Proteins In Breast Cancer Cells Research Articles

Identification of estrogen receptor proteins in breast cancer cells using matrix-assisted laser desorption/ionization time of flight mass spectrometry (Review).

Estrogen receptors [ERs (subtypes α and β)], classified as a nuclear receptor super family, are intracellular proteins with an important biological role as the transcription factors for estrogen target genes. For ER-induced transcription, an interaction must exist between ligand and coregulators. Coregulators may stimulate (coactivators) or inhibit (corepressors) transcription, following binding with a specific region of the gene, called the estrogen response element. Misbalanced activity of coregulators or higher ligand concentrations may cause increased cell proliferation, resulting in specific types of cancer. These are exhibited as overexpression of ER proteins. Breast cancer currently ranks first in the incidence and second in the mortality of cancer in females worldwide. In addition, 70% of breast tumors are ERα positive and the importance of these proteins for diagnostic use is indisputable. Early diagnosis of the tumor and its classification has a large influence on the selection of appropriate therapy, as ER-positive tumors demonstrate a positive response to hormonal therapy. Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI TOF MS) has been hypothesized to have great potential, as it offers reliable, robust and efficient analysis methods for biomarker monitoring and identification. The present review discusses ER protein analysis by MALDI TOF MS, including the crucial step of protein separation.

Hypoxia Regulates the Expression of the Neuromedin B Receptor through a Mechanism Dependent on Hypoxia-Inducible Factor-1α

The neuromedin B receptor (NMB-R), a member of the mammalian bombesin receptor family, is frequently overexpressed in various tumors. In the present study, we found that exposure to hypoxic conditions increases the levels of NMBR mRNA and protein in breast cancer cells, which are tightly regulated by hypoxia-inducible factor-1α (HIF-1α). We confirmed the effect of HIF-1α on NMBR transcription by performing an NMBR promoter-driven reporter assay and then identified a functional hypoxia-responsive element (HRE) in the human NMBR promoter region. Further, the binding of HIF-1α to the NMBR promoter was corroborated by electrophoretic mobility shift and chromatin immunoprecipitation assays, which showed that HIF-1α specifically and directly bound to the NMBR promoter in response to hypoxia. Immunohistochemical analysis of a xenograft and a human breast cancer tissue array revealed a significant correlation between NMB-R and HIF-1α expression. Taken together, our findings indicate that hypoxia induces NMB-R expression through a novel mechanism to regulate HIF-1α expression in breast cancer cells.

Hypoxia-driven osteopontin contributes to breast tumor growth through modulation of HIF1α-mediated VEGF-dependent angiogenesis

Hypoxia is a salient feature of most solid tumors, and hypoxic adaptation of cancer cells has crucial implications in propagation of malignant clonal cell population. Osteopontin (OPN) has been identified as a hypoxia-responsive gene, but the mechanistic and regulatory role of OPN under hypoxia is less characterized. The present study identifies the existence of a positive inter-regulatory loop between hypoxia and OPN. We have shown that hypoxia induces OPN expression in breast cancer cells; however, the expression was found to be HIF1α independent. OPN enabled transcriptional upregulation of HIF1α expression both under normoxia and hypoxia, whereas stability of HIF1α protein in breast cancer cells remained unaffected. Moreover, we have shown that OPN induces integrin-linked kinase (ILK)/Akt-mediated nuclear factor (NF)-κB p65 activation leading to HIF1α-dependent vascular endothelial growth factor (VEGF) expression and angiogenesis in response to hypoxia. These in vitro data are biologically important as OPN expressing cells induce greater tumor growth and angiogenesis through enhanced expressions of proangiogenic molecules as compared with control. Immunohistochemical analysis of human breast cancer specimens revealed significant correlation between OPN and HIF1α but not HIF2α. Elevated expression of HIF1α and OPN was observed in pre-neoplastic and early stage infiltrating ductal carcinoma implicating the role of these proteins in neoplastic progression of breast cancer. Together, our results substantiate the prime role of OPN in cellular adaptation through ILK and NF-κB-mediated HIF1α-dependent VEGF expression in response to hypoxia that ultimately controls breast cancer progression and angiogenesis. Our study reinforces the fact that targeting OPN and its regulated signaling network hold important therapeutic implications.

The Nuclear Import of Oncoprotein Hepatitis B X-interacting Protein Depends on Interacting with c-Fos and Phosphorylation of Both Proteins in Breast Cancer Cells

Aberrant nuclear localization of oncogenic transcription factors and coactivators always leads to the development of cancer. We have reported that the oncoprotein hepatitis B X-interacting protein (HBXIP) acts as a novel transcriptional coactivator to promote proliferation and migration of breast cancer cells. However, the mechanism of regulating the nuclear import of HBXIP remains unclear. In the present study, we found that HBXIP interacted with c-Fos through their leucine zipper domains in vitro and in vivo. Interestingly, the leucine zipper mutant of HBXIP (or c-Fos) was unavailable to bind to c-Fos (or HBXIP), resulting in the disappearance of nuclear localization of HBXIP. Moreover, we revealed that the nuclear import of HBXIP was required for phosphorylation of c-Fos at Thr(232), Thr(325), Thr(331), and Ser(374) by ERK1/2. In addition, the mutant of HBXIP at the Ser(108) phosphorylation site failed to import into the nucleus. Strikingly, we found that the kinase ataxia telangiectasia mutated (ATM) phosphorylated HBXIP at Ser(108). The knockdown of ATM by siRNA remarkably decreased the levels of serine phosphorylation and blocked the nuclear import of HBXIP. Then, we identified that ATM could bind to HBXIP. Moreover, we validated that the nuclear import of HBXIP contributed to its nuclear function. Therefore, we conclude that the nuclear import of the oncoprotein HBXIP requires interaction with c-Fos through their leucine zipper domains and phosphorylation of both proteins in breast cancer cells. Thus, our findings provide new insights into the mechanism of the nuclear import of HBXIP. Therapeutically, the block of the nuclear import of HBXIP is significant in breast cancer.

Targeted inhibition of Hsp90 by ganetespib is effective across a broad spectrum of breast cancer subtypes

SummaryHeat shock protein 90 (Hsp90) is a molecular chaperone essential for the stability and function of multiple cellular client proteins, a number of which have been implicated in the pathogenesis of breast cancer. Here we undertook a comprehensive evaluation of the activity of ganetespib, a selective Hsp90 inhibitor, in this malignancy. With low nanomolar potency, ganetespib reduced cell viability in a panel of hormone receptor-positive, HER2-overexpressing, triple-negative and inflammatory breast cancer cell lines in vitro. Ganetespib treatment induced a rapid and sustained destabilization of multiple client proteins and oncogenic signaling pathways and even brief exposure was sufficient to induce and maintain suppression of HER2 levels in cells driven by this receptor. Indeed, HER2-overexpressing BT-474 cells were comparatively more sensitive to ganetespib than the dual HER2/EGFR tyrosine kinase inhibitor lapatinib in three-dimensional culture. Ganetespib exposure caused pleiotropic effects in the inflammatory breast cancer line SUM149, including receptor tyrosine kinases, MAPK, AKT and mTOR signaling, transcription factors and proteins involved in cell cycle, stress and apoptotic regulation, as well as providing combinatorial benefit with lapatinib in these cells. This multimodal activity translated to potent antitumor efficacy in vivo, suppressing tumor growth in MCF-7 and MDA-MB-231 xenografts and inducing tumor regression in the BT-474 model. Thus, ganetespib potently inhibits Hsp90 leading to the degradation of multiple clinically-validated oncogenic client proteins in breast cancer cells, encompassing the broad spectrum of molecularly-defined subtypes. This preclinical activity profile suggests that ganetespib may offer considerable promise as a new therapeutic candidate for patients with advanced breast cancers.Electronic supplementary materialThe online version of this article (doi:10.1007/s10637-013-9971-6) contains supplementary material, which is available to authorized users.

Knockdown of interferon-induced transmembrane protein 3 expression suppresses breast cancer cell growth and colony formation and affects the cell cycle

Interferon-induced transmembrane protein 3 (IFITM3) is an important anti-virus protein and has been recently shown to play a role in human cancer development. Thus, the present study aimed to assess the expression of the IFITM3 protein in breast cancer tissues and to investigate the in vitro effects of IFITM3 knockdown in the regulation of breast cancer cell growth and cell cycle distributions. A total of 64 patients of breast cancer and the matched normal tissue specimens were obtained for immunohistochemical analysis of IFITM3 expression. Lentivirus-carrying IFITM3 shRNA was used to knock down IFITM3 expression in breast cancer cell lines. Phenotypic changes of cell viability, growth, colony formation and cell cycle distribution was also assayed using flow cytometry, MTT, BrdU incorporation and colony formation assays. The IFITM3 protein was highly expressed in invasive breast cancer compared to normal tissues and was significantly associated with estrogen receptor and progesterone receptor status. The lentivirus-carried IFITM3 shRNA significantly reduced the expression of IFITM3 mRNA and protein in breast cancer cells, inhibiting tumor cell viability, growth and colony formation, arrested tumor cells at the G0/G1 phase of the cell cycle and reduced the number of cells in the S phase of the cell cycle. Expression of IFITM3 protein could be a potential therapeutic target in future treatment of breast cancer.

Abstract LB-37: P-REX1 is a novel PTEN-interacting protein that activates PI3K signaling in breast cancer.

Abstract The mechanism(s) underlying regulation of the tumor suppressor phosphatase PTEN, which negatively regulates phosphatidylinositol 3-kinase (PI3K) signaling, remains unclear. The PI3K/AKT pathway regulates cancer cell growth, survival, and metabolism. Since PTEN is expressed and PI3K is hyperactivated in the majority of breast cancers, we hypothesized that PTEN is inhibited by protein interactions, thus derepressing PI3K signaling. A proteomic screen to identify novel PTEN-binding proteins in breast cancer cells revealed interaction with P-REX1, a known Rac guanine exchange factor (GEF). A separate proteomic experiment to identify PTEN/PI3K-regulated proteins showed that P-REX1 levels are decreased upon loss of PTEN and increased upon inhibition of PI3K. PTEN/P-REX1 interaction and the effects of pharmacological PI3K pathway inhibitors on P-REX1 levels were validated by immunoprecipitation/immunoblot analyses. In reverse-phase protein array analysis of lysates from 597 primary human breast tumors, levels of P-REX1 protein were positively correlated with PTEN protein, and inversely correlated with phospho-AKT-T308 (both p<0.005). Comparing diverse types of carcinomas (n=2,009 from International Genomics Consortium's expO) and cancer cell lines (n=807 from Cancer Cell Line Encyclopedia), PREX1 mRNA levels were highest in ER+ and HER2+ breast cancer. In another series of 1,293 epithelial tumors (from The Cancer Genome Atlas), PREX1 is amplified or mutated in 6.2% of cases, and in 5% of breast cancers. Since multiple genomic lesions that can activate the PI3K pathway are known to co-exist in cancer cells, we tested whether PREX1 lesions co-exist with other PI3K pathway-activating lesions. Among genes encoding proteins implicated in growth factor receptor/PI3K/PTEN signaling and phosphatidylinositol metabolism, we found a significant enrichment for PREX1 mutation/amplification in 54/79 (68%) genes across 1,523 carcinomas. Overexpression and RNAi knockdown experiments revealed that P-REX1 increases steady-state and insulin-like growth factor-1 (IGF-1)-induced PI3K-dependent AKT phosphorylation in ER+ breast cancer cells. Studies are underway to identify the mechanistic role of P-REX1 in PI3K/PTEN signaling, and to determine whether P-REX1 directly inhibits PTEN. Our findings suggest that 1) P-REX1 is an activator of the PI3K/AKT pathway, 2) expression and mutation patterns implicate PREX1 in PI3K activation, and 3) neutralizing P-REX1 effects on PTEN/PI3K may be a novel therapeutic approach to selectively abrogate PI3K signaling in ER+ and HER2+ breast cancers while sparing normal tissues. Citation Format: Lloye M. Dillon, Jennifer R. Bean, Justin M. Balko, W. Hayes McDonald, David B. Friedman, Ana M. Gonzalez-Angulo, Gordon B. Mills, Carlos L. Arteaga, Todd W. Miller. P-REX1 is a novel PTEN-interacting protein that activates PI3K signaling in breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-37. doi:10.1158/1538-7445.AM2013-LB-37

Abstract 579: The interaction of ZSCAN4 with TRF1: effects on regulation of telomere elongation in cancer cells.

Abstract Telomeres are repetitive sequences at the ends of chromosomes protected by DNA binding proteins of shelterin complex that form capping structures. One of these proteins, TRF1, plays a role in the regulation of telomere length through its interaction with other proteins in the shelterin complex. High levels of TRF1 expression have been shown to accelerate telomere shortening, whereas dominant-negative inhibition of TRF1 leads to telomere elongation. Recently, the newly identified Zinc finger and SCAN domain containing4 gene (ZSCAN4) plays a key role in genomic stability by regulating telomere elongation and was shown to co-localize with TRF1 foci in mouse embryonic stem cell. Here, we show that ZSCAN4 is a TRF1 association protein in breast cancer cells. We hypothesized that ZSCAN4 would affect levels of TRF1expression and correspondingly control the function of TRF1 through negative regulation and dominant-negative inhibition. Using Co-immunoprecipitation and TNT Pull-down assay, we demonstrate that ZSCAN4 binds with TRF1 in vitro. Localization and visualization of interacting protein-protein interactions in living cells are crucial to elucidate between TRF1 and ZSCAN4. Using a bimolecular fluorescence complementation (BiFC) assay, we show the directly visualize TRF1 and ZSCAN4 interactions in living cells. Using telomere length assays, we demonstrate that ZSCAN4 is an association protein of TRF1 to regulate telomere and affect in the telomere elongation of cancer. The ZSCAN4 mediated cooperative binding of TRF1 to telomeric DNA has important implications for understanding the mechanism of telomere extension. Citation Format: kyungwoo lee. The interaction of ZSCAN4 with TRF1: effects on regulation of telomere elongation in cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 579. doi:10.1158/1538-7445.AM2013-579

Investigation of SSEA-4 Binding Protein in Breast Cancer Cells

SSEA-4, a sialyl-glycolipid, has been commonly used as a pluripotent human embryonic stem cell marker, and its expression is correlated with the metastasis of some malignant tumors. However, there is no in-depth functional study related to the receptor and the role of this glycolipid. Here, we report the identification of an SSEA-4-binding protein in a breast cancer cell line, MCF-7. By using affinity capture and glycan microarray techniques, the intracellular FK-506 binding protein 4 (FKBP4) was identified to bind directly to SSEA-4. The biological significance of SSEA-4/FKBP4 interaction was investigated.

Withaferin a induces proteasome-dependent degradation of breast cancer susceptibility gene 1 and heat shock factor 1 proteins in breast cancer cells.

The purpose of this study was to examine the regulation of prosurvival factors heat shock factor 1 (HSF1) and breast cancer susceptibility gene 1 (BRCA1) by a natural withanolide withaferin A (WA) in triple negative breast cancer cell lines MDA-MB-231 and BT20. Western analysis was used to examine alternations in HSF1 and BRCA1 protein levels following WA treatment. A protein synthesis inhibitor cycloheximide and a proteasome inhibitor MG132 were used to investigate the mechanisms of HSF1 and BRCA1 regulation by WA. It was found that WA induced a dose-dependent decrease in HSF1 and BRCA1 protein levels. Further analysis showed that levels of HSF1 and BRCA1 proteins decreased rapidly after WA treatment, and this was attributed to WA-induced denaturation of HSF1 and BRCA1 proteins and subsequent degradation via proteasome-dependent, and protein-synthesis dependent mechanism. In summary, WA induces denaturation and proteasomal degradation of HSF1 and BRCA1 proteins. Further studies are warranted to examine the contribution of HSF1 and BRCA1 depletion to the anticancer effects of WA in breast cancer.

TOPOGRAPHY MEDIATED REGULATION OF HER-2 EXPRESSION IN BREAST CANCER CELLS

This article demonstrates that the surface micro-topography regulates the biology of breast cancer cells, including the expression of HER-2 gene and protein. The breast tumor microenvironment is made up of heterogenous mixture of pores, ridges and collagen fibers with well defined topographical features. Although, significant progress has been achieved towards elucidating the biochemical and molecular mechanisms that underlie breast cancer progression, quantitative characterization of the associated mechanical/topographical properties and their role in breast tumor progression remains largely unexplored. Therefore, the aim of this study is to investigate the effect of topography on the adhesion and biology of breast cancer cells in in vitro cultures. Polydimethylsiloxane (PDMS) surfaces containing different topographies were coated with polyelectrolyte multilayers (PEMs) to improve cell adhesion and maintain cell culture. HER-2 expressing breast cancer cells, BT-474 and SKBr3, were cultured on these PDMS surfaces. We demonstrate that micro-topography affects the cell adhesion and distribution depending on the topography on the PDMS surfaces. We also report for the first time that surface topography down-regulates the HER-2 gene transcription and protein expression in breast cancer cells when cultured on PDMS surfaces with micro-topographies compared to the tissue culture polystyrene surface (TCPS) control. Results from this study indicate that micro-topography modulates morphology of cells, their distribution and expression of HER-2 gene and protein in breast cancer cells. This study provides a novel platform for studying the role of native topography in the progression of breast cancer and has immense potential for understanding the breast cancer biology.

ITRAQ-Based Proteomic Profiling of Breast Cancer Cell Response to Doxorubicin and TRAIL

Breast cancer is a molecularly heterogeneous disease, and predicting response to chemotherapy remains a major clinical challenge. To minimize adverse side-effects or cumulative toxicity in patients unlikely to benefit from treatment, biomarkers indicating treatment efficacy are critically needed. iTRAQ labeling coupled with multidimensional LC-MS/MS of the enriched mitochondria and endoplasmic reticulum fraction, key organelles regulating apoptosis, has led to the discovery of several differentially abundant proteins in breast cancer cells treated with the chemotherapeutic agent doxorubicin followed by the death receptor ligand, TRAIL, among 571 and 801 unique proteins identified in ZR-75-1 and MDA-MB-231 breast cancer cell lines, respectively. The differentially abundant proteins represent diverse biological processes associated with cellular assembly and organization, molecular transport, oxidative stress, cell motility, cell death, and cancer. Despite many differences in molecular phenotype between the two breast cancer cell lines, a comparison of their subproteomes following drug treatment revealed three proteins displaying common regulation: PPIB, AHNAK, and SLC1A5. Changes in these proteins, detected by iTRAQ, were confirmed by immunofluorescence, visualized by confocal microscopy. These novel potential biomarkers may have clinical utility for assessing response to cancer treatment and may provide insight into new therapeutic targets for breast cancer.

Targeting Her-2/neu breast cancers with a plant derived natural compound, Withaferin A (53.15)

Abstract Over expression of oncogenic Her-2/neu protein in breast cancer cells correlates with poor prognosis and patients initially responsive to Her-2/neu targeted treatments often experience recurrence within a year of treatment. Thus, new strategies to combat these cancers should be explored. Our research focuses on the development of Withaferin A (WA), a steroidal lactone derived from the Indian ayurvedic medicinal plant Withania somnifera as an immunotherapeutic agent. The benefit of using natural plant derived products is their low toxicity, multimodal action and potential ability to enhance the immune system. Our goal is to establish WA as a chemoimmunotherapeutic agent in the treatment of Her-2/neu breast cancers. We provide evidence that WA is effective in vitro against both human and murine Her-2/neu breast cancer cells. WA induces cell death by apoptosis and increases expression of immunogenic stress proteins. Furthermore, WA up-regulated costimualtory molecules, cytokine production and antigen presentation in cultured bone marrow derived murine dendritic cells (BMDC). Additionally, in vivo administration of WA in a therapeutic setting significantly delayed tumor growth with a 10% survival benefit. Our long-term goal is to elucidate the anti-tumor immunomodulatory mechanisms of WA. Such agents that can deliver multimodal action towards cancer at both a chemotherapeutic and immunotherapeutic level might prove to be the best alternative to current treatment strategies.

Ginsenoside Rp1 from Panax ginseng Exhibits Anti-cancer Activity by Down-regulation of the IGF-1R/Akt Pathway in Breast Cancer Cells

Cancer prevention is effective and reduces health care costs because cancer is often a preventable disease that can be affected by lifestyle factors. Therefore, researchers are interested in discovering natural compounds that have anticancer activities, such as delaying the development of cancer and preventing its progression. One such natural agent is ginseng (Panax ginseng), which is traditionally used in some parts of the world as a popular remedy for various diseases including cancer. We hypothesized that the ginsenoside Rp1, a component of ginseng, reduces cancer cell proliferation through inhibition of the insulin-like growth factor 1 receptor (IGF-1R)/Akt pathway. We first tested the efficacy of Rp1 against human breast cancer cell lines. Treatment with Rp1 inhibited breast cancer cell proliferation and inhibited both anchorage-dependent and -independent breast cancer cell colony formation. In addition, treatment with 20 μM Rp1 induced cycle arrest and apoptosis-mediated cell growth suppression. Our findings further indicated that Rp1 decreased the stability of the IGF-1R protein in breast cancer cells. Therefore, we suggest that Rp1 has potential as an anticancer drug and that IGF-1R is an important target for treatment and prevention of breast cancer.

Reduced association of anti-apoptotic protein Mcl-1 with E3 ligase Mule increases the stability of Mcl-1 in breast cancer cells

Background:Mechanisms that increase resistance to apoptosis help promote cellular transformation. Cancer cells have deregulated apoptotic pathways, where increased expression and stability of anti-apoptotic proteins Mcl-1 and Bcl-2 increases resistance to apoptosis. Pathways that increase the stability of proteins in cancer cells remain poorly understood.Methods:Using human mammary epithelial and established breast cancer cell lines, we assessed the mechanisms that increase the stability of anti-apoptotic proteins in breast cancer cells by caspase assay, western blot, small-inhibitory RNA treatment and immunoprecipitation.Results:While breast cancer cells were resistant to de novo inhibition of protein synthesis, a rapid proteosome-mediated degradation of Mcl-1 and Bcl-2 induced apoptosis in mammary epithelial cells. Although Mule, an E3 ligase that targets Mcl-1 for degradation was expressed in mammary epithelial and breast cancer cell lines, rapid increase of polyubiquitinated Mcl-1 and Bcl-2 was detected only in mammary epithelial cells. Only transient formation of the Mule–Mcl-1 complex was detected in breast cancer cells. Downregulation of pERK1/2 in breast cancer cells reduced Mcl-1 levels and increased Mcl-1/Mule complex.Conclusion:Our findings suggest that reduced Mule/Mcl-1 complex has a significant role in increasing the stability of Mcl-1 in breast cancer cells and increased resistance to apoptosis.

Abstract 2489: Targeting pro-survival BCL-2 family members increases radiation sensitivity in breast cancer cells

Abstract In previous studies we have shown that BCL-2 expression was associated with an increased risk of local recurrence in patients with early stage breast cancer (Yang Q, Breast Cancer Res Treat, 2009). Additionally, BCL-2-overexpressing lymphoma and prostate cancer cell lines were resistant to radiation-induced apoptosis. The BCL-2 family encompasses two groups: the pro-survival group (BCL-2, BCL-XL, BCL-w, MCL-1, A1) and pro-apoptotic group (BAK, BAX, BAD, BIK, etc.) ABT-737 is a BH3 mimetic compound that targets the pro-survival members excluding MCL-1. Targeting pro-survival BCL-2 family members presents an attractive novel adjuvant therapeutic option. The purpose of this study was to explore an approach to overcoming radiation resistance by targeting pro-survival BCL-2 family proteins in breast cancer cells. First, siRNAs were employed to target BCL-2 in MCF-7 and BT474 breast cancer cells. Apoptosis was determined by detecting cleaved PARP after siRNA and radiation treatment. Cleaved PARP in BCL-2 siRNA treated MCF-7 cells increased compared to non-targeting siRNA treated cells 72 hours after 8Gy of radiation, indicating the targeting BCL-2 alone increased apoptotic cell death. Despite evidence of increased radiation related apoptosis, cell viability showed there was no significant difference between BCL-2 siRNA and non-targeting siRNA treated MCF-7 cells at 2, 4, 6 and 8Gy, p>0.05, indicating that targeting the BCL-2 alone did not enhance radiation sensitivity. However, the combination of radiation (8Gy) and ABT-737 (variable doses) enhanced BAK and cleaved PARP at 24hr, 48hr and 72hr. Further, the cell viability of the combination of radiation (2Gy) and ABT-737 (5µM) significantly decreased 16.5%, p<0.01 and 26.5%, p<0.01, compared with radiation alone or ABT-737 alone treated MCF-7 cells, respectively. Similar results were observed in T47D and MDA-MB231 cells. Finally, after knocking down MCL-1 by siRNA, cell viability went down in 14.5% (p<0.05) in MCL-1 siRNA treated MCF-7 cells compared with non-targeting siRNA treated cells at 5µm of ABT-737. In addition, MCL-1 protein level decreased after treatment of radation alone and the combination of radiation and ABT-737 in MCF-7, MDA-MB 231 and ZR75-1 cells. Together, our results showed that synergistic effect of radiation and ABT-737 on breast cancer cell lines through down-regulating MCL-1 and activating bak-apoptotic pathway. These results support the combination of radiation and pro-survival BCL-2 family inhibitor as a potential novel therapeutic strategy in breast cancer treatment. Supported by the Breast Cancer Research Foundation Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2489. doi:10.1158/1538-7445.AM2011-2489

Hsp-27 induction requires POU4F2/Brn-3b TF in doxorubicin-treated breast cancer cells, whereas phosphorylation alters its cellular localisation following drug treatment

POU4F2/Brn-3b transcription factor (referred to as Brn-3b) is elevated in >60% of breast cancers and profoundly alters growth and behaviour of cancer cells by regulating distinct subsets of target genes. Previous studies showed that Brn-3b was required to maximally transactivate small heat shock protein, HSPB1/Hsp-27 (referred to as Hsp-27), and consequently, Brn-3b expression correlated well with Hsp27 levels in human breast biopsies. In these studies, we showed that Brn-3b is increased in MCF7 breast cancer cells that survive following treatment with chemotherapeutic drug doxorubicin (Dox) with concomitant increases in Hsp-27 expression. Targeting of Brn-3b using short interfering RNA reduced Hsp-27 in Dox-treated cells, suggesting that Brn-3b regulates Hsp-27 expression under these conditions. Wound healing assays showed increased Brn-3b in Dox-treated migratory cells that also express Hsp-27. Interestingly, Hsp-27 phosphorylation and cellular localisation are also significantly altered at different times following Dox treatment. Thus, phospho-Hsp-27 (p-Hsp27) protein displayed widespread distribution after 24 hrs of Dox treatment but was restricted to the nucleus after 5 days. However, in drug-resistant cells (grown in Dox for > 1 month), p-Hsp-27 was excluded from nuclei and most of the cytoplasm and appeared to be associated with the cell membrane. Studies to determine how this protein promotes survival and migration in breast cancer cells showed that the protective effects were conferred by unphosphorylated Hsp-27 protein. Thus, complex and dynamic mechanisms underlie effects of Hsp-27 protein in breast cancer cells following treatment with chemotherapeutic drugs such as Dox, and this may contribute to invasiveness and drug resistance following chemotherapy.

Significance of Bax Expression in Breast Cancer Patients

Bax protein, the proapoptotic member of Bcl-2 protein family, plays the key role in apoptosis pathway. THE AIM OF THE STUDY was to assess the expression of Bax protein in breast cancer cells. Sixty-two breast cancer patients were included in the study. The control group encompassed 11 fibroadenoma patients. Single cells were isolated from defrosted samples and prepared for flow cytometry measurement. Median expression of Bax protein in study group was 7.9% (range: 0-49.4%) and was significantly lower than in control (median expression 15.8%; range 4.9-30.9%; p=0.034). Expression of Bax correlated with expression of p53 and caspase-3 proteins (p<0.01, rank Spearman test). In patients under 70 years old and with positive estrogen receptors status the expression of Bax protein was significantly higher (p=0.03 and p=0.01 respectively). Lower expression of Bax protein in breast cancer cells may suggest the potential way of apoptosis avoidance of tumor cells. Correlations among Bax protein, p53 and caspase-3 are likely associated with active apoptotic mechanism in breast cancer cells expressing Bax protein. Further investigation with long time follow-up should be performed to establish the prognostic role of Bax protein expression in breast cancer patients.

Targeting Prosurvival Bcl-2 Family Members Increases Radiation Sensitivity in Breast Cancer Cells

Targeting Prosurvival Bcl-2 Family Members Increases Radiation Sensitivity in Breast Cancer Cells

Overexpression of PPARγ can down-regulate Skp2 expression in MDA-MB-231 breast tumor cells

Skp2 is frequent amplified and overexpressed in breast cancer, making it a potential molecular target for cancer therapy. The objective of this study was to examine the effect of PPARγ overexpression on Skp2 expression in breast cancer cell lines. First, we investigated the role of PPARγ and Skp2 in human breast cancer progression. Immunohistochemical analysis of 70 specimens on formalin-fixed paraffin sections was performed. Furthermore in vitro, Western blot analysis was used to study the relationship between PPARγ and Skp2. We found that the expression of PPARγ and Skp2 expression was inverse correlation whether in vivo or in vitro. In addition, PPARγ overexpression can down-regulate the expression of Skp2 mRNA and protein in breast cancer cells. PPARγ overexpression decreased breast cancer cell proliferation and induced spontaneous apoptosis even in the absence of exogenous ligand. These PPARγ-overexpressing cells were dramatically more sensitive to PPARγ ligand-induced apoptosis compared with parental or Myc-control transfected cells. Overexpressing of Skp2 partially reversed PPARγ's pro-apoptotic and anti-proliferative abilities. These results suggested that PPARγ's pro-apoptotic and anti-proliferative abilities appear to be triggered at least in part by the modulation of Skp2.