Estrogen Receptor-negative Breast Cancer Cells Research Articles

Retracted Manuscript: The WNT-5a derived peptide, Foxy-5, possesses dual properties that impair progression of ERα negative breast cancer

Retraction for: “The WNT-5a derived peptide, Foxy-5, possesses dual properties that impair progression of ERa negative breast cancer,” by Caroline E. Ford, Elin J. Ekström, Jillian Howlin and Tommy Andersson, which appeared in the June 15, 2009 issue of Cell Cycle (Ford CE, et al. Cell Cycle 2009; 8:1838-1842; 10.4161/cc.8.12.8863). The authors wish to note the following: “Recently a paper, on which I was the senior author and that was published in the Proceedings of the National Academy of Sciences titled “Wnt-5a signaling restores tamoxifen sensitivity in estrogen receptor-negative breast cancer cells” (Ford CE, Ekström EJ, Andersson T. Proc Natl Acad Sci USA 2009; 106:3919-24) was retracted. The fact that this paper was the direct reason for our review article in the Cell Cycle journal makes it logical that I also retract the cited review article published in the Cell Cycle journal, the other authors approve this retraction. We apologize for any inconvenience this may have caused.”

The G Protein-coupled Receptor 30 Is Up-regulated by Hypoxia-inducible Factor-1α (HIF-1α) in Breast Cancer Cells and Cardiomyocytes

GPR30, also known as GPER, has been suggested to mediate rapid effects induced by estrogens in diverse normal and cancer tissues. Hypoxia is a common feature of solid tumors involved in apoptosis, cell survival, and proliferation. The response to low oxygen environment is mainly mediated by the hypoxia-inducible factor named HIF-1α, which activates signaling pathways leading to adaptive mechanisms in tumor cells. Here, we demonstrate that the hypoxia induces HIF-1α expression, which in turn mediates the up-regulation of GPER and its downstream target CTGF in estrogen receptor-negative SkBr3 breast cancer cells and in HL-1 cardiomyocytes. Moreover, we show that HIF-1α-responsive elements located within the promoter region of GPER are involved in hypoxia-dependent transcription of GPER, which requires the ROS-induced activation of EGFR/ERK signaling in both SkBr3 and HL-1 and cells. Interestingly, the apoptotic response to hypoxia was prevented by estrogens through GPER in SkBr3 cells. Taken together, our data suggest that the hypoxia-induced expression of GPER may be included among the mechanisms involved in the anti-apoptotic effects elicited by estrogens, particularly in a low oxygen microenvironment.

Shift in cytotoxic target from estrogen receptor-positive to estrogen receptor-negative breast cancer cells by trastuzumab in combination with taxane-based chemotherapy

Trastuzumab has shown significant clinical benefits in patients with operable and metastatic HER2-positive breast cancer. However, the biological mechanism of the additional effect of trastuzumab administered in combination with conventional chemotherapy is poorly understood. We performed a retrospective analysis of 55 patients with HER2-positive breast cancer treated with anthracycline and taxane (chemotherapy alone; CT), or trastuzumab in combination with taxane-based chemotherapy (CT+T) for neoadjuvant chemotherapy. We determined the therapeutic efficacies [clinical (CR) and pathological complete responses (pCR)] and changes in the proportion of positive cells for each biomarker pre- to post-neoadjuvant chemotherapy for each treatment regimen. Clinical-CR and quasi-pCR rates defined as the absence of invasive tumors or only a few remaining invasive tumor cells were 6.9 and 31.0% in the CT group and 46.2 and 65.4% in the CT+T group, respectively. In the CT group, the proportion of estrogen receptor (ER)-/progesterone receptor (PgR)-positive cells decreased significantly following treatment (ER, 73.5 vs. 50.9%; P=0.02). Changes in the proportion of ER-/PgR-positive cells were not noted in the CT+T group (ER, 81.9 vs. 80.3%; P=0.61), although a relatively greater decrease in the proportion of Ki-67-positive cells was found in the CT+T group than that in the CT group (-26.5 vs. -13.7%). These findings indicate that CT+T inhibits ER-negative and Ki-67-positive breast cancer cells. In conclusion, trastuzumab sensitized ER-negative proliferative cells to cytotoxic chemotherapy. This finding may indicate an additional clinical effect of trastuzumab when administered in combination with conventional chemotherapy as neoadjuvant chemotherapy for HER2-positive breast cancer.

Preparation and Evaluation of Carborane Analogues of Tamoxifen

A stereoselective synthesis of closo carborane analogues of tamoxifen was developed where the products represent a new approach to developing metabolically robust SERMs. The A-ring found in the backbone of tamoxifen was replaced with an ortho carborane cluster; the product was determined to be the desired Z isomer, which showed superior chemical stability to tamoxifen both in solution and in the solid state. By use of microwave heating, it was possible to convert some of the Z carborane tamoxifen analogue to the corresponding E isomer. Cell growth assays using both isomers and a carborane that is known to target the ER were conducted using estrogen receptor (ER) positive and ER negative human breast cancer cells with and without the presence of estradiol (E2). The Z carborane isomer was able to inhibit cell proliferation better than tamoxifen in an E2 free environment, while the E isomer inhibited cell growth better than tamoxifen when E2 was present.

Estradiol-dependent regulation of angiopoietin expression in breast cancer cells

Angiopoietin-1 (Ang-1) is a ligand for Tie-2 receptors and a promoter of angiogenesis. Angiogenesis plays an important role in breast cancer, as it is one of the critical events required for tumors to grow and metastasize. In this study, we investigated the influence of estradiol (E2) on the expression of angiopoietins in breast cancer cell lines. Ang-1 mRNA and protein expressions were significantly higher in estrogen receptor-negative (ERα−) breast cancer cells than in estrogen receptor-positive (ERα+) cells. Exposure of ERα+ cells to E2 resulted in further reductions of Ang-1 levels. In mouse mammary pads inoculated with breast cancer cells, both tumor size and Ang-1 production were significantly lower in ERα+ cell-derived xenografts, as compared to those derived from ERα− cells. Reduction of circulating levels of E2 by ovariectomy eliminated this response. Overall, these results indicate that Ang-1 mRNA and protein expressions: (1) negatively correlate with the level of ERα in breast cancer cell lines; (2) are downregulated by E2 in an ERα dependent manner; and (3) positively correlate with the degree of angiogenesis in vivo. We conclude that Ang-1 is an important modulator of growth and progression of ERα− breast cancers.

Troglitazone suppresses telomerase activity independently of PPARγ in estrogen-receptor negative breast cancer cells

BackgroundBreast cancer is one the highest causes of female cancer death worldwide. Many standard chemotherapeutic agents currently used to treat breast cancer are relatively non-specific and act on all rapidly dividing cells. In recent years, more specific targeted therapies have been introduced. It is known that telomerase is active in over 90% of breast cancer tumors but inactive in adjacent normal tissues. The prevalence of active telomerase in breast cancer patients makes telomerase an attractive therapeutic target. Recent evidence suggests that telomerase activity can be suppressed by peroxisome proliferator activated receptor gamma (PPARγ). However, its effect on telomerase regulation in breast cancer has not been investigated.MethodsIn this study, we investigated the effect of the PPARγ ligand, troglitazone, on telomerase activity in the MDA-MB-231 breast cancer cell line. Real time RT-PCR and telomerase activity assays were used to evaluate the effect of troglitazone. MDA-MB-231 cells had PPARγ expression silenced using shRNA interference.ResultsWe demonstrated that troglitazone reduced the mRNA expression of hTERT and telomerase activity in the MDA-MB-231 breast cancer cell line. Troglitazone reduced telomerase activity even in the absence of PPARγ. In agreement with this result, we found no correlation between PPARγ and hTERT mRNA transcript levels in breast cancer patients. Statistical significance was determined using Pearson correlation and the paired Student's t test.ConclusionsTo our knowledge, this is the first time that the effect of troglitazone on telomerase activity in breast cancer cells has been investigated. Our data suggest that troglitazone may be used as an anti-telomerase agent; however, the mechanism underlying this inhibitory effect remains to be determined.

Role and mechanism of hypoxia-inducible factor-1 in cell growth and apoptosis of breast cancer cell line MDA-MB-231

The role of hypoxia-inducible factor-1 (HIF-1) in tumor development and progression is well-established but its effect on tumor growth remains controversial. The present study investigated the effect of HIF-1 on tumor growth using the estrogen receptor-negative breast cancer cell line, MDA-MB-231. Using Western blotting, we detected a higher level of HIF-1α protein in MDA-MB-231 cells than in any other breast cancer cell lines analyzed, and this was accompanied by a more rapid growth pattern. Interruption of HIF-1α expression using small interference RNA (siRNA) significantly suppressed cell growth and increased apoptosis, but the cell cycle was not affected. Activated fragments with increased caspase 3 activity and a mobility shift of B cell lymphoma (Bcl-2) were also detected in cells treated with HIF-1α siRNA. HIF-1 allows breast cancer cells to grow under long-term serum deprivation by inactivation of the caspase cascade and thus inhibition of apoptosis. Blocking HIF-1α protein resulted in loss of Bcl-2 function, which may contribute to the activation of the caspase cascade.

Epigenetic Repression of PDZ-LIM Domain-containing Protein 2: IMPLICATIONS FOR THE BIOLOGY AND TREATMENT OF BREAST CANCER

The NF-kappaB transcription factor plays a pivotal role in breast cancer progression and therapy resistance. However, the mechanisms by which the tightly regulated NF-kappaB becomes constitutively activated during breast cancer pathogenesis remain obscure. Here, we report that PDZ-LIM domain-containing protein 2 (PDLIM2), an essential terminator of NF-kappaB activation, is repressed in both estrogen receptor-positive and estrogen receptor-negative breast cancer cells, suggesting one important mechanism for the constitutive activation of NF-kappaB. Indeed, PDLIM2 reexpression inhibited constitutive NF-kappaB activation and expression of NF-kappaB-targeted genes in those breast cancer cells. Importantly, PDLIM2, but not its mutants defective in NF-kappaB termination, could suppress in vitro anchorage-independent growth and in vivo tumor formation of those malignant breast cells. In addition, we have shown that PDLIM2 repression involves promoter methylation. Accordingly, treatment of the breast cancer cells with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine reverses the methylation of the PDLIM2 promoter, restored PDLIM2 expression, and suppressed tumorigenicities of human breast cancer cells both in vitro and in vivo. These studies thus provide important mechanistic insights into breast cancer pathogenesis. These studies also suggest a tumor suppression function of PDLIM2 and a therapeutic strategy for breast cancer.

Genistein inhibits osteolytic bone metastasis and enhances bone mineral in nude mice

In this study, the effective activity of genistein on osteolytic bone metastasis and bone mineral was investigated. Female BALB/c-nu/nu mice were injected with estrogen receptor-negative human breast cancer cells, MDA-MB-231, into left cardiac ventricle to form osteolytic bone metastases, and administered genistein subcutaneously after radiologically small but defined osteolytic metastases had been observed (protocol 1), simultaneously with cancer cells inoculation (protocol 2) and prophylactically 7 days before inoculation of cancer cells (protocol 3). In all protocols, genistein (10 mg/kg/day) markedly reduced the number and volume of osteolytic bone metastases assessed by radiography and the number of osteoclasts. Furthermore, histomorphometrical analysis revealed that genistein markedly increased trabecular area (Tb.Ar%), trabecular thickness (Tb.Th) and trabecular number (Tb.N), and decreased trabecular separation (Tb.Sp). These results thus demonstrate that genistein could inhibit osteolytic bone metastases, suppress bone resorption, increase bone mass and improve bone microstructure in bone metastases of breast cancer.

Estriol acts as a GPR30 antagonist in estrogen receptor-negative breast cancer cells

Estrogens are structurally related steroids that regulate important physiological processes. 17β-estradiol (E2) is reversibly oxidized to estrone (E1) and both E2 and E1 can be irreversibly converted to estriol (E3), which also originates directly from androstenedione. The action of E2 has been traditionally explained by the binding to the estrogen receptor (ER) α and ERβ, however the G protein-coupled receptor (GPR) 30 has been recently involved in the rapid signaling triggered by estrogens. Although the role of E2 in the development of breast cancer has been largely documented, the contribution of E3 still remains to be completely evaluated. Here, we demonstrate for the first time that E3 acts as a GPR30 antagonist since it was able to inhibit the GPR30-mediated responses such as the rapid ERK activation, the up-regulation of target genes like c-fos and connective tissue growth factor, the proliferative effects observed in ER-negative SkBr3 cells.

P130Cas substrate domain signaling promotes migration, invasion, and survival of estrogen receptor-negative breast cancer cells

Elevated Src tyrosine kinase activity is commonly observed in breast cancer and likely contributes to neoplasia and malignancy. p130Cas ("Crk-associated substrate") is a major Src substrate found at the sites where integrins mediate cell adhesion to the extracellular matrix. Src phosphorylates multiple tyrosines in the p130Cas "substrate domain" (SD) and this signaling event has been implicated in the promotion of cell motility, primarily from studies on fibroblasts. In breast cancer, studies on p130Cas have focused on its role in conferring antiestrogen resistance to cells that express the estrogen receptor (ER+). However, little is known regarding the role of p130Cas in the more aggressive estrogen receptor negative (ER-) breast cancers for which there is a need for development of effective targeted therapies. We found high levels of p130Cas SD tyrosine phosphorylation to be a common characteristic of ER- breast cancer cell lines, with particularly high levels observed for the BT-549 cell line. Using RNA interference to knock down p130Cas expression in BT-549 cells, combined with rescue by WT p130Cas versus a signaling-deficient control, we provide evidence that p130Cas SD tyrosine phosphorylation is an important signaling event in the migration, invasion, proliferation, and survival of this ER-breast cancer cell line.

Modification of survival pathway gene expression in human breast cancer cells by tetraiodothyroacetic acid (tetrac)

Tetraiodothyroacetic acid (tetrac) inhibits the cellular actions of thyroid hormone initiated at the hormone receptor on plasma membrane integrin αvβ3. Via interaction with the integrin, tetrac is also capable of inhibiting the angiogenic effects of vascular endothelial growth factor and basic fibroblast growth factor. MDA-MB-231 cells are estrogen receptor-negative human breast cancer cells shown to be responsive to tetrac in terms of decreased cell proliferation. Here we describe actions initiated at the cell surface receptor by unmodified tetrac and nanoparticulate tetrac on a panel of survival pathway genes in estrogen receptor-negative human breast cancer (MDA-MB-231) cells. Nanoparticulate tetrac is excluded from the cell interior. Expression of apoptosis inhibitors XIAP (X-linked inhibitor of apoptosis) and MCL1 (myeloid cell leukemia sequence 1) was downregulated by nanoparticulate tetrac in these breast cancer cells whereas apoptosis-promoting CASP2 and BCL2L14 were upregulated by the nanoparticulate formulation. Unmodified tetrac affected only XIAP expression. Expression of the angiogenesis inhibitor thrombospondin 1 (THBS1) gene was increased by both formulations of tetrac, as was the expression of CBY1, a nuclear inhibitor of catenin activity. The majority of differentially regulated Ras-oncogene family members were downregulated by nanoparticulate tetrac. The latter downregulated expression of epidermal growth factor receptor gene and unmodified tetrac did not. Nanoparticulate tetrac has coherent anti-cancer actions on expression of differentially-regulated genes important to survival of MDA-MB-231 cells.

EGFR over-expression and activation in high HER2, ER negative breast cancer cell line induces trastuzumab resistance

HER2 is gene amplified or over-expressed in 20-25% of breast cancers resulting in elevated HER2 activation. Trastuzumab (Herceptin), a humanized monoclonal antibody, targets activated HER2 and is clinically effective in HER2-over-expressing breast cancers. However, despite prolonged survival, treated breast cancer patients develop resistance. Resistance to trastuzumab occurs upon inactivation of HER2 regulatory proteins or upon up-regulation of alternative receptors. In particular, elevated levels of EGFR, present in estrogen receptor (ER) positive, trastuzumab-resistant BT-474 xenografts caused, a trastuzumab-resistant phenotype (Ritter et al. Clin Cancer Res 13:4909-4919, 2007). However, the role of EGFR in acquired trastuzumab resistance in ER negative cell models is not well defined. In this study, SKBR3 cell line clones expressing EGFR were generated to examine the role of EGFR over-expression on trastuzumab sensitivity in an, ER-negative breast carcinoma cell line. A stable clone, SKBR3/EGFR (clone 4) expressing moderate levels of EGFR remained sensitive to trastuzumab, whereas a stable clone, SKBR3/EGFR (clone 5) expressing high levels of EGFR, became resistant to trastuzumab. Depletion of EGFR by EGFR small-interfering RNAs in the SKBR3/EGFR (clone 5) reversed trastuzumab resistance. However, the SKBR3/EGFR (clone 5) cell line remained sensitive to lapatinib, an EGFR/HER2 inhibitor. Biochemical analysis using co-immunoprecipitation and proximity-based quantitative VeraTag assays demonstrated that high levels of EGFR phosphorylation, EGFR/EGFR homo-dimerization, and EGFR/HER2 hetero-dimerization were present in the trastuzumab-resistant cells. We conclude that EGFR over-expression can mediate trastuzumab resistance in both ER positive and ER negative cells and hypothesize that a threshold level of EGFR, in the absence of autocrine ligand production, is required to induce the resistant phenotype.

Pro-survival of estrogen receptor-negative breast cancer cells is regulated by a BLT2–reactive oxygen species-linked signaling pathway

Leukotriene B4 (LTB4) is an inflammatory mediator with potent biological activities in the pathogenesis of many inflammatory diseases. In the present study, we found that expression of BLT2, a low-affinity LTB4 receptor, is significantly upregulated in breast cancer cells. In addition, we observed that inhibition of BLT2 by a specific antagonist, LY255283, or by siBLT2 RNA interference caused dramatic apoptotic cell death in breast cancer cells, especially in the estrogen receptor (ER)-negative MDA-MB-468 and MDA-MB-453 cells, suggesting a role for BLT2 in survival of these breast cancer cells. In an approach to understand the downstream mechanism by which BLT2 mediates the potential pro-survival signaling, we found that the elevated reactive oxygen species (ROS) generation is associated with BLT2-mediated survival. Expression of Nox1, a member of the NADPH oxidase family, is also highly upregulated in a BLT2-dependent manner in these breast cancer cells, suggesting that 'Nox1-derived ROS' lie downstream of BLT2. Consistent with the proposed role of 'Nox1-ROS' in pro-survival signaling, knockdown of Nox1 with siNox1 or treatment with a ROS scavenging agent caused dramatic apoptotic death in these breast cancer cells. Taken together, our results demonstrate, for the first time, that the 'BLT2-Nox1-ROS'-linked cascade is involved in the pro-survival signaling, especially in ER-negative breast cancer cells.

Isolation and characterization of a highly metastatic hybrid cell line generated between estrogen receptor (ER) negative and ER positive breast cancer cells in mouse bone marrow.

Abstract Abstract #2053 Background: Breast cancer cells in a patient are heterogeneous. How the presence of these heterogeneous cancer cells may contribute to cancer progression is not clear. Experimental evidence indicates that soluble factors produced by aggressive cancer cells may generate metastasis niches for less aggressive cancer cells. In this study, we sought to determine how a growing orthotopic tumor formed by an aggressive ER negative breast cancer cell line might affect the metastatic potential of a less aggressive ER positive breast cancer cell line.
 Methods and Results: Five-week-old female nude mice were injected orthotopically with metastasis-competent ER negative human breast cancer MDA-MB-231/GFP/Neo cells. After three weeks, metastasis-incompetent human breast cancer ER positive ZR-75-1/GFP/puro cells were inoculated into these mice via intra-cardiac (IC) route and puromycin resistant metastatic ZR-75-1/GFP/puro cells were flushed out of the bone marrow of one mouse and established as a variant cell line called B6. B6 cells have both estrogen-dependent and -independent cells when they were orthotopically injected into female nude mice with or without estrogen supplementation. The cells isolated from an estrogen-dependent tumor were called B6TE and the cells isolated from an estrogen-independent tumor were called B6TC. Further characterization revealed that B6TC cell was more tumorigenic without estrogen supplementation and has a unique metastatic property. More interestingly, B6TC cell was resistant to both puromycin and G418 suggesting it was derived from the fusion of MDA-MB-231/GFP/Neo and ZR-75-1/GFP/puro in the mouse bone marrow. When compared with parental MDA-MB-231/GFP/Neo, the B6TC cells were found to be more metastatic to lung and bone after IC inoculation as detected by GFP imaging, with significant development of paraplegia in the hind limbs after four weeks. More significantly, B6TC mice also developed metastases to brain, which was not observed in the MDA-MB-231/GFP/Neo group. B6TC cell has a low expression of ER alpha and CD24, and high expression of p-ERK, CD44, ALDH. It also expresses vimentin, CXCR4 and Integrin-β1. Its growth is refractory to the anti-estrogens, raloxifene and tamoxifen.
 Conclusion: Our study indicates that B6 cells generated in the bone marrow were a heterogeneous population of ER positive and ER negative cells with marked differences in their proliferative potential and malignancy. The low ER-expressing B6TC is a novel hybrid cell line generated spontaneously in a metastatic site, which has propensity to metastasize to brain in addition to lung and bone. The expression of CD44 and ALDH indicates that it has stem cell-like features. Further characterization and molecular profiling is underway to reveal the novel biomarkers. This cell line should be a useful model for the investigation of the molecular mechanism of brain metastasis and the therapeutic strategies for the treatment of metastatic breast cancer resistant to anti-estrogens. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2053.

Timing is everything: Order of administration of 5-aza 2′ deoxycytidine, trichostatin A and tamoxifen changes estrogen receptor mRNA expression and cell sensitivity

Restoration of estrogen receptor (ER) expression using epigenetic inhibitors re-establishes expression of the estrogen receptor (ER) and restores tamoxifen sensitivity in ER negative breast cancer cells. We tested if order of administration of the DNMT (5-aza 2′ deoxycytidine/AZA) or HDAC (trichostatin A/TSA) inhibitors and tamoxifen affected ER re-expression and tamoxifen sensitivity. Treatment with AZA followed by co-administration of TSA plus tamoxifen resulted in the greatest ER re-expression and tamoxifen sensitivity, although sensitivity was not increased as robustly as expected. This could be due to increased cytoplasmic levels of HuR, suggesting that cytoplasmic HuR levels are central to tamoxifen responsiveness.

Cadmium regulation of apoptotic and stress response genes in tumoral and immortalized epithelial cells of the human breast

Cadmium (Cd) is a widely-disseminated metal which can be imported and accumulated in living cells thereby drastically interfering with their biological mechanisms. Increasing interest has been recently focused on the elucidation of the cellular and molecular aspects of Cd-dependent regulation of gene expression and signal transduction pathways in different model system. Concerning breast cancer, very limited studies have been produced so far on the role played by Cd on estrogen receptor-negative human breast cancer cells, that are expected to be insensitive to the already-proven metallo-estrogenic effect exerted by Cd on the estrogen receptor-positive cell counterparts. Here, we have examined the effects of long-term (96 h) exposure of estrogen receptor-negative MDA-MB231 malignant adenocarcinoma cells to CdCl 2 at 5 μM concentration, corresponding to the IC 50 for this time of incubation, by evaluating the expression levels of genes coding for stress response factors (e.g. heat shock proteins and metallothioneins), and for apoptosis-related factors and enzymes. In parallel, we tested the gene expression pattern of immortalized HB2 breast epithelial cells, taken as non-tumoral counterpart, after the same exposure to the metal which instead did not exert any change in their cell number with respect to controls. Our cumulative results indicate that, whilst HB2 cells appear to activate defense mechanisms against metal stress principally via metallothionein massive up-regulation and appearance of the spliced form of XBP-1 message, MDA-MB231 cells seem to couple the onset of a protective reaction (e.g. up-regulation of hsp27 and metallothioneins) to the switching-on of new intracellular pathways directing cells to a kind of death which shares several aspects with the apoptotic program, such as down-regulation of Bcl-2 and over-expression of Dap kinase and several caspases.

CCAAT/Enhancer Binding Protein δ Up-regulates Aromatase Promoters I.3/II in Breast Cancer Epithelial Cells

Aromatase is the enzyme responsible for the last step of estrogen synthesis. The female hormone, estrogen, is known to stimulate breast cancer cell growth. Because the expression of aromatase in breast cancer tissues is driven by unique promoters I.3 and II, a more complete understanding of the regulatory mechanism of aromatase expression through promoters I.3/II in breast tumors should be valuable in developing targeted therapies, which selectively suppress estrogen production in breast tumor tissue. Results from in vivo footprinting analyses revealed several protein binding sites, numbered 1 to 5. When site 2 (-124/-112 bp, exon I.3 start site as +1) was mutated, promoters I.3/II activity was dramatically reduced, suggesting that site 2 is a positive regulatory element. Yeast one-hybrid screening revealed that a potential protein binding to site 2 was CCAAT/enhancer binding protein delta (C/EBP delta). C/EBP delta was shown to bind to site 2 of aromatase promoters I.3/II in vitro and in vivo. C/EBP delta up-regulated promoters I.3/II activity through this site and, as a result, it also up-regulated aromatase transcription and enzymatic activity. p65, a subunit of nuclear factor-kappaB (NF-kappaB) transcription factor, inhibited C/EBP delta-up-regulated aromatase promoters I.3/II and enzymatic activity. This inhibitory effect of p65 was mediated, in part, through prevention of the C/EBP delta binding to site 2. This C/EBP delta binding site in aromatase promoters I.3/II seems to act as a positive regulatory element in non-p65-overexpressing breast cancer epithelial cells, whereas it is possibly inactive in p65 overexpressing cancer epithelial cells, such as estrogen receptor-negative breast cancer cells.

Sensitizing estrogen receptor–negative breast cancer cells to tamoxifen with OSU-03012, a novel celecoxib-derived phosphoinositide-dependent protein kinase-1/Akt signaling inhibitor

Tamoxifen is a mainstay in the treatment of estrogen receptor (ER)-positive breast cancer patients. Although the efficacy of tamoxifen has been attributed to induction of tumor cell growth arrest and apoptosis by inhibition of ER signaling, recent evidence indicates that tamoxifen possesses ER-independent antitumor activities. Here, we use OSU-03012, a small-molecule inhibitor of phosphoinositide-dependent protein kinase-1 (PDK-1) to address the hypothesis that PDK-1/Akt signaling represents a therapeutically relevant target to sensitize ER-negative breast cancer to tamoxifen. OSU-03012 sensitized both ER-positive MCF-7 and ER-negative MDA-MB-231 cells to the antiproliferative effects of tamoxifen in an ER-independent manner. Flow cytometric analysis of phosphatidylserine externalization revealed that this augmented suppression of cell viability was attributable to a marked enhancement of tamoxifen-induced apoptosis by OSU-03012. Mechanistically, this OSU-03012-mediated sensitization was associated with suppression of a transient tamoxifen-induced elevation of Akt phosphorylation and enhanced modulation of the functional status of multiple Akt downstream effectors, including FOXO3a, GSK3alpha/beta, and p27. The growth of established MDA-MB-231 tumor xenografts was suppressed by 50% after oral treatment with the combination of tamoxifen (60 mg/kg) and OSU-03012 (100 mg/kg), whereas OSU-03012 and tamoxifen alone suppressed growth by 30% and 0%, respectively. These findings indicate that the inhibition of PDK-1/Akt signaling to sensitize ER-negative breast cancer cells to the ER-independent antitumor activities of tamoxifen represents a feasible approach to extending the use of tamoxifen to a broader population of breast cancer patients. Considering the urgent need for novel therapeutic strategies for ER-negative breast cancer patients, this combinatorial approach is worthy of continued investigation.

The Effects of betulinic acid on microRNA‐27a regulated target genes in MDA‐MB‐231 breast cancer cells

Betulinic acid (BA), a pentacyclic triterpene isolated from tree bark is cytotoxic to cancer cells and tumors by inhibiting their growth and inducing apoptosis. There is evidence that specificity proteins (Sps), such as Sp1, Sp3 and Sp4, are overexpressed in tumors and contribute to the proliferative and angiogenic phenotype associated with cancer cells. This objective of this study was to determine the efficacy of BA in decreasing Sps expression and underlying mechanisms. Our results show that BA decreased proliferation of estrogen‐receptor negative breast cancer cell line MDA‐MB‐231 within a concentration‐range of 2.5–10 μmMol/L. BA also decreased Sp1, Sp3, and Sp4 expression and this was accompanied by increased zinc finger ZBTB10 gene expression, a putative Sp‐repressor (ZBTB10) and decreased microRNA‐27a levels, a microRNA involved in the regulation of ZBTB10. Similar results were observed in MDA‐MB‐231 cells transfected with antisense miR‐27a and ZBTB10 expression plasmid. Both as‐miR‐27a and ZBTB10 overexpression decreased the percentage of cells in S phase of the cell cycle; however, ZBTB10 increased the percentage of cells in G0/G1, whereas as‐miR‐27a increased the percentage in G2/M. This latter response was associated with induction of Myt‐1 (a miR‐27a target gene) which causes inhibition in G2/M by phosphorylation of cdc2. The effects to BA can be reversed by transient transfection with a mimic of miR‐27a. These results show that part of the underlying mechanism of the anti‐cancer effect of BA is based on a decreased miR‐27a, accompanied by increased expression of Myt‐1 and ZBTB10 resulting in ZBTB10‐dependent suppression of Sp proteins and dependent genes.