Sensitive MCF-7 Cells Research Articles

A let-7b binding site SNP in the 3'-UTR of the Bcl-xL gene enhances resistance to 5-fluorouracil and doxorubicin in breast cancer cells.

The development of acquired resistance to chemotherapy is a major obstacle in the successful treatment of cancer. In breast cancer cells, B-cell lymphoma-extra large (Bcl-xL) is involved in the development of resistance to various chemotherapeutic agents; therefore, preliminary biological prediction was performed to identify a putative binding site for let-7b in the 3′-untranslated region (UTR) of the Bcl-xL gene and a single nucleotide polymorphism (SNP) within this binding region. The present study investigated the association between the SNP rs3208684 A>C and chemotherapeutic agent resistance in breast cancer cells. The data indicated that let-7b negatively regulates the expression of Bcl-xL and appears to sensitize MCF-7 cells to the chemotherapeutic agents 5-fluorouracil (5-FU) and doxorubicin. Furthermore, the SNP rs3208684 A>C was demonstrated to enhance Bcl-xL protein expression by disrupting the binding of let-7b to the 3′-UTR of Bcl-xL and, in MCF-7 cells, overexpression of let-7b in the presence of a mutant Bcl-xL 3′-UTR (C allele) significantly increased 5-FU and doxorubicin resistance. Thus, the results of the present study demonstrate that the SNP rs3208684 A>C may upregulate Bcl-xL protein expression and enhance the resistance of the MCF-7 cells to 5-FU and doxorubicin by decreasing the binding of let-7b to the 3′-UTR of Bcl-xL.

Codelivery of DNA and siRNA via arginine-rich PEI-based polyplexes.

In this study, we formulated polyplexes with different compositions for codelivery of DNA and small-interfering RNA (siRNA). Since DNA and siRNA have distinctive and complementary morphological characteristics (DNA is long and winding and siRNA is short and rigid), we hypothesized that their codelivery using polyplex would enhance each other's transfection. To test this hypothesis, cationic polymer branched polyethylenimine (bPEI) as a standard transfecting agent and its derivative arginine-rich oligopeptide-grafted bPEI modified with polyethylene glycol (P(SiDAAr)5P3), synthesized in our laboratory, were used as carriers for transfection. Polyplexes at different nucleic acid to polymer weight ratios were characterized for transfection in breast cancer sensitive (MCF-7) and resistant (MCF-7/Adr) cell lines. Gene silencing effect of polyplexes was determined in MDA-MB-231-luc-D3H2LN cell line. The results demonstrated that the polyplexes formed with derivative P(SiDAAr)5P3 show significantly lower toxicity compared to polyplexes formed using bPEI. Further, codelivery resulted in 20-fold higher DNA transfection and 2-fold higher siRNA transfection as compared to the respective single nucleotide delivery. DNA transfection was ∼100-fold lower in resistant MCF-7/Adr cells than in sensitive MCF-7 cells. Confocal imaging and flow cytometry data demonstrated that enhanced transfection does not solely depend on DNA's cellular uptake, suggesting that other mechanisms contribute to increased transfection. DNA-co-siRNA delivery could be a promising therapeutic approach to achieve synergistic effects because it can simultaneously target and interfere with multiple regulatory levels in a cell to halt and reverse disease progression.

Combination of survivin siRNA with neoadjuvant chemotherapy enhances apoptosis and reverses drug resistance in breast cancer MCF-7 cells

Chemotherapeutic resistance is a main problem in clinical breast cancer therapy. The purpose of our study is to investigate whether the combination of neoadjuvant chemotherapy and survivin siRNA treatment could enhance the therapeutic effect of neoadjuvant chemotherapy using paclitaxel or epirubicin. The molecular cloning technique was applied to construct the expression vector of siRNA against survivin. Effectene Transfection Reagent was used to transfect plasmids to MCF-7 cells. Survivin expressions were detected by quantitative real-time PCR (qRT-PCR) and Western blot methods. The effect of paclitaxel or epirubicin, with or without the combination of survivin siRNA treatment, on drug susceptibility of MCF-7 cells was detected by CCK-8 assay. MCF-7 cell apoptosis was detected by Flow Cytometry. Survivin siRNA effectively inhibited the expression of Survivin RNA and protein levels (P < 0.05). Both paclitaxel and epirubicin can suppress the proliferation of MCF-7 cells and induce apoptosis to a certain degree respectively. The combination of survivin siRNA with the two chemotherapy drugs significantly enhanced both effects of the two chemotherapeutics respectively (P < 0.05). Survivin siRNA combined with the neoadjuvant chemotherapy can significantly enhance the sensitivity of MCF-7 cells to chemotherapeutics and cell apoptosis. This technology has important potential value in the therapeutic study of breast cancer.

Autophagy inhibitor sensitizes MCF-7 breast cancer cells to novel cyclic tetrapeptide CTS203-induced caspase-9-dependent apoptotic cell death.

Histone deacetylase (HDAC) inhibitors have been demonstrated to be effective anti-cancer candidates against aggressive malignancies. In previous study, anovel hydroxamic acid derivate, CTS203 (cyclo(-l-Asu(NHOH)-l-A3mc6c-l-Phe-d-Pro-)), demonstrated promising HDAC inhibitory activity. Herein, more biological evaluations including cell viability, cell cycle distribution, cellular morphology, expression quantification as well as protein-protein interactions were measured to investigate its cytotoxic mechanism. Corresponding with its significant HDAC inhibitory activity, CTS203 led to increased acetylation of H3K14, cell cycle arrest as well as consequent apoptotic cell death, with bearable influence on the viability of normal cells. However, schedule-dependent cytotoxicity against MCF-7 breast cancer cells revealed adelayed cellular response to chemo-stimuli. Within this corresponding period, autophagy was rapidly triggered once exposure started, whereas autophagy inhibitor sensitized MCF-7 cells to CTS203, exhibiting synergistically anti-proliferative effects. The expression variation in MCF-7 cells revealed that the cleavage of Beclin 1 mediated by caspase-8 resulted in disabled autophagy, thus ultimately facilitated and fastened caspase-9-dependent apoptotic cell death. Taken together, these findings elucidated the mechanism of CTS203-induced cytotoxicity as well as suggested that appropriate manipulation of autophagy would be an adjunctive strategy to enhance HDAC inhibitor-induced cell death. cyclic tetrapeptide, apoptosis, autophagy, HDAC inhibitor, Beclin 1 cleavage.

Ionizing radiation sensitizes breast cancer cells to Bcl-2 inhibitor, ABT-737, through regulating Mcl-1.

Breast-conserving surgery followed by radiation therapy has become the standard of care for early stage breast cancer. However, there are some patients that develop a local failure. We have previously shown that Bcl-2 overexpression was associated with an increased risk of local recurrence in patients with early stage breast cancer. The purpose of this study was to explore an approach to overcome radiation resistance by targeting pro-survival Bcl-2 family proteins in breast cancer cells. The breast cancer cell lines MCF-7, ZR-75-1 and MDA-MB231 were used in this study. siRNAs were employed to silence myeloid cell leukemia 1 (Mcl-1). A small molecule inhibitor of Bcl-2, ABT-737, was used to target anti-apoptotic Bcl-2 family proteins. Apoptosis was identified by FITC Annexin V, PI staining and Western blot analysis. The sensitivity to ionizing radiation and ABT-737 were measured by clonogenic assays. The effect of radiation and ABT-737 was also tested in a MCF-7 xenograft mouse model. Our data demonstrate that the combination of ABT-737 and radiation-induced apoptosis had an inhibitory effect on breast cancer cell proliferation. However, treatment with ABT-737 resulted in elevated Mcl-1 in breast cancer cell lines. Targeting Mcl-1 by siRNA sensitized MCF-7 cells to ABT-737. We revealed that radiation blunted Mcl-1 elevation induced by ABT-737, and that radiation downregulated Mcl-1 by promoting its degradation. Our results indicate that radiation and ABT-737 exert a synergistic effect on breast cancer cell lines through downregulating Mcl-1 and activating the bak-apoptotic pathway. These results support the combination of radiation and pro-survival Bcl-2 family inhibitor as a potential novel therapeutic strategy in the local-regional management of breast cancer.

Identification of a Putative Tdp1 Inhibitor (CD00509) by in Vitro and Cell-Based Assays

Mutations of DNA repair pathways contribute to tumorigenesis and provide a therapeutic target for synthetic lethal interactions in tumor cells. Given that tyrosyl-DNA phosphodiesterase 1 (Tdp1) repairs stalled topoisomerase-I DNA complexes, we hypothesized that inhibition of Tdp1 has synthetic lethal effects in some cancers. To test this, we screened tumor arrays for Tdp1 expression and observed that Tdp1 is expressed in many tumors, including more than 90% of human breast tumors. Subsequent chemical screening identified putative Tdp1 inhibitors. Treatment of control human mammary epithelial cells and the breast cancer cell line MCF-7 with compound CD00509 preferentially sensitized MCF-7 cells to camptothecin and decreased cell proliferation 25% more than camptothecin treatment alone. This suggests that CD00509 specifically targeted Tdp1 in vitro, and CD00509 increased the sensitivity of wild-type murine embryonic fibroblasts (MEFs) to camptothecin to a degree comparable to that of Tdp1–/– MEFs. In addition, consistent with poly ADP-ribose polymerase-1 (PARP-1) collaborating with Tdp1 in DNA repair, combined Tdp1 and PARP-1 inhibition was more detrimental to MCF-7 cells than either treatment alone, whereas the combination was not additively harmful to control mammary cells. We conclude that targeting Tdp1 in anticancer therapy preferentially enhances the sensitivity of some breast cancer cells to camptothecin and may be an effective adjuvant for breast cancer therapy.

Poly (DL-Lactic-Co-Glycolic Acid) Microparticle- Doxorubicin Formulations for Anti-cancer Drug Delivery

Cancer is a group of diseases in which normal cells are converted to cells capable of autonomous growth and invasion. In the chemotherapeutic control of cancer, drugs are usually given systemically so they reach toxic levels in cancer cells. This causes serious side effects in healthy cells. Another important problem with chemotherapy is resistance developed to cytotoxic drugs (multi drug resistance). As a possible solution to these problems, in the present study, two different microparticle fabrication methods were compared; double emulsion solvent evaporation (SE) method for encapsulation of Doxorubicin into PLGA microparticles. The most appropriate method was the SE techniquies which lead to higher encapsulation efficiencies. Processing factors were evaluated for their effects on encapsulation efficiency and results indicated that any change that hinder drug diffusion would result in increased drug content in microparticles. To asses the cytotoxicities of synthesized microparticles, cell proliferation assays were performed with XTT reagent on Doxorubicin resitant and sensitive breast cancer cell lines, MCF-7. DOX entrapped microparticles was effective on both sensitive and DOX resistant MCF-7 cells. The concentration of drug in resistant cancer cells was increased indicating a partial reversal of drug resistance. The results of this study will provide new insights to the development of new drug delivery systems for cancer therapy.

Abstract 5369: Effects of resveratrol on chemotherapeutic agent 5-fluorouracil induced DNA damage in prostate and breast cancer cells

Abstract Resveratrol is known as a powerful antioxidant present in many foods, especially in grapes and red wine. Recent studies have shown that diets rich in antioxidants, such as Resveratrol, may significantly lower an individual's risk for developing cancer and reduce the chances of metastasis in cancer patients. Previous studies have also shown that cancer patient exposure to 5-fluorouracil (5-FU), a widely used chemotherapeutic agent typically used to treat colon, rectum, head, and neck cancers, causes cell death and apoptosis in cancer cells through various mechanisms, including DNA damage. This study investigated the effects of high concentrations of Resveratrol in human prostate cancer (PC3) cells and human breast cancer (MCF-7) cells exposed to relatively large doses of 5-FU, through the use of the Alkaline Comet Assay. PC3 and MCF-7 cells were exposed to 5-FU at concentrations of 100 µM, 200 µM, 400 µM, and 500 µM for one hour, and then subsequently exposed to Resveratrol at concentrations of 25 µM, 50 µM, and 100 µM for another hour. DNA damage was quantitatively measured using the % DNA in Tail parameter. We found a dose-dependent positive correlation between 5-FU exposure and DNA damage in both cell lines. The addition of Resveratrol in PC3 cells also produced a mean reduction in DNA damage of 45.10% in 25 µM samples, 61.42% in 50 µM samples, and 72.86% in 100 µM samples, in comparison to samples exclusively exposed to 5-FU. Similar trends were also observed in MCF-7 cells, and there was a significant difference between the treated and negative control samples (p &amp;lt; 0.001). In addition, when comparing cell lines, we recorded higher DNA damage values in MCF-7 cells than in PC3 cells when exposed to 5-FU. There was also a greater reduction in DNA damage in PC3 cells than in MCF-7 cells with the addition of Resveratrol in all 5-FU treated samples. These results may possibly indicate higher sensitivity of MCF-7 cells to 5-FU and lower sensitivity to Resveratrol. To further investigate possible underlying mechanisms of Resveratrol in ameliorating DNA damage caused by 5-FU, we performed the Oxygen Radical Absorbance Capacity (ORAC) Assay to determine antioxidant capacity. The antioxidant capacity of various concentrations of resveratrol and 5-FU in conjunction were calculated as Trolox equivalents per liter (TE/L) using individually generated Trolox standard curves. We found that Resveratrol exhibits high antioxidant capacity levels, while 5-FU exhibits limited antioxidant capacity levels. There was a dose-dependent increase of antioxidant capacity measured by the ORAC Assay for both compounds. More research is necessary to further elucidate the mechanisms and pathways involved in DNA repair in cancer cells exposed to high doses of Resveratrol and 5-FU. Citation Format: Michael Xiao, Irene Yeung, Gaju Shrestha, Luis Camberos, Richard A. Robison, Kim L. O'Neill. Effects of resveratrol on chemotherapeutic agent 5-fluorouracil induced DNA damage in prostate and breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5369. doi:10.1158/1538-7445.AM2014-5369

Abstract 5488: Everolimus inhibits the proliferation of aromatase inhibitor-resistant breast cancer cells

Abstract Aromatase inhibitors (AIs) are currently the standard treatment for estrogen receptor positive breast cancer in postmenopausal women. Unfortunately, the majority of patients treated with AIs eventually develop resistance to these drugs. The mechanism by which AI resistance occurs is still not known, however, there is strong evidence that upregulation of the PI3K/Akt/mTOR pathway might play an important role. As such, mTOR inhibition is now under study in clinical trials as a breast cancer treatment. The newest mTOR inhibitor, Everolimus (Afinitor®), has been FDA approved for the treatment of metastatic breast cancer. We have previously reported the development of two AI-resistant breast cancer cell lines (MCF-7:5C and MCF-7:2A) which were clonally derived from parental MCF-7 cells following long term estrogen deprivation. Both MCF-7:5C and MCF-7:2A cells have been shown to overexpress members of the PI3K/Akt/mTOR pathway. In this study, we examined the therapeutic benefits of everolimus in AI-resistant MCF-7:5C and MCF-7:2A cells compared to endocrine sensitive MCF-7 breast cancer cells and normal breast epithelium MCF10A cells. Results from cell viability assay showed that everolimus markedly inhibited the proliferation of endocrine sensitive MCF-7 cells and AI-resistant MCF-7:5C and MCF-7:2A cells in a time-dependent and concentration-dependent manner with IC50s of 30 nM, 40 nM and 10 nM, respectively. Notably, the MCF-7:2A cells were the most sensitive to everolimus which caused a 70% reduction in proliferation at 24 hours whereas in MCF-7 and MCF-7:5C cells only 30% inhibition was observed at the same time point. The inhibitory effect of everolimus was due primarily to G1 arrest and was associated with significant downregulation of phospho-mTOR, phospho-p70S6K, phospho-Akt, GSK3β, cyclin D1 and p21. Colony formation assay in soft agar indicated that AI-resistant MCF-7:5C and MCF-7:2A cells formed significantly more colonies than MCF-7 cells and that treatment with everolimus notably reduced the size and number of colonies for all three cell lines. Finally, we found that exposure of AI-resistant MCF-7:5C and MCF-7:2A cells to a single dose of gamma-irradiation (6 Gy) significantly enhanced the anti-proliferative effect of everolimus on these cells. In summary, these studies showed that everolimus significantly inhibited the proliferation and tumorigenicity of AI-resistant breast cancer cells and that everolimus might be an effective treatment option for patients with AI-resistant breast cancer. Citation Format: Asona Lui, Joan Lewis-Wambi. Everolimus inhibits the proliferation of aromatase inhibitor-resistant breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5488. doi:10.1158/1538-7445.AM2014-5488

Indirubin derivatives alter DNA binding activity of the transcription factor NF-Y and inhibit MDR1 gene promoter

Indirubin derivatives exert antitumor activity. However, their effects on the expression of multidrug resistance gene 1 (MDR1) have not been investigated. Here we found three derivatives that inhibit the MDR1 gene promoter. To investigate the effects of indirubins on the DNA binding of NF-Y, a major MDR1 gene transcription factor that recognizes an inverted CCAAT element in the promoter, gel mobility shift assay was performed using the element as a probe with nuclear extracts from NG108-15, MCF7, HepG2, C2C12, and SK-N-SH cells. Among 17 compounds, 5-methoxyindirubin inhibited the DNA binding of NF-Y significantly, whereas indirubin-3′-oxime and 7-methoxyindirubin 3′-oxime increased the binding considerably. After evaluating a suitable concentration of each compound for transcription analysis using living tumor cells, we performed a reporter gene assay using a reporter DNA plasmid containing EGFP cDNA fused to the MDR1 gene promoter region. Indirubin-3′-oxime exerted a significant inhibitory effect on the MDR1 promoter activity in MCF7 and HepG2 cells, and 5-methoxyindirubin inhibited the activity only in MCF7 cells; 7-methoxyindirubin 3′-oxime suppressed the activity in all of the cell lines. We further confirmed that the compounds reduced endogenous MDR1 transcription without any inhibitory effect on NF-Y expression. Moreover, each compound increased the doxorubicin sensitivity of MCF7 cells. These results indicate that each indirubin derivative acts on the DNA binding of NF-Y and represses the MDR1 gene promoter with tumor cell-type specificity.

Ribosomal protein L19 overexpression activates the unfolded protein response and sensitizes MCF7 breast cancer cells to endoplasmic reticulum stress-induced cell death

Although first identified for their roles in protein synthesis, certain ribosomal proteins exert pleiotropic physiological functions in the cell. Ribosomal protein L19 is overexpressed in breast cancer cells by amplification and copy number variation. In this study, we examined the novel pro-apoptotic role of ribosomal protein L19 in the breast cancer cell line MCF7. Overexpression of RPL19 sensitized MCF7 cells to endoplasmic reticulum stress-induced cell death. RPL19 overexpression itself was not cytotoxic; however, cell death induction was enhanced when RPL19 overexpressing cells were incubated with endoplasmic reticulum stress-inducing agents, and this sensitizing effect was specific to MCF7 cells. Examination of the cell signaling pathways that mediate the unfolded protein response (UPR) revealed that overexpression of RPL19 induced pre-activation of the UPR, including phosphorylation of pERK-like ER kinase (PERK), phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α), and activation of p38 MAPK-associated stress signaling. Our findings suggest that upregulation of RPL19 induces ER stress, resulting in increased sensitivity to ER stress and enhanced cell death in MCF7 breast cancer cells.

Epigallocatechin-3-gallate potentiates the effect of curcumin in inducing growth inhibition and apoptosis of resistant breast cancer cells.

Drug resistance remains an on-going challenge in breast cancer chemotherapy. Combination of two or more drugs is an effective strategy to access context-specific multiple targets and overcome undesirable toxicity that is almost inevitable in single-drug chemotherapy. Many plant food-derived polyphenolic compounds have been proven to modulate many key factors responsible for cancer drug resistance, which makes them a promising group of low toxicity candidates for reversing cancer resistance. In this study, we analyzed the combination effect of two chemopreventive polyphenols, curcumin (Cur) and epigallocatechin-3-gallate (EGCG), in combating resistant breast cancer. Our present results showed that EGCG significantly enhanced the growth inhibition and apoptosis in both doxorubicin (DOX)-sensitive and resistant MCF-7 cells induced by Cur. The mechanism may be related to the further activation of caspase-dependent apoptotic signaling pathways and the enhanced cellular incorporation of Cur by inhibiting P-glycoprotein (P-gp) pump function. Moreover, Cur and EGCG in combination could enhance the toxicity of DOX and increase the intracellular level of DOX in resistant MCF-7 cells. Our findings with this practical combination of Cur and EGCG encourage us to move on to a promising strategy for successful treatment of human breast cancer resistance by combining two low-toxic chemotherapeutic agents from diet.

Combination of Ethanolic Extract of Citrus aurantifolia Peels with Doxorubicin Modulate Cell Cycle and Increase Apoptosis Induction on MCF-7 Cells.

New approach of breast cancer therapy is developed toward combination therapy with agents that have a specific molecular target. Our previous study showed that Citrus aurantifolia lime peels ethanolic extract (CPE) increased the sensitivity of MCF-7 cells againts doxorubicin. This study aims to observe the mechanism of combination CPE and doxorubicin in cell cycle modulation and apoptosis on MCF-7 cells. The assays were performed in the study were cell cycle assay, apoptosis induction, and immunocytochemistry of MCF-7 cells.The effect on the modulation of cell cycle and apoptosis were observed by flowcytometry assay in both single dose of CPE and its combination with Doxorubicin. Cell cycle distribution were observed with flowcytometer FACS-Calibur and its data was analyzed by Cell Quest program. Apoptotic induction in MCF-7 cells was examined using acrydine orange-ethidium bromide (AO-EtBr) double staining. Immunocytochemistry assay was done to observe the expression of apoptotic regulation protein p53 and Bcl-2. The result showed that CPE 6 μg/mL induced apoptosis and cell accumulation at G1 phase, while CPE 15 μg/mL induced apoptosis and cell accumulation at G2/M phase. The combination of doxorubicin 200 nM with CPE 6 μg/mL increased apoptosis induction than their single treatment, and cell accumulation at G2/M phase. Evidence of apoptosis and protein expression of p53 and Bcl-2 indicated that both single applications and combinations of CPE and doxorubicin are able to increase apoptotic bodies of MCF-7 cells by increasing the proteins expression. This result suggested that CPE could perform as co-chemotherapeutic agent with doxorubicin on breast cancer cells.

Cisplatin (CDDP) triggers cell death of MCF-7 cells following disruption of intracellular calcium ([Ca(2+)]i) homeostasis.

Breast cancer (BC) is a public health problem all over the world. Cisplatin (CDDP) is an antineoplastic agent with high rate of success in treating cancers. The down side of CDDP treatment is the development of chemo-resistance. Beside DNA damage and activation of p53 signaling pathway, CDDP induces tumor-cell death due to elevation in the intracellular calcium concentration ([Ca(2+)]i).However, the role of [Ca(2+)]i in CDDP induced apoptosis of breast cancer cells (MCF-7) is not well understood. Here we investigate the cytotoxic effects of CDDP in relation to [Ca(2+)]i homeostasis in MCF-7-sensitive and -resistant cell lines. Live-cell imaging using [Ca(2+)]i sensitive fluorescent dyes was employed to monitor [Ca(2+)]i CDDP treated MCF-7 cells (0.001-10 µM) and [Ca(2+)]i modulators i.e. Caffeine (10 mM); Nimodipine (10 µM); Ionomycin (10 µM); Thapsigargin (500 nM). A concentration-dependent increase of[Ca(2+)]i was observed in CDDP MCF-7 treated cells. From the concentration range tested 100 nM CDDP triggered the highest [Ca(2+)]i increase (120%; n = 19)while in drug resistant MCF-7 cells the effects of CDDP on [Ca(2+)]i were reduced as compared with the drug sensitive MCF-7 cells. Furthermore, the CDDP induced cell death correlates with the increase of [Ca(2+)]i, and thus, significantly lower in the CDDP desensitized cells (p < 0.05). Pre-application of the calcium channel blocker, Nimodipine reduced [Ca(2+)]i elevation significantly (46.6% increase; n = 26) as well as when a pre-application of Caffeine, Ionomycin or Thapsigargin occurred followed by the subsequent application of CDDP (n = 15; 37.8%, n = 32; 34.9%, n = 21; 53.7% increase respectively).

Abstract P3-03-01: The role of a newly discovered N-terminal methyltransferase in breast cancer

Abstract The recently discovered N-terminal methyltransferase, NRMT, is the first eukaryotic N-terminal methyltransferase to be identified. NRMT is a highly conserved, ubiquitously expressed, nuclear MT. After methionine cleavage, it methylates the X-P-K amino-terminal consensus sequence of its substrates. Loss of N-terminal methylation has been associated with decreased ability to bind to DNA and decreased protein stability. Many NRMT targets are known tumor suppressors or oncogenes, including DDB2, RB, SET, and NEMO. Knockdown of NRMT in HEK cells results in increased cell proliferation and multi-spindle formation, which are characteristics of malignant cells. In addition, microarray analysis has shown that NRMT expression can be either up or down regulated in a variety of human cancers. Our preliminary data in human breast cancer samples verifies the microarray data and shows that NRMT mRNA levels are both significantly increased or decreased in a patient specific manner. NRMT expression levels do not seem dependent on the type of tumor, therefore, we hypothesize that NRMT expression levels may be dependent on the endocrine sensitivity of the tumor. To test if NRMT activity differentially affects endocrine sensitive and endocrine resistant cell types in vitro, we knocked down NRMT expression in a panel of human breast cancer cell lines with decreasing endocrine sensitivity using shRNA. Cells expressing the shRNA against human NRMT (or mouse NRMT as a control) were then assayed for proliferation and migration potential. Knockdown of NRMT significantly increased proliferation of the endocrine sensitive MCF-7 and T47D cancer cell lines over a five-day time course. In contrast, NRMT knockdown only slightly inhibited LCC9 (an antiestrogen-resistant MCF-7 variant line) proliferation and had no effect on endocrine resistant MDA-MB-231 cells. NRMT knockdown also significantly inhibited MCF-7 cell migration at 72 hours but only slightly affected LCC9 cells and had no affect on the MDA-MD-231 cells. These data indicate that NRMT loss early in tumorigenesis has the potential to increase the growth rate of endocrine sensitive primary tumors but has little affect on endocrine resistant cells. We are now interested in determining if we can increase endocrine resistant cell proliferation and migration through NRMT overexpression, thus explaining the microarray profiles. Taken together these studies indicate that N-terminal methylation by NRMT is a biologically important post-translational modification with regard to cell division and tumorigenesis in breast cancer. Next, we will test if NRMT loss can confer endocrine resistance to endocrine sensitive cells and use a murine mammary gland transplantation and regeneration model to study the effect of NRMT knockdown on mammary tumorigenesis and metastasis in vivo. Understanding the general biological function of N-terminal methylation and its relationship to endocrine sensitivity could lead to the use of NRMT expression levels as a biomarker for tumors likely to develop resistance to endocrine therapies. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-03-01.

Abstract P6-04-03: ERb and breast cancer: A potential predictive and prognostic biomarker and novel therapeutic drug target

Abstract Background: Estrogen receptor beta (ERβ), unlike ERα, classically functions as a tumor suppressor in vitro. However, ERβ's biological functions in vivo and predictive/prognostic value in breast cancer are controversial. Methods: Expression of ERβ protein was determined using a well characterized and validated ERβ specific monoclonal antibody that only recognizes the full-length form of this receptor (PPG5/10) in the following 3 cohorts: 1) a cohort with all breast cancer subtypes (n = 182), 2) a prospective NCCTG adjuvant tamoxifen trial for postmenopausal women with ERα positive breast cancer with long-term follow-up (n = 198) and 3) a cohort of 80 triple negative breast cancers (TNBCs). To elucidate the biological functions of ERβ in breast cancer, novel ERβ expressing MCF7 and MDA-MB-231 cell lines were developed and characterized using multiple techniques and were examined for responsiveness to various ERβ targeted therapies. Results: About one-third of all breast tumors, regardless of sub-type, were shown to express nuclear ERβ and this expression was independent of ERα or HER2. In the NCCTG 89-30-52 cohort, breast cancer recurrence rates were significantly associated with ERβ protein expression with 10 year recurrence rates of 25%, 15% and 4% for zero, low or high levels of ERβ expression respectively. Interestingly, in TNBCs, nuclear ERβ was expressed at intermediate or high levels in 24% of tumors. In the triple negative MDA-MB-231 cell line, expression of ERβ led to inhibition of proliferation and induction of apoptosis in response to estrogen and multiple ERβ specific agonists. Conversely, these same treatments induced proliferation of ERβ-expressing MCF7 cells which endogenously express ERα. However, ERβ expression sensitized MCF7 cells to the anti-proliferative effects of anti-estrogens. Microarray analysis and RT-PCR profiling of MDA-MB-231-ERβ cells revealed that estrogen and ERβ agonists highly induced the expression of multiple cystatins, a family of small secreted cysteine protease inhibitors which function as tumor suppressors, and potently inhibited canonical TGFβ signaling. Conditioned media isolated from estrogen or ERβ agonist treated MDA-MB-231-ERβ cells suppressed the proliferation rates and inhibited TGFβ signaling in other TNBC cell lines, effects that were completely reversed following the depletion of cystatins from the conditioned media. Conclusions: These data demonstrate that ERβ is expressed in a substantial proportion of breast cancers and may have value as a predictive and/or prognostic biomarker. Therapeutic targeting of ERβ may have clinical benefit in multiple breast cancer sub-types; however, the specific drug of choice may vary based on ERα status. Specifically, we have demonstrated that ERβ expression in ERα+ MCF7 cells sensitizes them to the effectiveness of anti-estrogens, an observation that was confirmed in women enrolled in a prospective adjuvant tamoxifen trial. In TNBCs, where targeted therapies are lacking, our data suggest that targeting ERβ with either estrogen or ERβ specific agonists will elicit anti-tumor effects through the induction of cystatins and inhibition of TGFβ signaling resulting in tumor regression and improved patient outcomes. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-04-03.

Abstract P5-09-13: HDAC inhibition re-sensitizes acquired hormone resistant cells to the cytotoxic effect of tamoxifen

Abstract Breast cancer still continues to be a major cause of cancer related deaths in women, second only to lung cancer. Administering anti-estrogens and aromatase inhibitors, both in the adjuvant and metastatic settings, in estrogen receptor (ER) positive disease, is one of the most effective treatment strategies. However, prolonged exposure to these drugs leads to the emergence of resistance in about 40% of those who initially respond. Hence, the emphasis of a multitude of studies is to understand the underlying mechanisms of this acquired resistance and implementing means to overcome it. Recent studies have implicated epigenetic modulation of gene expression in the development of resistance to the anti-estrogen tamoxifen. A clinical trial conducted by our group has demonstrated the efficacy of combining the HDAC inhibitor vorinostat with tamoxifen in patients who had progressed on prior anti-estrogen therapies and showed a total 40% clinical benefit (19% objective response and 21% stable disease for more than 6 months). In an effort to identify patients most likely to benefit from this novel therapy, we sought to elucidate the mechanism behind the clinical efficacy of this combination. To this end, we have generated an in vitro tamoxifen resistant cell line (TAMR) by long-term exposure of MCF7 cells to 4-hydroxy tamoxifen. Significantly reduced anti-proliferative effect of tamoxifen and other anti-estrogens in TAMR compared to the sensitive MCF7 cells demonstrates the establishment of resistance to anti-estrogens in this cell line. In TAMR cells, addition of an HDAC inhibitor reverts resistance to tamoxifen. Although estrogen receptor (ER) expression in TAMR cells appears unaltered, the classical genomic signaling of ER in these resistant cells is suppressed and unresponsive to ligands, as deduced from transcription of ER response genes and luciferase assay of an ERE-luciferase construct. However, the ER remains important, since siRNA mediated depletion of ER inhibits cell growth in TAMR cells. Treatment with the dual EGFR/Her2 kinase inhibitor or an AKT inhibitor significantly inhibits the growth of these cells only when combined with tamoxifen, indicating the importance of crosstalk between pathways. Furthermore, Akt and mTOR protein and activities are down regulated by HDAC inhibition, which are further reduced when combined with tamoxifen. SiRNA mediated depletion of ER leads to reduced Akt and mTOR activities which suggests that ER may act as a driver, possibly through its non-genomic function at the plasma membrane activating members of the growth factor signaling pathways. Studies are ongoing to further characterize the interaction of HDAC and ER inhibition on these signaling pathways and to determine their significance to resistance. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-09-13.

Abstract PD5-2: Preclinical assessment of HDAC inhibitor entinostat combined with all trans retinoic acid (ATRA) in aromatase inhibitor resistant breast cancer

Abstract Treatment with aromatase inhibitors (AI’s) is highly effective against breast cancer in ER positive postmenopausal women. However, some patients eventually become resistant to AIs. Tumor initiating cells (TIC’s) represent a subpopulation of tumor cells, which show self-renewal capacity. We are focused towards discovering strategies to reduce the growth of breast cancer TICs, which may result in resistance. We have developed a xenograft model that mimics post-menopausal hormone responsive breast cancer. In this model, aromatase transfected human hormone sensitive MCF-7 cells (MCF-7Ca) are inoculated in ovariectomized athymic nude mice and allowed to grow in presence of D4A (aromatizable substrate of estrogen). Results obtained using this model have been confirmed by numerous clinical trials. Using this model, we have established that single agent AI is better than tamoxifen in controlling tumor growth. We also observed that although, AI letrozole provides a longer control over tumor growth, tumors eventually began to grow. In the current study, we investigated the effect of ATRA (All-trans Retinoic acid) (125μg/day, ip) and a histone deacetylase (HDAC) inhibitor entinostat (SNDX- 275) (50μg/day, po) with or without letrozole on letrozole resistant tumors in a xenograft model system. Ovariectomized athymic nude mice bearing xenografts of MCF-7Ca cells, were treated with letrozole till they became resistant (15 weeks). At this time, the mice were grouped to receive ATRA, entinostat plus ATRA or the combination of ATRA plus entinostat plus letrozole till week 23. The mice treated with entinostat plus ATRA letrozole showed a significant decrease in tumor growth rate compared to mice treated with single agents or entinostat plus ATRA (p&amp;lt;0.0001, p = 0.02). On week 20 weeks, 2 mice from each treatment group were euthanized and tumors were harvested. The tumors were digested enzymatically with collagenase and hyaluronidase and freed of debris using centrifugation and filtration. Mammosphere forming ability of TICs in the tumor tissue was measured by seeding 10,000 viable cells from each treated tumors under non-adherent conditions to access the self-renewal capacity. Quantitative PCR analysis of tumors cells showed a significant downregulation of the known TIC molecular markers, BCRP, ALDH, BMI-1 and Nanog compared to letrozole treated tumors. Similar results were also obtained when LTLT-Ca (long term letrozole treated MCF-7Ca) cells treated with ATRA and entinostat in combination with letrozole and then seeded in non-adherent conditions. The combination of ATRA plus ENT plus letrozole significantly (p&amp;lt;0.01) reduced number of mammospheres formed compared to single agents alone. We have shown previously, that LTLT-Ca cells have higher percentage of side population (cells expressing higher level of efflux pumps such as BCRP) compared to MCF-7Ca cells. The treatment of LTLT-Ca cells with the combination of ENT and ATRA with letrozole drastically reduced the percentage of side population. Overall, these studies indicate that the combination of ATRA, entinostat and letrozole is effective in reducing tumor recurrence in letrozole resistant tumors. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr PD5-2.

A novel long non-coding RNA-ARA: Adriamycin Resistance Associated

Long non-coding RNAs (lncRNAs) are emerging as an integral functional component of human genome and have been investigated as critical regulators in molecular biology of cancer. A recent study reported that lncRNA-UCA1 induced drug resistance in adriamycin chemotherapy. However, the contributions of lncRNAs to adriamycin resistance in cancers remain largely unknown. To address this issue, we performed a genome-wide lncRNA microarray analysis in adriamycin resistant MCF-7/ADR and parental MCF-7 cells, and revealed differential expression of lncRNAs in distinct category and chromosome distribution patterns. A specific differentially expressed lncRNA (Adriamycin Resistance Associated, termed ARA) was validated in MCF-7/ADR and HepG2/ADR cells. ARA is derived from an intron of PAK3 gene, predicted to contain several stable hairpins in secondary structure and has conservative sequences in primates. ARA expression is significantly associated with adriamycin sensitivity in a panel of breast and liver cancer cell lines and is markedly up-regulated in parental sensitive MCF-7 and HepG2 cell lines after receiving adriamycin treatment. The functions of ARA were assessed by silencing this lncRNA in vitro, and we found that ARA knockdown reduced the proliferation, induced cell death, G2/M arrest and migration defects. Furthermore, microarray transcriptomic analysis was carried out to comprehensively depict the ARA-regulated genes. We showed that ARA can modulate multiple signalling pathways, including MAPK signalling pathway, metabolism pathways, cell cycle and cell adhesion-related biological pathways, and regulate cellular processes, including transcriptional processes and protein binding function. Overall, our results indicate novel insights of adriamycin resistance in lncRNA level.

Benzo[a]pyrene sensitizes MCF7 breast cancer cells to induction of G1 arrest by the natural flavonoid eupatorin-5-methyl ether, via activation of cell signaling proteins and CYP1-mediated metabolism

Eupatorin-5-methyl ether (E5M) is a flavone containing 4 methoxy groups that is present in plants with medicinal activity, whereas luteolin (L) is a polyhydroxylated flavone commonly encountered in dietary products. In the present study we investigated the interaction of the two flavonoids with cytochrome P450 CYP1 enzymes in breast cancer MCF7 cells. Both compounds induced a dose dependent increase in CYP1A1 and CYP1B1 mRNA levels, as well as in EROD activity, a marker of CYP1 enzyme activity. Induction of cytochrome P450 CYP1 expression by E5M was accompanied by translocation of the ligand-activated transcription factor AhR to the nucleus, as demonstrated by confocal immunofluoresence. More importantly, although E5M was less active than L in inhibiting proliferation of MCF7 cells, when the cells were pretreated with the CYP1 inducer Benzo[a]pyrene (BaP) the potency of E5M was augmented. HPLC and LC–MS analysis revealed that E5M was metabolized to a major conversion product assigned E5M1 resulting from one step demethylation reaction in MCF7 cells whereas L metabolism by recombinant CYP1A1 did not reveal any metabolites. E5M1 production in BaP-induced MCF7 cells was attenuated in the presence of the CYP1A1 inhibitor α-napthoflavone. E5M further induced a dose dependent increase in the cell signaling proteins p21, JNK and p-JNK in MCF7 cells. This effect was enhanced in BaP pretreated cells and was associated with G1 arrest and a small percentage of apoptosis (3.5%). E5M antiproliferative effect in BaP pretreated cells was attenuated in the presence of the CYP1A1 inhibitor α-napthoflavone, as demonstrated by Western blotting and FACS analysis. Taken together the results demonstrate that BaP sensitizes MCF7 cells to E5M antiproliferative activity via enhanced induction of p21, JNK and p-JNK that in turn results by cytochrome P450 CYP1-mediated conversion to the metabolite E5M1.