Receptor-negative Breast Cancer Cell Lines Research Articles

Effect of combining CBD with standard breast cancer therapeutics

Breast cancer is the most common malignancy in women worldwide. Sixty-five percent of breast cancers are estrogen and/or progesterone receptor positive. Estrogen receptor expression is a prognostic and predictive biomarker of response to endocrine therapy, which consists of the selective estrogen receptor modulator tamoxifen, aromatase inhibitors, and the selective estrogen receptor degrader fulvestrant. Cannabidiol is a phytocannabinoid that is emerging as a potential therapeutic agent. The aim of this study was to investigate the effect of cannabidiol on estrogen receptor-positive and estrogen receptor-negative representative breast cancer cell lines in combination with standard therapeutic agents used in clinical practice. To compare the effects of cannabidiol on breast cancer cell viability, cancer cell lines were exposed to increasing concentrations of cannabidiol. The effects of cannabidiol in combination with the endocrine therapeutics tamoxifen, fulvestrant, and the cyclin-dependent kinase inhibitor palbociclib on breast cancer cell viability were examined. We demonstrated that cannabidiol dose-dependently decreased the viability of all breast cancer cell lines independent of estrogen receptor expression. The addition of cannabidiol to tamoxifen had an additive negative effect on cell viability in ER+ ​in estrogen receptor positive T-47D line. Cannabidiol did not attenuate the effect of standard treatment of hormone receptor-positive breast cancer with fulvestrant and palbociclib. In addition, cannabidiol did not attenuate the effect of standard treatment of triple-negative breast cancer and human epidermal growth factor receptor 2 positive breast cancer cell lines with trastuzumab and cisplatin.

Sevoflurane modulates breast cancer cell survival via modulation of intracellular calcium homeostasis

BackgroundSome retrospective and in vitro studies suggest that general anesthetics influence breast cancer recurrence and metastasis. We compared the effects of general anesthetics sevoflurane versus propofol on breast cancer cell survival, proliferation and invasion in vitro. The investigation focused on effects in intracellular Ca2+ homeostasis as a mechanism for general anesthetic-mediated effects on breast cancer cell survival and metastasis.MethodsEstrogen receptor-positive (MCF7) and estrogen receptor-negative (MDA-MB-436) human breast cancer cell lines along with normal breast tissue (MCF10A) were used. Cells were exposed to sevoflurane or propofol at clinically relevant and extreme doses and durations for dose- and time-dependence studies. Cell survival, proliferation and migration following anesthetic exposure were assessed. Intracellular and extracellular Ca2+ concentrations were modulated using Ca2+ chelation and a TRPV1 Ca2+ channel antagonist to examine the role of Ca2+ in mediating anesthetic effects.ResultsSevoflurane affected breast cancer cell survival in dose-, time- and cell type-dependent manners. Sevoflurane, but not propofol, at equipotent and clinically relevant doses (2% vs. 2 μM) for 6 h significantly promoted breast cell survival in all three types of cells. Paradoxically, extreme exposure to sevoflurane (4%, 24 h) decreased survival in all three cell lines. Chelation of cytosolic Ca2+ dramatically decreased cell survival in both breast cancer lines but not control cells. Inhibition of TRPV1 receptors significantly reduced cell survival in all cell types, an effect that was partially reversed by equipotent sevoflurane but not propofol. Six-hour exposure to sevoflurane or propofol did not affect cell proliferation, metastasis or TRPV1 protein expression in any type of cell.ConclusionSevoflurane, but not propofol, at clinically relevant concentrations and durations, increased survival of breast cancer cells in vitro but had no effect on cell proliferation, migration or TRPV1 expression. Breast cancer cells require higher cytoplasmic Ca2+ levels for survival than normal breast tissue. Sevoflurane affects breast cancer cell survival via modulation of intracellular Ca2+ homeostasis.

Pyruvate anaplerosis is a mechanism of resistance to pharmacological glutaminase inhibition in triple-receptor negative breast cancer

BackgroundGlutamine serves as an important nutrient with many cancer types displaying glutamine dependence. Following cellular uptake glutamine is converted to glutamate in a reaction catalysed by mitochondrial glutaminase. This glutamate has many uses, including acting as an anaplerotic substrate (via alpha-ketoglutarate) to replenish TCA cycle intermediates. CB-839 is a potent, selective, orally bioavailable inhibitor of glutaminase that has activity in Triple receptor-Negative Breast Cancer (TNBC) cell lines and evidence of efficacy in advanced TNBC patients.MethodsA panel of eleven breast cancer cell lines was used to investigate the anti-proliferative effects of the glutaminase inhibitors CB-839 and BPTES in different types of culture medium, with or without additional pyruvate supplementation. The abundance of the TCA cycle intermediate fumarate was quantified as a measure if TCA cycle anaplerosis. Pyruvate secretion by TNBC cultures was then assessed with or without AZD3965, a monocarboxylate transporter 1 (MCT1) inhibitor. Finally, two dimensional (2D) monolayer and three dimensional (3D) spheroid assays were used to compare the effect of microenvironmental growth conditions on CB-839 activity.ResultsThe anti-proliferative activity of CB-839 in a panel of breast cancer cell lines was similar to published reports, but with a major caveat; growth inhibition by CB-839 was strongly attenuated in culture medium containing pyruvate. This pyruvate-dependent attenuation was also observed with a related glutaminase inhibitor, BPTES. Studies demonstrated that exogenous pyruvate acted as an anaplerotic substrate preventing the decrease of fumarate in CB-839-treated conditions. Furthermore, endogenously produced pyruvate secreted by TNBC cell lines was able to act in a paracrine manner to significantly decrease the sensitivity of recipient cells to glutaminase inhibition. Suppression of pyruvate secretion using the MCT1 inhibitor AZD3965, antagonised this paracrine effect and increased CB-839 activity. Finally, CB-839 activity was significantly compromised in 3D compared with 2D TNBC culture models, suggesting that 3D microenvironmental features impair glutaminase inhibitor responsiveness.ConclusionThis study highlights the potential influence that both circulating and tumour-derived pyruvate can have on glutaminase inhibitor efficacy. Furthermore, it highlights the benefits of 3D spheroid cultures to model the features of the tumour microenvironment and improve the in vitro investigation of cancer metabolism-targeted therapeutics.

Abstract 4236: Evaluating alterations in mRNA biogenesis with TCGA breast cancer sequencing data

Abstract Alternative splicing leads to the production of multiple mRNA isoforms of a gene transcript with distinct functional characteristics. These events are frequently observed in rapidly dividing cancer cells and are broadly recognized as important signatures for oncogenesis pathways and treatment options. In this study, we utilized data from The Cancer Genome Atlas (TCGA) to identify differences in isoform expression between estrogen receptor-positive and estrogen receptor-negative breast cancer cell lines. RNA sequencing analysis combined with clinical annotation data revealed alterations in isoform expression that were associated with patient survival. These global changes in isoform expression occurred independently of total gene expression in both estrogen receptor-positive and -negative breast cancer. Furthermore, we identified multiple underrepresented isoforms including CDKAL1 and CSTF3 that were associated with favorable prognostic outcomes despite a lack of association observed with their traditional transcripts. These data suggest an underlying role mediated by alternative spliced isoforms in driving clinical outcome in breast cancer, warranting further investigation in delineating isoform expression profiles in previously established cancer pathways. Citation Format: Thomas J. Yan, Matthew E. Burow, Elizabeth C. Martin. Evaluating alterations in mRNA biogenesis with TCGA breast cancer sequencing data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4236.

Profiling of gene expression regulated by 17β-estradiol and tamoxifen in estrogen receptor-positive and estrogen receptor-negative human breast cancer cell lines.

One area of great importance in breast cancer (BC) research is the study of gene expression regulated by both estrogenic and antiestrogenic agents. Although many studies have been performed in this area, most of them have only addressed the effects of 17β-estradiol (E2) and tamoxifen (TAM) on MCF7 cells. This study aimed to determine the effect of low doses of E2 and TAM on the expression levels of 84 key genes, which are commonly involved in breast carcinogenesis, in four BC cell lines differentially expressing estrogen receptor (ER) α and HER2 (MCF7, T47D, BT474, and SKBR3). The results allowed us to determine the expression patterns modulated by E2 and TAM in ERα+ and ERα− cell lines, as well as to identify differences in expression patterns. Although the MCF7 cell line is the most frequently used model to determine gene expression profiles in response to E2 and TAM, the changes in gene expression patterns identified in ERα+ and ERα− cell lines could reflect distinctive properties of these cells. Our results could provide important markers to be validated in BC patient samples, and subsequently used for predicting the outcome in ERα+ and ERα− tumors after TAM or hormonal therapy. Considering that BC is a molecularly heterogeneous disease, it is important to understand how well, and which cell lines, best model that diversity.

Androgen receptor as a mediator and biomarker of radioresistance in triple-negative breast cancer

Increased rates of locoregional recurrence have been observed in triple-negative breast cancer despite chemotherapy and radiation therapy. Thus, approaches that combine therapies for radiosensitization in triple-negative breast cancer are critically needed. We characterized the radiation therapy response of 21 breast cancer cell lines and paired this radiation response data with high-throughput drug screen data to identify androgen receptor as a top target for radiosensitization. Our radiosensitizer screen nominated bicalutamide as the drug most effective in treating radiation therapy-resistant breast cancer cell lines. We subsequently evaluated the expression of androgen receptor in >2100 human breast tumor samples and 51 breast cancer cell lines and found significant heterogeneity in androgen receptor expression with enrichment at the protein and RNA level in triple-negative breast cancer. There was a strong correlation between androgen receptor RNA and protein expression across all breast cancer subtypes (R2 = 0.72, p < 0.01). In patients with triple-negative breast cancer, expression of androgen receptor above the median was associated with increased risk of locoregional recurrence after radiation therapy (hazard ratio for locoregional recurrence 2.9–3.2)) in two independent data sets, but there was no difference in locoregional recurrence in triple-negative breast cancer patients not treated with radiation therapy when stratified by androgen receptor expression. In multivariable analysis, androgen receptor expression was most significantly associated with worse local recurrence-free survival after radiation therapy (hazard ratio of 3.58) suggesting that androgen receptor expression may be a biomarker of radiation response in triple-negative breast cancer. Inhibition of androgen receptor with MDV3100 (enzalutamide) induced radiation sensitivity (enhancement ratios of 1.22–1.60) in androgen receptor-positive triple-negative breast cancer lines, but did not affect androgen receptor-negative triple-negative breast cancer or estrogen-receptor-positive, androgen receptor-negative breast cancer cell lines. androgen receptor inhibition with MDV3100 significantly radiosensitized triple-negative breast cancer xenografts in mouse models and markedly delayed tumor doubling/tripling time and tumor weight. Radiosensitization was at least partially dependent on impaired dsDNA break repair mediated by DNA protein kinase catalytic subunit. Our results implicate androgen receptor as a mediator of radioresistance in breast cancer and identify androgen receptor inhibition as a potentially effective strategy for the treatment of androgen receptor-positive radioresistant tumors.

INFLUENCE OF EXOGENOUS LACTOFERRIN ON THE OXIDANT/ ANTIOXIDANT BALANCE AND MOLECULAR PROFILE OF HORMONE RECEPTOR-POSITIvE AND -NEGATIvE HUMAN BREAST CANCER CELLS IN VITRO

To investigate the mechanisms of cytotoxic activity and pro-/antioxidant effect of lactoferrin on hormone receptor-positive and receptor-negative breast cancer cells in vitro. The study was performed on receptor-positive (MCF-7, T47D) and receptor-negative (MDA-MB-231, MDA-MB-468) human breast cancer cell lines. Immunocytochemical staining, flow cytometry, low-temperature electron paramagnetic resonance, and the Comet assay were used. Upon treatment with lactoferrin, the increased levels of reactive oxygen species (ROS) (p < 0.05), NO generation rate by inducible NO-synthase (p < 0.05) and the level of "free" iron (p < 0.05) were observed. Moreover, the effects of lactoferrin were more pronounced in receptor-negative MDA-MB-231and MDA-MB-468cells. These changes resulted in increased expression of proapoptotic Bax protein (p<0.05), reduced expression of the antiapoptotic Bcl-2 protein (p < 0.05) and level of not-oxidized mitochondrial cardiolipin (1.4-1.7-fold, p< 0.05). This, in turn, caused an increase in the percentage of apoptotic cells (by 14-24%, p < 0.05). Cytotoxic effects of lactoferrin were accompanied by an increase in the percentage of DNA in the comet tail and blocking cell cycle at G2/M phase, especially in receptor-negative cell lines. The study showed that exogenous lactoferrin causes a violation of an antioxidant balance by increasing the level of ROS, "free" iron and NO generation rate, resalting in the blocking of cell cycle at G2/M-phase and apoptosis of malignant cells.

Abstract 2729: Estrogen receptor-negative breast cancer cell lines exhibit hypersensitivity to the CHK1 inhibitor LY2606368

Abstract Checkpoint kinase 1 (CHK1) inhibitors are currently under investigation as chemopotentiating agents due to the role of CHK1 in establishing DNA damage checkpoints in the cell cycle. A novel CHK1/CHK2 inhibitor, LY2606368, as a single agent causes replication catastrophe, DNA double strand breaks and apoptosis (King C et al. Mol Cancer Ther. 2015). Accordingly, LY2606368 is currently in clinical development as a single agent and in combination with both cytotoxic and targeted agents. As subsets of breast cancers exhibit genomic instability and DNA repair deficiencies, we assessed the effect of LY2606368 as a single agent on breast cancer cell lines. We characterized the IC50's for growth inhibition by LY2606368 of a large panel of cell lines assembled on the basis of estrogen receptor (ER), progesterone receptor (PR), and HER2 expression status. Triple negative breast cancer (TNBC) is a subtype of breast cancer that is pathologically negative for expression of ER/PR and HER2 protein. As previously reported for other CHK1 inhibitors (Bryant C et al. BMC Cancer. 2014; Albiges L et al. Breast. 2014), TNBC cell lines exhibited high sensitivity to LY2606368. Interestingly, hypersensitivity to LY2606368 was observed in ER-negative cells, regardless of HER2 status. In addition, ER-positive cells were comparatively resistant suggesting that high sensitivity to LY2606368 occurs in the absence of ER and is not restricted to TNBC. No correlation was found between TP53 mutational status and sensitivity to LY2606368. Consistent with the observed hypersensitivity, ER-negative cell lines exposed to LY2606368 exhibited high levels of γH2AX and phospho-Ser1981-ATM demonstrating appearance of DNA double strand breaks. The concomitant appearance of phospho-Ser345-Chk1 marked aborted checkpoint activation by upstream detector/effector kinases (e.g. ATR, ATM). ER-positive cells did not engage in initiation of the DNA damage response or significant checkpoint activation when exposed to comparable doses of LY2606368. Collectively, these results suggest that the CHK1 inhibitor LY2606368 is likely to be more cytotoxic in ER-negative breast cancer types. Homologous recombination and other DNA repair deficiencies can explain the need for CHK1-dependent checkpoint activation during each replication cycle. On the other hand, ER-positive breast cancers might be less sensitive to monotherapy because of the absence of unresolved DNA damaging events that create checkpoint dependency. In summary, triple negative status does not directly determine the sensitivity of breast cancer cell lines to the CHK1 inhibitor LY2606368, although TNBC cells are in general hypersensitive. The absence of ER might be a more promising marker of sensitivity to LY2606368 as a monotherapy in breast cancer. Citation Format: Jean-Bernard Lazaro, Kalindi Parmar, Geoffrey I. Shapiro, Alan D. D’Andrea. Estrogen receptor-negative breast cancer cell lines exhibit hypersensitivity to the CHK1 inhibitor LY2606368. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2729.

A novel curcumin derivative increases the cytotoxicity of raloxifene in estrogen receptor-negative breast cancer cell lines.

There is a need for new, safe and efficacious drug therapies for the treatment of estrogen receptor (ER)-negative breast cancers. Raloxifene and the 2nd generation curcumin derivative 2,6-bis(pyridin-4-ylmethylene)-cyclohexanone (RL91) have been shown to inhibit the growth of ER-negative breast cancer cells invitro and invivo. We investigated whether RL91 could enhance the growth-suppressive effects mediated by raloxifene in MDA-MB-231, MDA-MB-468, Hs578t and SkBr3 human breast cancer cell lines. The cytotoxicity was consistent across the cell lines but RL91 was more potent. EC50 values for RL91 were 1.2-2µM while EC50 values for raloxifene were 9.6-11.2µM. When the cells were treated with raloxifene (15µM), RL91 (1µM) or a combination of the two for 6-72h, the combination treatment consistently elicited significantly greater cytotoxicity compared to all other treatments. In SkBr3 cells the combination treatment caused significantly more cells to undergo G1 arrest compared to raloxifene. In all cell lines apoptosis was synergistically induced by the combination treatment, as shown by both flow cytometery and cleaved caspase-3. Furthermore, the stress kinase p38 was increased and EFGR isoforms were decreased by both raloxifene and raloxifene + RL91. The anti-angiogenic anti-metastatic potential of raloxifene was not increased by RL91, as MDA-MB-231 cell migration and invasion as well as endothelial tube formation by HUVEC cells was not different between raloxifene (10µM) and the combination of raloxifene + RL91. Thus, our findings provide evidence that RL91 increases the ability of raloxifene to suppress ER-negative cancer cell growth by increasing the number of apoptotic cells. The broad effect of this drug combination across a range of ER-negative breast cancer cell lines indicates that this drug combination should be explored further in order to find a safe and efficacious therapy for ER-negative breast cancer.

Synthesis, characterization and biological evaluation of transition metal complexes derived from N, S bidentate ligands.

Two bidentate NS ligands were synthesized by the condensation reaction of S-2-methylbenzyldithiocarbazate (S2MBDTC) with 2-methoxybenzaldehyde (2MB) and 3-methoxybenzaldehyde (3MB). The ligands were reacted separately with acetates of Cu(II), Ni(II) and Zn(II) yielding 1:2 (metal:ligand) complexes. The metal complexes formed were expected to have a general formula of [M(NS)2] where M = Cu2+, Ni2+, and Zn2+. These compounds were characterized by elemental analysis, molar conductivity, magnetic susceptibility and various spectroscopic techniques. The magnetic susceptibility measurements and spectral results supported the predicted coordination geometry in which the Schiff bases behaved as bidentate NS donor ligands coordinating via the azomethine nitrogen and thiolate sulfur. The molecular structures of the isomeric S2M2MBH (1) and S2M3MBH (2) were established by X-ray crystallography to have very similar l-shaped structures. The Schiff bases and their metal complexes were evaluated for their biological activities against estrogen receptor-positive (MCF-7) and estrogen receptor-negative (MDA-MB-231) breast cancer cell lines. Only the Cu(II) complexes showed marked cytotoxicity against the cancer cell lines. Both Schiff bases and other metal complexes were found to be inactive. In concordance with the cytotoxicity studies, the DNA binding studies indicated that Cu(II) complexes have a strong DNA binding affinity.

English

The identification of medicinal plants and derived natural products for development as anti-cancer agents is of long standing interest. We have investigated the anti-proliferative properties of Lupinus angustifolius on breast cancer and, in particular, whether the extracts of roots and shoots of L. angustifolius can be considered as candidates for primary and secondary cancer prevention. Ethanolic extracts of roots and shoots of L. angustifolius were analysed for their substance classes by pyrolysis-field ionization mass spectrometry. Various concentrations of these extracts (0.1, 1, 10, 25, 50, 100 and 200 &micro;g/ml, incubation period: 24 h) were studied with respect to their effects on cell proliferation and cytotoxicity against the breast cancer cell lines MCF-7 (ER-&alpha;+, ER-&beta;+) and BT20 (ER-&alpha;-, ER-&beta;-). L. angustifolius ethanolic root and shoot extracts inhibited cell proliferation in the MCF-7 and BT20 cells; root extract: strongest inhibition MCF-7 (200 &mu;g/ml 71.03 &plusmn; 12.62%); BT20 (200 &mu;g/ml 99.72 &plusmn; 6.48%), IC50-values: MCF-7 (52.3 &plusmn; 9.39 &mu;g/ml); BT20 (66.86 &plusmn; 7.02 &mu;g/ml); shoot extract: strongest inhibition MCF-7 (200 &mu;g/ml 64.7 &plusmn; 10.07 %); BT20 (200 &mu;g/ml 86.32 &plusmn; 9.19%), IC50-values: MCF-7 (18.06 &plusmn;4.49 &mu;g/ml); BT20 (70.27 &plusmn; 0.76 &mu;g/ml). Thus, extracts of L. angustifolius roots and shoots have anti-tumour activity against receptor-positive and receptor-negative breast cancer cell lines. &nbsp; Key words: Anti-tumour activity, Lupinus angustifolius, breast cancer, MCF-7, BT20

Cdx2 polymorphism affects the activities of vitamin D receptor in human breast cancer cell lines and human breast carcinomas.

Vitamin D plays a role in cancer development and acts through the vitamin D receptor (VDR). It regulates the action of hormone responsive genes and is involved in cell cycle regulation, differentiation and apoptosis. VDR is a critical component of the vitamin D pathway and different common single nucleotide polymorphisms have been identified. Cdx2 VDR polymorphism can play an important role in breast cancer, modulating the activity of VDR. The objective of this study is to assess the relationship between the Cdx2 VDR polymorphism and the activities of VDR in human breast cancer cell lines and carcinomas breast patients. Cdx2 VDR polymorphism and antiproliferative effects of vitamin D treatment were investigated in a panel of estrogen receptor-positive (MCF7 and T-47D) and estrogen receptor-negative (MDA-MB-231, SUM 159PT, SK-BR-3, BT549, MDA-MB-468, HCC1143, BT20 and HCC1954) human breast cancer cell lines. Furthermore, the potential relationship among Cdx2 VDR polymorphism and a number of biomarkers used in clinical management of breast cancer was assessed in an ad hoc set of breast cancer cases. Vitamin D treatment efficacy was found to be strongly dependent on the Cdx2 VDR status in ER-negative breast cancer cell lines tested. In our series of breast cancer cases, the results indicated that patients with variant homozygote AA were associated with bio-pathological characteristics typical of more aggressive tumours, such as ER negative, HER2 positive and G3. Our results may suggest a potential effect of Cdx2 VDR polymorphism on the efficacy of vitamin D treatment in aggressive breast cancer cells (estrogen receptor negative). These results suggest that Cdx2 polymorphism may be a potential biomarker for vitamin D treatment in breast cancer, independently of the VDR receptor expression.

Differential effect of phosphorylation-defective survivin on radiation response in estrogen receptor-positive and -negative breast cancer.

Survivin is a key member of the inhibitor of apoptosis protein family, and is considered a promising therapeutic target due to its universal overexpression in cancers. Survivin is implicated in cellular radiation response through its role in apoptosis, cell division, and DNA damage response. In the present study, analysis of publically available data sets showed that survivin gene expression increased with breast cancer stage (p < 0.00001) and was significantly higher in estrogen receptor-negative cancers as compared to estrogen receptor-positive cancers (p = 9e-46). However, survivin was prognostic in estrogen receptor-positive tumors (p = 0.03) but not in estrogen receptor-negative tumors (p = 0.28). We assessed the effect of a survivin dominant-negative mutant on colony-formation (2D) and mammosphere-formation (3D) efficiency, and radiation response in the estrogen receptor-positive MCF7 and estrogen receptor-negative SUM149 breast cancer cell lines. The colony-formation efficiency was significantly lower in the dominant-negative survivin-transduced cells versus control MCF7 cells (0.42 vs. 0.58, p < 0.01), but it was significantly higher in dominant-negative population versus control-transduced SUM149 cells (0.29 vs. 0.20, p < 0.01). A similar, non-significant, trend in mammosphere-formation efficiency was observed. We compared the radiosensitivity of cells stably expressing dominant-negative survivin with their controls in both cell lines under 2D and 3D culture conditions following exposure to increasing doses of radiation. We found that the dominant-negative populations were radioprotective in MCF7 cells but radiosensitive in SUM149 cells compared to the control-transduced population; further, Taxol was synergistic with the survivin mutant in SUM149 but not MCF7. Our data suggests that survivin modulation influences radiation response differently in estrogen receptor-positive and estrogen receptor-negative breast cancer subtypes, warranting further investigation.

Dickkopf-1 is regulated by the mevalonate pathway in breast cancer

IntroductionAmino-bisphosphonates and statins inhibit the mevalonate pathway, and may exert anti-tumor effects. The Wnt inhibitor dickkopf-1 (DKK-1) promotes osteolytic bone lesions by inhibiting osteoblast functions and has been implicated as an adverse marker in multiple cancers. We assessed the effects of mevalonate pathway inhibition on DKK-1 expression in osteotropic breast cancer.MethodsRegulation of DKK-1 by bisphosphonates and statins was assessed in human breast cancer cell lines, and the role of the mevalonate pathway and downstream targets was analyzed. Moreover, the potential of breast cancer cells to modulate osteoblastogenesis via DKK-1 was studied in mC2C12 cells. Clinical relevance was validated by analyzing DKK-1 expression in the tissue and serum of women with breast cancer exposed to bisphosphonates.ResultsDKK-1 was highly expressed in receptor-negative breast cancer cell lines. Patients with receptor-negative tumors displayed elevated levels of DKK-1 at the tissue and serum level compared to healthy controls. Zoledronic acid and atorvastatin potently suppressed DKK-1 in vitro by inhibiting geranylgeranylation of CDC42 and Rho. Regulation of DKK-1 was strongest in osteolytic breast cancer cell lines with abundant DKK-1 expression. Suppression of DKK-1 inhibited the ability of breast cancer cells to block WNT3A-induced production of alkaline phosphates and bone-protective osteoprotegerin in preosteoblastic C2C12 cells. In line with the in vitro data, treatment of breast cancer patients with zoledronic acid decreased DKK-1 levels by a mean of 60% after 12 months of treatment.ConclusionDKK-1 is a novel target of the mevalonate pathway that is suppressed by zoledronic acid and atorvastatin in breast cancer.

Sensitivity Evaluation of Two Human Breast Cancer Cell Lines to Tamoxifen through Apoptosis Induction

Tamoxifen citrate (TAM) has been used to treat breast cancer in women for many years. The com-parative effects of TAM in inducing apoptosis were evaluated in estrogen receptor-positive (ER- positive MCF-7) and estrogen receptor-negative (ER-negative MDA-MB-231) human breast cancer cell lines in vitro in order to determine if these two cell lines differ in their sensitivity to TAM. Mi-tochondrial membrane permeability potential disruption was assessed in both cell lines by a lip-ophilic cationic dye (DePsipher assay, Trevigen, Inc.) utilizing fluorescence microscopy. Using this specific fluorochrome, we were able to associate mitochondrial membrane disruption to early, mid-, and late apoptotic cells. TAM induced cell death via apoptosis in both ER-positive and ER- negative cells, however, apoptosis induction was more pronounced in ER-positive MCF-7 compared to ER-negative MDA-MB-231 breast cancer cells. These findings may have some therapeutic use in the treatment of estrogen dependent and estrogen independent breast cancer.

The role of insulin-like growth factor binding protein-3 in the breast cancer cell response to DNA-damaging agents

Following exposure to radiation and chemotherapeutic agents, the epidermal growth factor receptor (EGFR) can modulate the repair of DNA double-strand breaks (DSB) by forming protein complexes that include the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). This is one of the key mechanism by which tumors become resistant to DNA-damaging therapies. Our previous studies have shown that insulin-like growth factor binding protein-3 (IGFBP-3) is a substrate for DNA-PKcs, and can transactivate EGFR. We therefore questioned whether IGFBP-3 might interact with the EGFR-DNA-PK complex that regulates the DNA damage response. The aim of this study was to delineate the role of IGFBP-3 in the response of breast cancer cells to DSB-inducing chemotherapeutic agents. In the estrogen receptor-negative breast cancer cell lines MDA-MB-468 and Hs578T, which express IGFBP-3 highly, nuclear localization of EGFR and IGFBP-3 was enhanced by treatment with cytotoxic drugs etoposide or doxorubicin and reduced by the EGFR kinase inhibitor gefitinib. Enhanced association among IGFBP-3, EGFR and DNA-PKcs, following the exposure to DNA-damaging drugs was supported by both co-immunoprecipitation analysis and direct visualization by proximity ligation assay. The activation of DNA-PKcs at Ser2056, DNA repair as measured by a nonhomologous end-joining assay, and the increase in EGFR and DNA-PKcs interaction induced by DNA-damaging agents, were all decreased by IGFBP-3 silencing, suggesting that IGFBP-3 has an obligatory role in the DNA repair response to DNA-damaging therapy. In conclusion, IGFBP-3 co-translocation to the nucleus of breast cancer cells and its formation of a complex with DNA-PKcs and EGFR in response to DNA damage shows its potential involvement in the regulation of DNA repair. This suggests the possibility of a therapeutic approach for sensitizing breast cancer to chemo- or radiotherapy by targeting the DNA repair function of IGFBP-3.

The inflammation-resolving tri-hydroxy docosahexaenoic acid derivative, resolvin D2 increases MCF-7 cell number

. • Formyl peptide receptors (FPRs) are G-protein coupled receptors. • FPRs can bind a wide range of structurally diverse ligands including proteins, small peptides and bioactive lipid mediators. They are expressed in both estrogen receptor-positive (MCF-7) and estrogen receptor-negative (MDA-MB-231) breast cancer cell lines. • Some of the endogenous inflammation- resolving FPRs ligands (annexin A1 and its N terminal AC2-26 and the trihydroxy lipid mediator, lipoxin A

Activation of Src and transformation by an RPTPα splice mutant found in human tumours

Receptor protein tyrosine phosphatase α (RPTPα)-mediated Src activation is required for survival of tested human colon and oestrogen receptor-negative breast cancer cell lines. To explore whether mutated RPTPα participates in human carcinogenesis, we sequenced RPTPα cDNAs from five types of human tumours and found splice mutants in ∼30% of colon, breast, and liver tumours. RPTPα245, a mutant expressed in all three tumour types, was studied further. Although it lacks any catalytic domain, RPTPα245 expression in the tumours correlated with Src tyrosine dephosphorylation, and its expression in rodent fibroblasts activated Src by a novel mechanism. This involved RPTPα245 binding to endogenous RPTPα (eRPTPα), which decreased eRPTPα-Grb2 binding and increased eRPTPα dephosphorylation of Src without increasing non-specific eRPTPα activity. RPTPα245-eRPTPα binding was blocked by Pro210 → Leu/Pro211 → Leu mutation, consistent with the involvement of the structural 'wedge' that contributes to eRPTPα homodimerization. RPTPα245-induced fibroblast transformation was blocked by either Src or eRPTPα RNAi, indicating that this required the dephosphorylation of Src by eRPTPα. The transformed cells were tumourigenic in nude mice, suggesting that RPTPα245-induced activation of Src in the human tumours may have contributed to carcinogenesis.

Frondoside A inhibits human breast cancer cell survival, migration, invasion and the growth of breast tumor xenografts

Breast cancer is a major challenge for pharmacologists to develop new drugs to improve the survival of cancer patients. Frondoside A is a triterpenoid glycoside isolated from the sea cucumber, Cucumaria frondosa. It has been demonstrated that Frondoside A inhibited the growth of pancreatic cancer cells in vitro and in vivo. We investigated the impact of Frondoside A on human breast cancer cell survival, migration and invasion in vitro, and on tumor growth in nude mice, using the human estrogen receptor-negative breast cancer cell line MDA-MB-231. The non-tumorigenic MCF10-A cell line derived from normal human mammary epithelium was used as control. Frondoside A (0.01–5μM) decreased the viability of breast cancer cells in a concentration- and time-dependent manner, with 50%-effective concentration (EC50) of 2.5μM at 24h. MCF10-A cells were more resistant to the cytotoxic effect of Frondoside A (EC50 superior to 5μM at 24h). In the MDA-MB-231 cells, Frondoside A effectively increased the sub-G1 (apoptotic) cell fraction through the activation of p53, and subsequently the caspases 9 and 3/7 cell death pathways. In addition, Frondoside A induced a concentration-dependent inhibition of MDA-MB-231 cell migration and invasion. In vivo, Frondoside A (100μg/kg/dayi.p. for 24days) strongly decreased the growth of MDA-MB-231 tumor xenografts in athymic mice, without manifest toxic side-effects. Moreover, we found that Frondoside A could enhance the killing of breast cancer cells induced by the chemotherapeutic agent paclitaxel. These findings identify Frondoside A as a promising novel therapeutic agent for breast cancer.

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.