Receptor-positive MCF-7 Breast Cancer Research Articles

Abstract 5977: Evaluation of the potential combination regimens for q901, a clinical stage selective cdk7 inhibitor, as a DNA damage repair inhibitor

Abstract Background; Q901 is an extremely selective CDK7 inhibitor with potent single agent activity in the multiple in vivo tumor models including small cell lung cancer, cholangiocarcinoma, colon cancer, pancreatic cancer, hormone receptor positive breast cancer, castrate-resistant prostate cancer, and ovarian cancer. Q901 is currently in Phase 1/2 clinical trial for patients with selected advanced solid tumors (ClinicalTrials.gov: NCT05394103). In the preclinical models, Q901 treatment induces DNA damage response (DDR) impairment markers, particularly ones involved in the double-strand break repair mechanisms. This opens up broad range of possibilities for new combination regimen that could benefit from the accumulated DNA damages caused by Q901 treatment. Methods: Various combinations have been tested to evaluate potential clinical regimens in multiple solid tumor models. Two different BRCA wild-type ovarian cancer A2780 and OVCAR3 models, an estrogen receptor positive MCF7 breast cancer model, or a syngeneic RENCA mouse renal cancer model was tested with Q901 in combination with poly ADP-ribose polymerase (PARP) inhibitor, selective estrogen receptor degrader (SERD), an estrogen receptor (ER) PROTAC degrader, or an anti-PD-1 antibody, respectively. Results: In the A2780 model, the PARPi alone group showed 15% TGI (tumor growth inhibition), but significant tumor regression (104% TGI) was observed in the Q901 and PARPi combination. In the OVCAR3 model, the Q901 and PARPi combination group showed 105% TGI, whereas the PARPi alone group showed 38% TGI. In the MCF7 model, SERD alone showed 64% TGI and Q901 combination with SERD showed 94% TGI. Remarkable tumor regression (111% TGI) was observed in the MCF7 model combining ER PROTAC and Q901, whereas ER PROTAC alone group showed 55% TGI. In the RENCA model, the anti-PD-1 antibody alone group showed 13.2% TGI and the combination of Q901 and anti-PD-1 antibody showed 66% TGI. In summary, these preclinical model studies demonstrated significant additive/synergistic effects of the Q901 combination on tumor growth inhibition. Conclusion: The non-clinical data demonstrate that Q901 not only has a potential as a single agent,but can also be used in combination with other therapeutic strategies to develop next-generation cancer therapeutics to potentially address unmet medical needs. Citation Format: Donghoon Yu, Yeejin Jeon, YoonJi Lee, Seung-Joo Lee, Jaeseung Kim, Hyerim Jung, Tae-Kyung Kim, Kiyean Nam. Evaluation of the potential combination regimens for q901, a clinical stage selective cdk7 inhibitor, as a DNA damage repair inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5977.

ADİPONEKTİN’İN MCF-7 PROLİFERASYONU VE GİRELİN SEVİYESİ ÜZERİNE ETKİSİ

Amaç
 Lokal ve sistemik faktörlerin, östrojen reseptörü pozitif
 meme kanseri riskinde artış olan postmenopozal
 obez kadınlarda meme kanseri hücrelerinin büyümesini
 yönlendirdiği gösterilmiştir. Postmenapozal
 dönemdeki obez kadınlarda, dolaşımdaki girelin hormonunun
 azaldığı, bazı tümör hücreleri tarafından
 üretildiği ve ayrıca tümör büyümesini etkilediği gösterilmiştir.
 Bu çalışmada, adiponektin uygulamasının
 östrojen reseptörü pozitif MCF-7 meme kanseri hücre
 hattındaki rolünü ve girelin seviyeleri üzerine olan etkilerini
 araştırmayı amaçladık.
 Gereç ve Yöntem
 MCF-7 meme kanseri hücre hattı 2 boyutlu kültürde
 çoğaltıldı. MCF-7 meme kanseri hücrelerine artan
 konsantrasyonlarda adiponektin (50-5000 ng/mL) ile
 muamele edilerek inhibisyon dozu belirlendi. MCF-7
 meme kanseri hücre hattına 50, 100, 250 ve 500 ng/
 mL konsantrasyonda adiponektin uygulanarak 24, 48
 ve 72. Saatlerde örneklerde girelin seviyeleri enzim
 bağlı immünosorbent analizi (ELISA) ile ölçüldü.
 Bulgular
 24, 48 ve 72. saate alınan MCF-7 meme kanseri hücre
 hattı kültür örneklerinde 50, 100, 250 ve 500 ng/mL
 adiponektin konsantrasyonuna bağlı olarak proliferasyonun
 azaldığı bulundu. 24, 48 ve 72. saate alınan
 MCF-7 meme kanseri hücre hattı kültür örneklerinde
 50, 100, 250 ve 500 ng/mL adiponektin konsantrasyonuna
 bağlı olarak girelin miktarında artış oluştuğu
 bulundu.
 Sonuç
 Çalışmamızda MCF-7 meme kanseri hücre hattında
 (ER/PR pozitif) anti-kanserojenik özelliği kanıtlanmış
 olan ve aynı zamanda obezite ile negatif korelasyon
 gösteren adiponektinin, girelin ile karşılıklı etkileşimlerinin,
 ER pozitif meme kanserlerindeki rolünü ortaya
 koymuş olduk.

STAT3 as a Potential Target for Tumor Suppressive Effects of 15-Deoxy-Δ12,14-prostaglandin J2 in Triple Negative Breast Cancer.

STAT3 plays a prominent role in proliferation and survival of tumor cells. Thus, STAT3 has been considered to be a prime target for development of anti-cancer therapeutics. The electrophilic cyclopentenone prostaglandin,15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) has been well recognized for its capability to modulate intracellular signaling pathways involved in cancer cell growth and progression. We previously reported that 15d-PGJ2 had potent cytotoxicity against harvey-ras transformed human mammary epithelial cells through direct interaction with STAT3. In this study, we have attempted to verify the inhibitory effects of 15d-PGJ2 on STAT3 signaling in human breast tumor cells. The triple negative breast cancer cell lines, MDA-MB-231 and MDA-MB-468 displaying constitutive phosphorylation of STAT3 on the tyrosine 705 (Tyr705) residue, underwent apoptosis upon inhibition of STAT3 by 15d-PGJ2. In contrast, estrogen receptor positive MCF-7 breast cancer cells that do not exhibit elevated STAT3 phosphorylation were much less susceptible to 15d-PGJ2-induced apoptosis as assessed by PARP cleavage. Furthermore, 15d-PGJ2 inhibited interleukin-6-induced tyrosine phosphorylation of STAT3 in LNCaP cells. According to molecular docking studies, 15d-PGJ2 may preferentially bind to the cysteine 259 residue (Cys259) present in the coiled-coil domain of STAT3. Site-directed mutagenesis of STAT3 identified Cys259 to be the critical amino acid for the 15d-PGJ2-induced apoptosis as well as epithelial-to-mesenchymal transition. Taken together, these findings suggest STAT3 inactivation through direct chemical modification of its Cys259 as a potential therapeutic approach for treatment of triple negative breast cancer treatment.

Requirement for cleavage factor IIm in the control of alternative polyadenylation in breast cancer cells.

Alternative polyadenylation (APA) determines stability, localization and translation potential of the majority of mRNA in eukaryotic cells. The heterodimeric mammalian cleavage factor II (CF IIm) is required for pre-mRNA 3′ end cleavage and is composed of the RNA kinase hClp1 and the termination factor hPcf11; the latter protein binds to RNA and the RNA polymerase II carboxy-terminal domain. Here, we used siRNA mediated knockdown and poly(A) targeted RNA sequencing to analyze the role of CF IIm in gene expression and APA in estrogen receptor positive MCF7 breast cancer cells. Identified gene ontology terms link CF IIm function to regulation of growth factor activity, protein heterodimerization and the cell cycle. An overlapping requirement for hClp1 and hPcf11 suggested that CF IIm protein complex was involved in the selection of proximal poly(A) sites. In addition to APA shifts within 3′ untranslated regions (3′-UTRs), we observed shifts from promoter proximal regions to the 3′-UTR facilitating synthesis of full-length mRNAs. Moreover, we show that several truncated mRNAs that resulted from APA within introns in MCF7 cells cosedimented with ribosomal components in an EDTA sensitive manner suggesting that those are translated into protein. We propose that CF IIm contributes to the regulation of mRNA function in breast cancer.

Lemon Balm Extracts Prevent Breast Cancer Progression In Vitro and In Ovo on Chorioallantoic Membrane Assay.

Breast cancer is the most frequently diagnosed malignant pathology, representing the primary cause of cancer death in women. Natural products are an appealing strategy to limit the progression of the disease. Targeting angiogenesis in breast cancer may positively impact on poor prognosis of breast cancer. As source of natural compounds, we investigated the leaves of Melissa officinalis L. (MO), known as lemon balm, an aromatic plant that spontaneously grows in the South and Western areas of Romania, being traditionally recommended as anxiolytic, antispasmodic, or as digestive remedy. Our aim was to investigate the phytochemical profiling and the antiangiogenic and chemopreventive bioactivity of MO from Banat region, on breast cancer. Two ethanolic extracts of MO (MOE96 and MOE70) and one methanolic extract (MOM80) were subjected to polyphenol and triterpene profiling by HPLC-MS, and the antioxidant capacity was evaluated. The antiangiogenic potential was investigated using the chorioallantoic membrane assay (CAM). The MTT(3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide) assay was used to investigate the cytotoxic effects on MCF-7 and MDA-MB-231breast cancer cells, as well as on MCF-10A normal breast epithelial cells, while apoptosis was performed by DAPI staining. Rosmarinic acid (RA) and ursolic acid (UA) were revealed as dominant phytocompounds. The highest concentration in phytochemicals were found in MOM80; MOE96 was more concentrated in UA, while MOE70 extracted more RA. MOE96 inhibited cancer progression and angiogenesis in the in ovo CAM model using MDA-MB-231 cells, inhibiting breast cancer progression and angiogenesis for the MDA-MB-231 breast cancer cell line; no secondary tumoral areas were registered, indicative for a preventive effect against breast tumor cell invasiveness. The highest cell inhibitory activity was also exhibited by MOE96, in particular against the estrogen receptor positive MCF7 breast cancer cell line, with no cytotoxic effect on healthy cells. The estrogen receptor positive MCF7 cell line proved to be more sensitive to the extract antiproliferative activity than the triple negative MDA-MB-231 breast cancer cell line. Nevertheless, the chemopreventive potential of MOE96 extract is phenotype-dependent and is rather related to the apoptosis and antiangiogenic effects suggesting a multitargeted mechanism of action due to its multiple compound composition next to a concentration ratio of RA : UA in favor of UA.

Synergistic Cytotoxicity of Renieramycin M and Doxorubicin in MCF-7 Breast Cancer Cells.

Renieramycin M (RM) is a KCN-stabilized tetrahydroisoquinoline purified from the blue sponge Xestospongia sp., with nanomolar IC50s against several cancer cell lines. Our goal is to evaluate its combination effects with doxorubicin (DOX) in estrogen receptor positive MCF-7 breast cancer cells. MCF-7 cells were treated simultaneously or sequentially with various combination ratios of RM and DOX for 72 h. Cell viability was determined using the MTT assay. Synergism or antagonism was determined using curve-shift analysis, combination index method and isobologram analysis. Synergism was observed with pharmacologically achievable concentrations of DOX when administered simultaneously, but not sequentially. The IC95 values of RM and DOX after combination were reduced by up to four-fold and eight-fold, respectively. To gain insights on the mechanism of synergy, real-time profiling, cell cycle analysis, apoptosis assays, and transcriptome analysis were conducted. The combination treatment displayed a similar profile with DNA-damaging agents and induced a greater and faster cell killing. The combination treatment also showed an increase in apoptosis. DOX induced S and G2/M arrest while RM did not induce significant changes in the cell cycle. DNA replication and repair genes were downregulated commonly by RM and DOX. p53 signaling and cell cycle checkpoints were regulated by DOX while ErbB/PI3K-Akt, integrin and focal adhesion signaling were regulated by RM upon combination. Genes involved in cytochrome C release and interferon gamma signaling were regulated specifically in the combination treatment. This study serves as a basis for in vivo studies and provides a rationale for using RM in combination with other anticancer drugs.

Abstract 2862: Inhibition of DNA repair pathways in breast cancer is a potential mechanism of action of IT-139

Abstract IT-139 is a novel small-molecule drug that demonstrated a manageable safety profile at the maximum tolerated dose and modest antitumor activity as a monotherapy in a phase I clinical trial. A high affinity for albumin and unique chemistry allow increased uptake and activation of IT-139 within tumor cells, respectively. To date, IT-139 has been reported to inhibit the endoplasmic reticulum stress-sensing protein GRP78. However, different cancer cells show disparate responses to IT-139 and the precise mechanism of action in high-response versus low-response cancer cells remains unclear. In vitro studies have indicated that IT-139 induces cytostatic rather than cytotoxic effects as a monotherapy. In this study, to understand IT-139 mediated signaling mechanism in breast cancer cells, we used estrogen receptor-positive (ER+) MCF7 breast cancer cells to obtain gene-metabolite integrated models following IT-139 treatment (10 or 100 μM for 72 h). Our data showed that IT-139 significantly reduced expression of genes involved in the DNA repair pathway, including BRCA1 and RAD51, following treatment with IT-139 in MCF7 cells. Validation of our findings in other breast cancer cell lines including MDA-MB-175 (ER+), MDA-MB-231 and MDA-MB-468 (both ER-) cells also showed increased phosphorylation of H2AX (Ser139) following IT-139 treatment, suggesting lack of proper DNA surveillance and subsequent accumulation of DNA double-stranded breaks (DSB). Consequently, we investigated the effects of combination treatment approaches with IT-139 and other clinical anticancer therapies that interfere with nucleotide synthesis or DNA damage repair pathways, particularly in ER- breast cancers. Our data showed additive effects of IT-139 in combination with capecitabine (5-FU prodrug) or olaparib (PARP inhibitor) and a synergistic effect with carboplatin in ER- breast cancer cell lines. Further analysis of IT-139 mediated signal transduction in breast cancer cells will reveal whether it can be combined with aforementioned chemotherapies to circumvent resistance. Citation Format: Isabel Conde, Yassi Fallah, Diane Demas, Suzanne Bakewell, Filipa Lynce, Ayesha N. Shajahan-Haq. Inhibition of DNA repair pathways in breast cancer is a potential mechanism of action of IT-139 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2862.

Regulation of tamoxifen resistance in estrogen receptor positive MCF-7 breast cancer cells by microRNA-10b targeting histone deacetylase 4

Objective To investigate the effect of microRNA-10b (miR-10b) targeting histone deacetylase 4 (HDAC4) on the resistance of estrogen receptor positive (ER+ ) MCF-7 breast cancer cells to tamoxifen. Methods ER+ and tamoxifen resistant MCF-7 cell line MCF-7-TR was established, and miR-10b expression level in MCF-7 cells and MCF-7-TR cells was detected by reverse transcription real-time quantitative polymerase chain reaction (Real-time PCR). Methyl thiazol tetrazolium (MTT) and trypan blue were used to detect the proliferation and activity of MCF-7 cells and MCF-7-TR cells transfected with pre-miR-10b, miR-10b inhibitor and siHDAC4 at different tamoxifen concentrations (0, 5, 10, 15, 20, 30 μmol/L), respectively. The expression of HDAC4 protein was detected by Western blotting. Results Taken expression level and invasive ability of miR-10b in MCF-7 breast cancer cells as the standard, the miR-10b expression level and invasion ability of MCF-7-TR cell breast cancer cells were 7.2±1.1 and 5.3±1.3 respectively. With the increase of the concentration of tamoxifen, decreased the rate of cell proliferation in MCF-7 cells, which is most obvious, followed by MCF-7-TR+ miR-10b inhibitor + siHDAC4 and MCF-7+ pre-miR-10b+ HDAC4 cells, and the proliferation of MCF-7-TR+ siHDAC4 cells and MCF-7-TR cells decreased the rate of the lowest. The migration ability of MCF-7 cells and MCF-7-TR+ miR-10b cells of the inhibitor decreased most, MCF-7+ pre-miR-10b+ and tamoxifen (10 μmol/L) MCF-7-TR+ cells and tamoxifen (10 μmol/L) had no significant difference in the ability of cell migration (t=1.116, P=0.272), but was significantly higher than that of MCF-7 cells and MCF-7-TR+ miR-10b cells (t=0.124, 10.769, 14.569, 11.577, P=0.000). The expression level of HDAC4 protein in MCF-7+ pre-miR-10b cells was lower than that in MCF-7 cells, MCF-7-TR cells, HDAC4 protein expression level, MCF-7-TR+ miR-10b inhibitor cells, MCF-7+ siHDAC4 cells HDAC4 protein expression level was lower than MCF-7 cells. Conclusion The miR-10b-HDAC4 signaling pathway may be a molecular mechanism of tamoxifen resistance in breast cancer. Key words: Breast cancer; Estrogen receptor positive; Tamoxifen resistance; MicroRNA-10b; Histone deacetylase 4

SUMOylation regulates nuclear accumulation and signaling activity of the soluble intracellular domain of the ErbB4 receptor tyrosine kinase

Erb-B2 receptor tyrosine kinase 4 (ErbB4) is a kinase that can signal via a proteolytically released intracellular domain (ICD) in addition to classical receptor tyrosine kinase-activated signaling cascades. Previously, we have demonstrated that ErbB4 ICD is posttranslationally modified by the small ubiquitin-like modifier (SUMO) and functionally interacts with the PIAS3 SUMO E3 ligase. However, direct evidence of SUMO modification in ErbB4 signaling has remained elusive. Here, we report that the conserved lysine residue 714 in the ErbB4 ICD undergoes SUMO modification, which was reversed by sentrin-specific proteases (SENPs) 1, 2, and 5. Although ErbB4 kinase activity was not necessary for the SUMOylation, the SUMOylated ErbB4 ICD was tyrosine phosphorylated to a higher extent than unmodified ErbB4 ICD. Mutation of the SUMOylation site compromised neither ErbB4-induced phosphorylation of the canonical signaling pathway effectors Erk1/2, Akt, or STAT5 nor ErbB4 stability. In contrast, SUMOylation was required for nuclear accumulation of the ErbB4 ICD. We also found that Lys-714 was located within a leucine-rich stretch, which resembles a nuclear export signal, and could be inactivated by site-directed mutagenesis. Furthermore, SUMOylation modulated the interaction of ErbB4 with chromosomal region maintenance 1 (CRM1), the major nuclear export receptor for proteins. Finally, the SUMO acceptor lysine was functionally required for ErbB4 ICD-mediated inhibition of mammary epithelial cell differentiation in a three-dimensional cell culture model. Our findings indicate that a SUMOylation-mediated mechanism regulates nuclear localization and function of the ICD of ErbB4 receptor tyrosine kinase.

Kinome-Wide RNA Interference Screen Reveals a Role for PDK1 in Acquired Resistance to CDK4/6 Inhibition in ER-Positive Breast Cancer.

Acquired resistance to cyclin-dependent kinases 4 and 6 (CDK4/6) small-molecule inhibitors in breast cancer arises through mechanisms that are yet uncharacterized. In this study, we used a kinome-wide siRNA screen to identify kinases that, when downregulated, yield sensitivity to the CDK4/6 inhibitor ribociclib. In this manner, we identified 3-phosphoinositide-dependent protein kinase 1 (PDK1) as a key modifier of ribociclib sensitivity in estrogen receptor-positive MCF-7 breast cancer cells. Pharmacologic inhibition of PDK1 with GSK2334470 in combination with ribociclib or palbociclib, another CDK4/6 inhibitor, synergistically inhibited proliferation and increased apoptosis in a panel of ER-positive breast cancer cell lines. Ribociclib-resistant breast cancer cells selected by chronic drug exposure displayed a relative increase in the levels of PDK1 and activation of the AKT pathway. Analysis of these cells revealed that CDK4/6 inhibition failed to induce cell-cycle arrest or senescence. Mechanistic investigations showed that resistant cells coordinately upregulated expression of cyclins A, E, and D1, activated phospho-CDK2, and phospho-S477/T479 AKT. Treatment with GSK2334470 or the CDK2 inhibitor dinaciclib was sufficient to reverse these events and to restore the sensitivity of ribociclib-resistant cells to CDK4/6 inhibitors. Ribociclib, in combination with GSK2334470 or the PI3Kα inhibitor alpelisib, decreased xenograft tumor growth more potently than each drug alone. Taken together, our results highlight a role for the PI3K-PDK1 signaling pathway in mediating acquired resistance to CDK4/6 inhibitors. Cancer Res; 77(9); 2488-99. ©2017 AACR.

Effects of Electroporation on Tamoxifen Delivery in Estrogen Receptor Positive (ER+) Human Breast Carcinoma Cells.

Estrogen receptor positive breast cancer is the most common type of breast cancer and in most cases, hormone therapy is considered complementary to surgery. Tamoxifen is one of the most common drugs used in hormone therapy for treating estrogen receptor positive breast cancer cells. However, it has severe side-effects depending on the duration of treating breast cancer and amount of tamoxifen used. In this study, we examined the effects of electroporation on the tamoxifen uptake in estrogen receptor positive MCF-7 breast cancer cells. The survival rate of MCF-7 cells had a negative relationship with energy dissipation in cells. Similarly, the electrical charge delivered to cells during electroporation was inversely proportional to survival rate. The combined application of electroporation and tamoxifen is much more effective than the usage of tamoxifen alone in the treatment of estrogen receptor positive breast cancer. The application of electroporation increased the uptake of tamoxifen into MCF-7 cells and reduced the minimal tamoxifen dosage which, is needed for the treatment of estrogen receptor positive breast cancer.

Synthesis and cancer cell growth inhibitory activity of icaritin derivatives

A series of icaritin derivatives bearing carboxylic acid or carboxylic ester groups are synthesized, and their in vitro cytotoxic activity against three cancer cell lines, MCF-7, MDA-MB-435s, and A549, are evaluated by MTT assay. Several derivatives including 2h, 2j, 5b and 5d show higher cytotoxic activity than the parent compound icaritin against these cancer cell lines. Compounds 5b and 5d are even more cytotoxic to MCF-7 cells than the clinic drug tamoxifen. Moreover, compound 5b is found to be non-toxic to normal cells (Vero) and both 5b and 5d exhibit good selectivity towards estrogen receptor positive MCF-7 breast cancer cells over estrogen receptor negative MDA-MB-435s breast cancer cells. The structure activity relationship analysis has revealed that mono-substitution at either C-3 or C-7 hydroxyl group of icaritin could improve the cytotoxicity of icaritin, and the C-3 hydroxyl group may be a preferable site for chemical modification. In addition, the length, the flexibility and the additional branching substituent group of the substitution chain(s) at both C-3 and C-7 hydroxyl groups can all affect the anti-cancer activity of these derivatives.

The potential in breast tissue engineering for the combined effects of linoleic acid and tamoxifen

Literature to date has strongly suggested that linoleic acid (LA) enhances mammary cancer cell proliferation when used in the range of 0·5–20 μg/ml. In contrast, the current in vitro studies show that deoxyribonucleic acid (DNA) levels of estrogen receptor-positive (ER+) MCF7 breast cancer cells were diminished following treatment with 30 μg/ml (100 μM) LA. MCF7 cells, when treated for 6 days with LA, consumed significantly less glucose than untreated controls (P-value<0·01). MFC7 cells treated with 30 μg/ml LA and 10 μM tamoxifen had even lower levels of DNA and lower glucose uptake. The same combination treatment of tamoxifen and LA was applied to a noncancerous mammary cell line, MCF10A, and did not have a significant effect on MCF10A DNA concentrations. Linoleic acid and its byproducts are important to cell proliferation and tissue reconstruction and, in combination with tamoxifen, can provide a uniquely different approach to soft tissue engineering. That is, LA and tamoxifen-loaded scaffolds may provide a viable reconstructive approach for patients undergoing lumpectomy and mastectomy by encouraging natural breast healing while limiting the ability of ER+ cancer cells to regrow. This approach could allow the suppression of estrogen-dependent mammary carcinomas while allowing natural cell proliferation after a mastectomy or lumpectomy.

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.

Oxidative Stress-Mediated Apoptosis Induced by Ethanolic Mango Seed Extract in Cultured Estrogen Receptor Positive Breast Cancer MCF-7 Cells

Breast cancer has become a global health issue requiring huge expenditures for care and treatment of patients. There is a need to discover newer cost-effective alternatives for current therapeutic regimes. Mango kernel is a waste product with potential as a source of anti-cancer phytochemicals, especially since it is non-toxic towards normal breast cell lines at concentrations for which it induces cell death in breast cancer cells. In this study, the anti-cancer effect of mango kernel extract was determined on estrogen receptor-positive human breast carcinoma (MCF-7) cells. The MCF-7 cells were cultured and treated with 5, 10 and 50 μg/mL of mango kernel extract for 12 and 24 h. In response to treatment, there were time- and dose-dependent increases in oxidative stress markers and pro-apoptotic factors; Bcl-2-like protein 4 (BAX), p53, cytochrome c and caspases (7, 8 and 9) in the MCF-7 cells treated with the extract. At the same time, there were decreases in pro-survival markers (Bcl-2 and glutathione) as the result of the treatments. The changes induced in the MCF-7 cells by mango kernel extract treatment suggest that the extract can induce cancer cell apoptosis, likely via the activation of oxidative stress. These findings need to be evaluated further to determine whether mango kernel extract can be developed as an anti-breast cancer agent.

Anti-cancer Effects of Costunolide in Estrogen Receptor Positive MCF-7 Breast Cancer Cells

Costunolide (C<SUB>15</SUB>H<SUB>20</SUB>O<SUB>2</SUB>) is a sesquiterpene lactone that was isolated from many herbal medicines and it has diverse effects (anti-viral, anti-fungal, and anti-inflammatory) according to previous reports. However, the anti-cancer effects of Costunolide and its mechanism of actions are not well known in estrogen receptor positive breast cancer. In this study, we observed that costunolide suppresses cell growth in estrogen receptor positive MCF-7 breast cancer cells as shown by MTT assay and soft agar colony formation assay. To examine the mechanism by which costunolide inhibits MCF-7 cell growth, we performed FACS analysis. We found that costunolide induced G2/M and S cell cycle arrest, and regulated cycle-related protein expression. In addition, costunolide inhibited ERK signaling pathway and induced autophagy. Therefore, costunolide might be a good and useful chemotherapy agent for estrogen receptor positive breast cancer patients.

Insulin-Like Growth Factor-1 Signaling Regulates miRNA Expression in MCF-7 Breast Cancer Cell Line

In breast carcinomas, increased levels of insulin-like growth factor 1 (IGF-1) can act as a mitogen to augment tumorigenesis through the regulation of MAPK and AKT signaling pathways. Signaling through these two pathways allows IGF-1 to employ mechanisms that favor proliferation and cellular survival. Here we demonstrate a subset of previously described tumor suppressor and oncogenic microRNAs (miRNAs) that are under the direct regulation of IGF-1 signaling. Additionally, we show that the selective inhibition of either the MAPK or AKT pathways prior to IGF-1 stimulation prevents the expression of previously described tumor suppressor miRNAs that are family and cluster specific. Here we have defined, for the first time, specific miRNAs under the direct regulation of IGF-1 signaling in the estrogen receptor positive MCF-7 breast cancer cell line and demonstrate kinase signaling as a modulator of expression for a small subset of microRNAs. Taken together, these data give new insights into mechanisms governing IGF-1 signaling in breast cancer.

92P Tff1 Trefoil Protein Involves in Resistance to Doxorubicin-Induced Apoptosis in Breast Cancer Cell

ABSTRACT TFF1 trefoil protein is a small secretory protein expressed in a various type of carcinomas including breast cancer. TFF1 gene contained estrogen responsive element and its expression could be regulated by estrogen. In addition, estrogen has been demonstrated its ability to promote resistance to doxorubicin in estrogen receptor positive MCF-7 breast cancer cell line. Moreover, it has been reported that TFF1 can protect conjunctival cells from UV-induced apoptosis. In this study, we demonstrated the role of TFF1 in estrogen-promoted resistance to doxorubicin-induced apoptosis using MCF-7 model. Permanent knockdown of TFF1 gene in MCF-7 cell had been generated and used to test the sensitivity to doxorubicin treatment compared to parental cell in the conditions present or absent of 17b-estradiol, a potent estrogenic agent. The apoptosis cells were measured by fluorescence staining and flow cytometry method. The results showed that among the stimulation of apoptosis by doxorubicin, 17b-estradiol could suppress this process in parental MCF-7 cell but not in TFF1 knockdown MCF-7 cell. Moreover, by trypan blue staining method, it has been shown that anti-TFF1 antibody could reverse the anti-apoptotic effect of estrogen in parental MCF-7 cell and recombinant TFF1 could recover TFF1 knockdown MCF-7 cell death induced by doxorubicin. However, this process could not be inhibited by fulvestrant, an estrogen antagonist. Apoptosis protein array experiment reflected the role of the anti-oxidative enzymes catalase and heme oxygenase 1 in estrogen and TFF1 modulated apoptosis. These phenomena determine the role of TFF1 in estrogen-promoted resistance to apoptosis induced by doxorubicin in MCF-7 breast cancer cell. TFF1 gene may be a target for enhancing of sensitivity to chemotherapy in breast cancer treatment. Disclosure All authors have declared no conflicts of interest.

Shu-Gan-Liang-Xue decoction simultaneously down-regulates expressions of aromatase and steroid sulfatase in estrogen receptor positive breast cancer cells

Estradiol (E2) plays an important role in the development of breast cancer. In postmenopausal women, the estrogen can be synthesized via aromatase (CYP19) pathway and steroid-sulfatase (STS) pathway in peripheral tissues, when the production in ovary has ceased. The objective of our study was to explore the effects of Shu-Gan-Liang-Xue Decoction (SGLXD) on the expressions of CYP19 and STS in estrogen receptor positive breast cancer MCF-7 and T47D cells. The effects of SGLXD on the cell viability of MCF-7 and T47D were analyzed by MTT assay. By quantitative real-time RT-PCR and Western blot, we evaluated the mRNA and protein expressions of CYP19 and STS in MCF-7 and T47D cells after SGLXD treatment. By MTT assay, the cell viability rates of MCF-7 and T47D were significantly inhibited by SGLXD in a dose-dependent manner, the IC50 values were 40.07 mg/ml for MCF-7 cells and 25.62 mg/ml for T47D cells, respectively. As evidenced by real-time PCR and Western blot, the high concentrations of SGLXD significantly down-regulated the expressions of CYP19 and STS both in the transcript level and the protein level. The results suggest that SGLXD is a potential dual aromatase-sulfatase inhibitor by simultaneously down-regulating the expressions of CYP19 and STS in MCF-7 and T47D cells.

Abstract 3787: Serotonin signaling plays a role in regulation of autophagy in breast cancer cells

Abstract Breast cancer is the second most common cause of cancer death in women in the United States, 40,000 dying of this disease each year. One out of eight women will be diagnosed with breast cancer in their lifetime. There are several treatments for breast cancer, such as surgery, radiation therapy, chemotherapy, and hormone therapy, but still 5 year survival is less than 40% in advance cases. Due to the high incidence and mortality associated with this cancer better understanding of the biology of the disease and development of new treatments are urgently needed. The actions of local physiological mediators in the context of breast cancer are poorly understood. Serotonin (5HT, 5-hydroxytryptamine) is a critical local regulator of epithelial homeostasis in the breast and other organs. Serotonin is more commonly known as a monoamine neurotransmitter and a mediator of depression if the levels drop in the brain. Serotonin has been implicated in epithelial homeostasis of the breast, lung, pancreas, liver, and prostate. Deregulation of serotonin signaling in these epithelia may be associated with various pathologies, including cancer. Previous and our studies suggested serotonin can induce proliferation of some cancer cell lines. On the other hand serotonin can induce apoptosis or induce cell proliferation depending on the cancer type. Serotonin is believed to function as a growth factor working as an autocrin manner. Also tryptophan hydroxylase 1 (TPH1), rate-limiting step in 5-HT synthesis, has been shown to be overexpressed in breast cancer cell lines and biopsy samples from patients. The effect of serotonin signaling and its role in survival of breast cancer cells has not been well understood. We tried to address the questions that what is the role of serotonin signaling in breast cancer survival and if serotonin inhibits apoptosis or autophagy? We found that serotonin slightly induced cell proliferation in estrogen receptor positive MCF-7 breast cancer cells. Because autophagy is implicated as a survival pathway in some cancers we also examined if serotonin induces autophagy in MCF7 ER+ breast cancer cells. We found that serotonin induces autophagy that is detected Acridine orange staining and FACS analysis and LC-3 II induction, a hallmark of autophagy, by western blot analysis in MCF7 cells after serotonin treatment for 4 hours. We found that serotonin (10 uM) induces expression of cyclinD1, NF-kB and mTOR and leads induction of ERK and AMPK pathway as indicated by increased phosphorylation of ERK1/2 and AMPK (Thr 172), both of which are known to mediate autophagy. We are currently testing if induction ERK and AMPk signaling is involved in induction autophagy in these cells using specific inhibitors of MEK. In conclusion our studies showed for the first that induction of serotonin signaling induces autophagy in ER+ breast cancer cells. 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 3787. doi:10.1158/1538-7445.AM2011-3787