which increased to values of 3.6 +/- 0.6 and 4.3 +/- 0.5 at 4 h and 16 h, respectively. In the subsequent 3 h following transfer to medium lacking [3H]E2, the concentration of E2-L declined to 3.7 +/- 0.3 pmol/mg DNA. The subfraction of E2-L composed of E2-17 beta arachidonate, linolenate and docosahexaenoate, was seen to decline in relative abundance after 0.5 h and to reach significantly lower relative levels at 16 h, and again in the 3 h period following estrogen withdrawal. The data suggests that these components, derived from essential fatty acids, are more metabolically active. This may then provide a new lead to link these novel estrogen derivatives with the established relationship between unsaturated fatty acids and an increased mammary cancer incidence.     

Formation and turnover of long-chain fatty acid esters of 5-androstene-3 beta, 17 beta -diol in estrogen receptor positive and negative human mammary cancer cell lines in culture

Microsomal preparations derived from bovine placenta cotyledons, previously investigated as a convenient source of fatty acyl coenzyme A: estradiol-17 beta-acyl transferase, have been shown to acylate other steroids bearing 3 beta- or 17 beta-hydroxyl groups. In the presence of 0.1 mM oleoyl CoA, the apparent Km values for dehydroepiandrosterone, testosterone, and 5-androstene-3 beta,17 beta-diol (delta 5-DIOL) were 45, 67, and 20 microM, respectively. Acylation of delta 5-DIOL occurred at either the 3 beta- or 17 beta-positions to give monoesters. Testosterone, estradiol-17 beta, and delta 5-DIOL acted as competitive inhibitors for the acylation of the 3 beta-hydroxyl group of dehydroepiandrosterone (Ki values 71, 75, and 41 microM, respectively). Such data indicate that a single enzyme of wide substrate specificity may be involved in these acylation reactions. When estrogen receptor (ER) positive and negative human mammary cancer cell lines were incubated with 10 nM [3H]delta 5-DIOL, intracellular accumulation of delta 5-DIOL long-chain fatty acid esters occurred; rates being higher (p less than 0.001) in ER negative cells (MDA-MB-231 and MDA-MB-330) compared to MCF-7 cells (ER positive), and higher (P less than 0.005) in MDA-MB-231 cells compared to ZR-75-1 cells (ER positive). After exposure to 10 nM [3H]delta 5-DIOL for 16 h, the total labeled steroid fatty acid fraction was composed predominantly of delta 5-DIOL-3 beta- and 17 beta-monoesters (approximately 85%), the remainder containing approximately equal amounts of delta 5-DIOL-diesters and dehydroepiandrosterone-3 beta-esters. Subsequent transfer to medium lacking delta 5-DIOL was accompanied by a breakdown of the labeled esters, which was more rapid in the ER positive cell lines. During this period, intracellular free delta 5-DIOL levels rapidly declined in MDA-MB-330 cells but were maintained in MCF-7 cells, presumably by binding to ER. This behavior parallels that of estradiol-17 beta previously observed in these cell lines and further emphasizes the potential importance of the adrenal-derived estrogen delta 5-DIOL in consideration of a hormone-based etiology of human breast cancer.     

Concentration-dependent mitogenic and antiproliferative actions of 2-methoxyestradiol in estrogen receptor-positive human breast cancer cells

We compared in this study the effects of 2-methoxyestradiol (2-MeO-E(2)) on the growth of two estrogen receptor (ER)-negative human breast cancer cell lines (MDA-MB-231 and MDA-MB-435s) and two ER-positive human breast cancer cell lines (MCF-7 and T-47D). 2-MeO-E(2) exerted a concentration-dependent antiproliferative action in the ER-negative MDA-MB-231 and MDA-MB-435s cells. The presence or absence of exogenous 17beta-estradiol (E(2)) in the culture medium did not affect the potency and efficacy of 2-MeO-E(2)'s antiproliferative action in these ER-negative cells. When the ER-positive MCF-7 and T-47D cells were cultured in a medium supplemented with 10nM of exogenous E(2), 2-MeO-E(2) at 750 nM to 2 microM concentrations exerted a similar antiproliferative effect. However, when the ER-positive cell lines were cultured in the absence of exogenous E(2), 2-MeO-E(2) at relatively low concentrations (10-750 nM) had a moderate mitogenic effect, with its apparent efficacy 75-80% of that of E(2). This mitogenic effect of 2-MeO-E(2) was ER-mediated and largely attributable to 2-MeO-E(2)'s residual estrogenic activity on the basis of our following findings: (i) its effect was only manifested in the ER-positive cells but not in the ER-negative cells; (ii) its effect in the ER-positive cells was partially or fully abolished when exogenous E(2) was concomitantly present in the culture medium; (iii) 2-MeO-E(2) retained 1-2% of E(2)'s binding affinity for the human ERalpha and ERbeta, and its mitogenic effect was inhibited in a concentration-dependent manner by ICI-182,780, a pure ER antagonist; and (iv) its effect was not due to its metabolic conversion to 2-hydroxyestradiol. Our timely findings are of importance to the on-going clinical trials designed to evaluate 2-MeO-E(2)'s effectiveness for the treatment of different types (ER-positive or ER-negative) of human breast cancer. This knowledge will improve the design of clinical trials as well as the interpretation of clinical outcomes when 2-MeO-E(2) is used as a single agent therapy or as part of a combination therapy for human breast cancer.     

The DHEA metabolite 7β-hydroxy-epiandrosterone exerts anti-estrogenic effects on breast cancer cell lines

7β-Hydroxy-epiandrosterone (7β-OH-EpiA), an endogenous androgenic derivative of dehydroepiandrosterone, has previously been shown to exert anti-inflammatory action in vitro and in vivo via a shift from prostaglandin E2 (PGE2) to 15-deoxy-Δ(12,14)-PGJ2 production. This modulation in prostaglandin production was obtained with low concentrations of 7β-OH-EpiA (1-100nM) and suggested that it might act through a specific receptor. Inflammation and prostaglandin synthesis is important in the development and survival of estrogen-dependent mammary cancers. Estrogen induced PGE2 production and cell proliferation via its binding to estrogen receptors (ERs) in these tumors. Our objective was to test the effects of 7β-OH-EpiA on the proliferation (by counting with trypan blue exclusion), cell cycle and cell apoptosis (by flow cytometry) of breast cancer cell lines MCF-7 (ERα+, ERβ+, G-protein coupled receptor 30: GPR30+) and MDA-MB-231 (ERα-, ERβ+, GPR30+) and to identify a potential target of this steroid in these cell lineages (by transactivations) and in the nuclear ER-negative SKBr3 cells (GPR30+) (by proliferation assays). 7β-OH-EpiA exerted anti-estrogenic effects in MCF-7 and MDA-MB-231 cells associated with cell proliferation inhibition and cell cycle arrest. Moreover, transactivation and proliferation with ER agonists assays indicated that 7β-OH-EpiA interacted with ERβ. Data from proliferation assays on the MCF-7, MDA-MB-231 and SKBr3 cell lines suggested that 7β-OH-EpiA may also act through the membrane GPR30 receptor. These results support that this androgenic steroid acts as an anti-estrogenic compound. Moreover, this is the first evidence that low doses of androgenic steroid exert antiproliferative effects in these mammary cancer cells. Further investigations are needed to improve understanding of the observed actions of endogenous 7β-OH-EpiA.     

Involvement of p53 in gemcitabine mediated cytotoxicity and radiosensitivity in breast cancer cell lines

Gemcitabine (2',2'-difluoro-2'-deoxycytidine; dFdCyd) is one of the anti-metabolites drugs that target DNA replication. We evaluated dFdCyd cytotoxicity and its radiosensitizing ability in human breast cancer cell lines, MCF-7 (wild-type p53) and MDA-MB-231 (mutant-type p53) along with normal mammary epithelial cell line (MCF-12) for comparison. Radiosensitivity and cytotoxicity were measured by the clonogenic survival assays. DNA DSBs was studied by Pulse Field Gel Electrophoresis (PFGE) and cell cycle distribution was analyzed by flow cytometry. MDA-MB-231 cells were the most sensitive to the cytotoxicity of dFdCyd (IC(50) 5 nM) then MCF-7 (IC(50) 10nM), whereas MCF-12 cells were the most resistant to the cytotoxicity of dFdCyd (IC(50) 70 nM). MCF-12 and MCF-7 cell lines did not show any radiosensitization to dFdCyd, whereas the MDA-MB-231 cells showed significantly increased radioresistant to dFdCyd at equimolar concentration (p=0.002) and at IC(50) concentration (p<0.001). The DNA double strand breaks (DSBs) repair showed that dFdCyd neither increases DNA DSBs nor decreases the rate of their repair in MCF-12 and MCF-7 cell lines, while the same treatment in MDA-MB-231 cell line led to decrease the rate of DSBs or increase the rate of DNA repair (p=0.034). Therefore, dFdCyd is a cytotoxic agent, especially in the cancer cells irrespective of having wild-type or mutated p53 protein, but it is not effective as radiosensitizer in the cell lines used in this study. dFdCyd combined with radiation reduces the efficacy of chemo-radiotherapy in p53 mutated cells. Therefore, p53-mutated cancer could be a counter-indication for radiation-gemcitabine combined treatment.     

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on cell growth and the secretion of the estrogen-induced 34-, 52- and 160-kDa proteins in human breast cancer cells

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the growth of estrogen-responsive MCF-7 human breast cancer cells in the presence of 17 beta-estradiol was determined. After treatment with 17 beta-estradiol (1 nM), TCDD (10 nM) and 17 beta-estradiol (1 nM) plus TCDD (10 nM) the cells were monitored daily for cell growth and DNA content for 7 days. The results showed that TCDD inhibited cell proliferation and DNA content of untreated cells and inhibited the 17 beta-estradiol-stimulated cell proliferation and increase in cellular DNA content. In contrast, TCDD did not effect the growth of estrogen non-responsive MDA-MB-231 human breast cancer cells. TCDD (0.1-10 nM) also caused a concentration-dependent decrease in the 17 beta-estradiol-induced proliferation in MCF-7 cells. The effects of TCDD on the 17 beta-estradiol-induced secretion of the 52-kDa protein (i.e. procathepsin D), the 34-kDa (cathepsin D) and 160-kDa proteins were also determined in the MCF-7 and MDA-MB-231 human breast cancer cell lines. The levels of the proteins were determined by autoradiographic analysis of the incorporation of [35S]methionine into the secreted proteins which were separated by denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Treatment of MCF-7 cells with 17 beta-estradiol (1 nM), TCDD (10 and 100 nM) and 17 beta-estradiol (1 nM) plus TCDD (10 nM) resulted in levels of the 52-kDa protein which were 497, 63.6, 98.1 and 66.3%, respectively, of the corresponding levels observed in control (untreated) cells. Using the same concentrations, the levels of the 34-kDa protein secreted into the media were 372, 42.3, 64.0 and 43.8% of control values, respectively, and the corresponding levels of the 160-kDa protein were 381, 52.9, 71.2 and 76.6% of the control values, respectively. In contrast, treatment of MDA-MB-231 cells with 17 beta-estradiol (1 nM), TCDD (10 and 100 nM) and 17 beta-estradiol (1 nM) plus TCDD (10 nM) resulted in a 31-39% reduction in the secretion of the 52-kDa protein however these effects were not statistically different from the control values. In addition, the treatments did not cause any significant effects on the secretion of the 34- and 160-kDa proteins by MDA-MB-231 cells. These results clearly confirm and extend the range of antiestrogenic effects caused by TCDD in estrogen-responsive MCF-7 cells and indicate that the MDA-MB-231 cells are not responsive to the antiestrogenic effects of TCDD.     

Functional and molecular characteristics of system L in human breast cancer cells

The functional and molecular properties of system L in human mammary cancer cells (MDA-MB-231 and MCF-7) have been examined. All transport experiments were conducted under Na(+)-free conditions. alpha-Aminoisobutyric acid (AIB) uptake by MDA-MB-231 and MCF-7 cells was almost abolished by BCH (2-amino-2-norbornane-carboxylic acid). AIB uptake by MDA-MB-231 cells was also inhibited by L-alanine (83.6%), L-lysine (75.6%) but not by L-proline. Similarly, L-lysine and L-alanine, respectively, reduced AIB influx into MCF-7 cells by 45.3% and 63.7%. The K(m) of AIB uptake into MDA-MB-231 and MCF-7 cells was, respectively, 1.6 and 8.8 mM, whereas the V(max) was, respectively, 9.7 and 110.0 nmol/mg protein/10 min. AIB efflux from MDA-MB-231 and MCF-7 cells was trans-stimulated by BCH, L-glutamine, L-alanine, L-leucine, L-lysine and AIB (all at 2 mM). In contrast, L-glutamate, L-proline, L-arginine and MeAIB had no effect. The interaction between L-lysine and AIB efflux was one of low affinity. The fractional release of AIB from MDA-MB-231 cells was trans-accelerated by D-leucine and D-tryptophan but not by D-alanine. MDA-MB-231 and MCF-7 cells expressed LAT1 and CD98 mRNA. MCF-7 cells also expressed LAT2 mRNA. The results suggest that AIB transport in mammary cancer cells under Na(+)-free conditions is predominantly via system L which acts as an exchange mechanism. The differences in the kinetics of AIB transport between MDA-MB-231 and MCF-7 cells may be due to the differential expression of LAT2.     

Long-chain fatty acid esters of 5-androstene-3 beta, 17 beta-diol: composition and turnover in human mammary cancer cells in culture

Long-chain fatty acid esters of the adrenal-derived estrogen 5-androstene-3 beta, 17 beta-diol (ADIOL) were found to accumulate in four human mammary cancer cell lines (MCF-7, ZR-75-1, MDA-MB-231 and MDA-MB-330) when explosed to 10-30 nM ADIOL for variable time periods. At each time point examined, the monoester fraction, which represented the major component of the total lipoidal fraction, contained fatty acids linked to either the 3 beta- or 17 beta-positions. However, there was considerable variation in the ratio of 3 beta- to 17 beta-monoesters in the four cell lines. By means of reverse phase HPLC and referral to authentic synthesized compounds, each monoester fraction was found to contain a number of long-chain fatty acid components whose composition resembled that previously determined for the fatty acid esters formed from 17 beta-estradiol. A specific and measurable turnover of a subfraction of ADIOL-17 beta-monoesters composed of essential fatty acids (22:6, 20:4, 18:3) occurred in MCF-7 cells, and to a lesser extent in ZR-75-1 cells. No changes were observed with time in any of the components of the 3 beta- or 17 beta-monoester fractions in MDA-MB-231 and MDA-MB-330 cells. These results, coupled with other studies, now suggest that a very rapid turnover of some components of these lipoidal derivatives may be occurring. If so, it is possible that the system of acylation-deacylation may be involved in a transport mechanism for estrogens and perhaps other steroid hormones.     

Up-regulation of PI3K/Akt signaling by 17beta-estradiol through activation of estrogen receptor-alpha, but not estrogen receptor-beta, and stimulates cell growth in breast cancer cells

Estrogen stimulates cell proliferation in breast cancer. The biological effects of estrogen are mediated through two intracellular receptors, estrogen receptor-alpha (ERalpha) and estrogen receptor-beta (ERbeta). However, the role of ERs in the proliferative action of estrogen is not well established. Recently, it has been known that ER activates phosphatidylinositol-3-OH kinase (PI3K) through binding with the p85 regulatory subunit of PI3K. Therefore, possible mechanisms may include ER-mediated phosphoinositide metabolism with subsequent formation of phosphatidylinositol-3,4,5-trisphosphate (PIP(3)), which is generated from phosphatidylinositol 4,5-bisphosphate via PI3K activation. The present study demonstrates that 17beta-estradiol (E2) up-regulates PI3K in an ERalpha-dependent manner, but not ERbeta, and stimulates cell growth in breast cancer cells. In order to study this phenomenon, we have treated ERalpha-positive MCF-7 cells and ERalpha-negative MDA-MB-231 cells with 10nM E2. Treatment of MCF-7 cells with E2 resulted in a marked increase in PI3K (p85) expression, which paralleled an increase in phospho-Akt (Ser-473) and PIP(3) level. These observations also correlated with an increased activity to E2-induced cell proliferation. However, these effects of E2 on breast cancer cells were not observed in the MDA-MB-231 cell line, indicating that the E2-mediated up-regulation of PI3K/Akt pathway is ERalpha-dependent. These results suggest that estrogen activates PI3K/Akt signaling through ERalpha-dependent mechanism in MCF-7 cells.     

GPER mediated estradiol reduces miR-148a to promote HLA-G expression in breast cancer

Breast cancer is the most common malignant diseases in women. miR-148a plays an important role in regulation of cancer cell proliferation and cancer invasion and down-regulation of miR-148a has been reported in both estrogen receptor (ER) positive and triple-negative (TN) breast cancer. However, the regulation mechanism of miR-148a is unclear. The role of estrogen signaling, a signaling pathway is important in development and progression of breast cancer. Therefore, we speculated that E2 may regulate miR-148a through G-protein-coupled estrogen receptor-1 (GPER). To test our hypothesis, we checked the effects of E2 on miR-148a expression in ER positive breast cancer cell MCF-7 and TN cancer cell MDA-MB-231. Then we used GPER inhibitor G15 to investigate whether GPER is involved in regulation of E2 on miR-148a. Furthermore, we analyzed whether E2 affects the expression of HLA-G, which is a miR-148a target gene through GPER. The results showed that E2 induces the level of miR-148a in MCF-7 and MDA-MB-231 cells, GPER mediates the E2-induced increase in miR-148a expression in MCF-7 and MDA-MB-231 cells and E2-GPER regulates the expression of HLA-G by miR-148a. In conclusion, our findings offer important new insights into the ability of estrogenic GPER signaling to trigger HLA-G expression through inhibiting miR-148a that supports immune evasion in breast cancer.     

Effect of fetal bovine serum on erythropoietin receptor expression and viability of breast cancer cells

Erythropoietin receptors (EPORs) are present not only in erythrocyte precursors but also in non-hematopoietic cells including cancer cells. In this study, we determined the effect of fetal bovine serum (FBS) in culture medium on the EPOR expression and viability of the estrogen receptor (ER)-positive MCF-7 and ER-negative MDA-MB-231 breast cancer cells. Using flow cytometry, we showed that the inclusion of 10% FBS in the medium increased the EPOR expressions and viabilities of MDA-MB-231 and MCF-7 cells. The MDA-MB-231 showed greater EPOR expression than MCF-7 cells, suggesting that the presence of ERs on cells is associated with poor expression of EPOR. Culture medium containing 10% FBS also caused increased number of breast cancer cells entering the synthesis phase of the cell cycle. The study also showed that rHuEPO treatment did not affect viability of breast cancer cells. In conclusion, it was shown that the inclusion of FBS in culture medium increased expression of EPOR in breast cancer cells and rHuEPO treatment had no effect on the proliferation of these cancer cells.     

Microenvironment Promotes Tumor Cell Reprogramming in Human Breast Cancer Cell Lines

The microenvironment drives mammary gland development and function, and may influence significantly both malignant behavior and cell growth of mammary cancer cells. By restoring context, and forcing cells to properly interpret native signals from the microenvironment, the cancer cell aberrant behavior can be quelled, and organization re-established. In order to restore functional and morphological differentiation, human mammary MCF-7 and MDA-MB-231 cancer cells were allowed to grow in a culture medium filled with a 10% of the albumen (EW, Egg White) from unfertilized chicken egg. That unique microenvironment behaves akin a 3D culture and induces MCF-7 cells to produce acini and branching duct-like structures, distinctive of mammary gland differentiation. EW-treated MDA-MB-231 cells developed buds of acini and duct-like structures. Both MCF-7 and MDA-MB-231 cells produced β-casein, a key milk component. Furthermore, E-cadherin expression was reactivated in MDA-MB-231 cells, as a consequence of the increased cdh1 expression; meanwhile β-catenin – a key cytoskeleton component – was displaced behind the inner cell membrane. Such modification hinders the epithelial-mesenchymal transition in MDA-MB-231 cells. This differentiating pathway is supported by the contemporary down-regulation of canonical pluripotency markers (Klf4, Nanog). Given that egg-conditioned medium behaves as a 3D-medium, it is likely that cancer phenotype reversion could be ascribed to the changed interactions between cells and their microenvironment.     

Estrogen receptor alpha mediated induction of the transforming growth factor alpha gene by estradiol and 4-hydroxytamoxifen in MDA-MB-231 breast cancer cells

The selective estrogen receptor modulator, 4-hydroxytamoxifen (4-OHT) is a full agonist at the transforming growth factor (TGF) alpha gene in ER negative breast cancer cells stably transfected with ER alpha cDNA (Levenson et al., Br. J. Cancer 77 (1998) 1812-1819). E(2) and 4-OHT increase TGF alpha mRNA and protein in a concentration dependent manner. The responses to E(2) and 4-OHT are blocked by the pure antiestrogen ICI 182,780, which does not induce TGF alpha. Transfected MDA-MB-231 cells contain functional ER alpha but no ER beta function was detected. Neo transfected cells that did not express ER alpha or cells stably transfected with the DNA binding domain mutant C202R/E203V which prevents gene activation did not induce TGF alpha mRNA after either E(2) or 4-OHT treatment. An examination of the time course for either 10 nM E(2) or 1 microM 4-OHT for MDA-MB-231 cells stably transfected with cDNA for ER alpha showed increases in TGF alpha mRNA within 2 or 3 h respectively. Cells pretreated with cycloheximide (1 microg/ml) showed induced TGF alpha mRNA in response to E(2) or 4-OHT but TGF alpha mRNA induction was blocked by actinomycin D (1 microg/ml). We conclude that both E(2) and 4-OHT induce TGF alpha by direct interaction of ER alpha with DNA and that ER beta is not involved in the estrogen-like response to 4-OHT in the MDA-MB-231 cells.     

Estrogen down-regulates uncoupling proteins and increases oxidative stress in breast cancer

Oxidative stress has been postulated as one of the mechanisms underlying the estrogen carcinogenic effect in breast cancer. Estrogens are known to increase mitochondrial-derived reactive oxygen species (ROS) by an unknown mechanism. Given that uncoupling proteins (UCPs) are key regulators of mitochondrial energy efficiency and ROS production, our aim was to check the presence and activity of UCPs in estrogen receptor (ER)-positive and ER-negative breast cancer cells and tumors, as well as their relation to oxidative stress. Estrogen (1 nM) induced higher oxidative stress in the ER-positive MCF-7 cell line, showing increased mitochondrial membrane potential, H₂O₂ levels, and DNA and protein damage compared to ER-negative MDA-MB-231 cells. All isoforms of uncoupling proteins were highly expressed in ER-positive breast cancer cells and tumors compared to negative ones. ROS production in mitochondria isolated from MCF-7 was increased by inhibition of UCPs with GDP, but not in mitochondria from MDA-MB-231. Estrogen treatment decreased uncoupling protein and catalase levels in MCF-7 and decreased GDP-dependent ROS production in isolated mitochondria. On the whole, these results suggest that estrogens, through an ER-dependent mechanism, may increase mitochondrial ROS production by repressing uncoupling proteins, which offers a new perspective on the understanding of why estrogens are a risk factor for breast cancer.     

A novel gene STYK1/NOK is upregulated in estrogen receptor-alpha negative estrogen receptor-beta positive breast cancer cells following estrogen treatment

The human STYK1/NOK protein is approximately 30–35% similar to mouse fibroblast growth factor receptor 3 and a kinase homologue in D. melanogaster in the tyrosine protein kinase region. STYK1/NOK was identified as being up regulated in MDA-MB-231, an estrogen receptor-alpha negative breast cancer cell line, following 12 h of estrogen treatment at 1 × 10⁻⁹ M. On further investigation of STYK1/NOK in estrogen treated cell line MDA-MB-231, STYK1/NOK was up regulated at 6 h post treatment when compared to untreated cells. We also investigated the expression levels of STYK1/NOK in other breast cancer cell lines MCF-7, MDA-MB-231, BT-549, and MDA-MB-435S using QRT-PCR. In addition, the analysis of message accumulation was increased with other synthetic estrogen response modifiers. We propose that the regulation of STYK1/NOK is achieved independent of ERα and suggests further investigation to the relevance of this kinase in breast cancer progression.     

Placental Kisspeptins Differentially Modulate Vital Parameters of Estrogen Receptor-Positive and -Negative Breast Cancer Cells

Kisspeptins (KPs) are major regulators of trophoblast and cancer invasion. Thus far, limited and conflicting data are available on KP-mediated modulation of breast cancer (BC) metastasis; mostly based on synthetic KP-10, the most active fragment of KP. Here, we report for the first time comprehensive functional effects of term placental KPs on proliferation, adhesion, Matrigel invasion, motility, MMP activity and pro-inflammatory cytokine production in MDA-MB-231 (estrogen receptor-negative) and MCF-7 (estrogen receptor-positive). KPs were expressed at high level by term placental syncytiotrophoblasts and released in soluble form. Placental explant conditioned medium containing KPs (CM) significantly reduced proliferation of both cell types compared to CM without (w/o) KP (CM-w/o KP) in a dose- and time-dependent manner. In MDA-MB-231 cells, placental KPs significantly reduced adhesive properties, while increased MMP9 and MMP2 activity and stimulated invasion. Increased invasiveness of MDA-MB-231 cells after CM treatment was inhibited by KP receptor antagonist, P-234. CM significantly reduced motility of MCF-7 cells at all time points (2–30 hr), while it stimulated motility of MDA-MB-231 cells. These effects were reversed by P-234. Co-treatment with selective ER modulators, Tamoxifen and Raloxifene, inhibited the effect of CM on motility of MCF-7 cells. The level of IL-6 in supernatant of MCF-7 cells treated with CM was higher compared to those treated with CM-w/o KP. Both cell types produced more IL-8 after treatment with CM compared to those treated with CM-w/o KP. Taken together, our observations suggest that placental KPs differentially modulate vital parameters of estrogen receptor-positive and -negative BC cells possibly through modulation of pro-inflammatory cytokine production.