Target For Breast Cancer Therapy Research Articles

Predictive features of ligand-specific signaling through the estrogen receptor.

Some estrogen receptor‐α (ERα)‐targeted breast cancer therapies such as tamoxifen have tissue‐selective or cell‐specific activities, while others have similar activities in different cell types. To identify biophysical determinants of cell‐specific signaling and breast cancer cell proliferation, we synthesized 241 ERα ligands based on 19 chemical scaffolds, and compared ligand response using quantitative bioassays for canonical ERα activities and X‐ray crystallography. Ligands that regulate the dynamics and stability of the coactivator‐binding site in the C‐terminal ligand‐binding domain, called activation function‐2 (AF‐2), showed similar activity profiles in different cell types. Such ligands induced breast cancer cell proliferation in a manner that was predicted by the canonical recruitment of the coactivators NCOA1/2/3 and induction of the GREB1 proliferative gene. For some ligand series, a single inter‐atomic distance in the ligand‐binding domain predicted their proliferative effects. In contrast, the N‐terminal coactivator‐binding site, activation function‐1 (AF‐1), determined cell‐specific signaling induced by ligands that used alternate mechanisms to control cell proliferation. Thus, incorporating systems structural analyses with quantitative chemical biology reveals how ligands can achieve distinct allosteric signaling outcomes through ERα.

LncRNA PANDAR regulates the G1/S transition of breast cancer cells by suppressing p16(INK4A) expression.

It has been reported that lncRNA PANDAR (promoter of CDKN1A antisense DNA damage-activated RNA) is induced as a result of DNA damage, and it regulates the reparation of DNA damage. In this study, we investigated the role of lncRNA PANDAR in the progression of breast cancer and found that PANDAR was up-regulated in breast cancer tissues and cell lines. The knockdown of PANDAR suppresses G1/S transition of breast cancer cells. We demonstrated mechanistically that the regulation of G1/S transition by PANDAR was partly due to the transcriptional modulation of p16INK4A. Moreover, we showed that PANDAR impacted p16INK4A expression by regulating the recruitment Bmi1 to p16INK4A promoter. To our knowledge, this is the first study which showed the functional roles and mechanisms of PANDAR in regulating the progression of breast cancer. The PANDAR/Bmi1/p16INK4A axis could serve as novel targets for breast cancer therapy.

Identification and frequency of the rs12516 and rs8176318 BRCA1 gene polymorphisms among different populations.

Genetic mutation of breast cancer 1 (BRCA1) is one of the most notable factors responsible for a proportion of breast cancer cases. BRCA1 encodes a 1,863-amino acid protein and functions as a negative regulator of tumor growth. Thus, investigation of the underlying mechanisms that regulate BRCA1 gene expression provide further insight into possible targets for breast cancer therapy. Previous studies have demonstrated that the genetic variants in the BRCA1 3' untranslated region (3'UTR), in addition to the cytosine-phosphate-guanine (CpG) islands in the promoter region, are significantly associated with breast cancer risk; however, the role of single nucleotide polymorphisms (SNPs) in the BRCA1 3'UTR remains unclear. The present study aimed to investigate the association between SNPs and BRCA1 mRNA expression levels. Bioinformatics analysis demonstrated that 2 SNPs in the BRCA1 3'UTR (rs12516 and rs8176318 with putative microRNA binding sites) were significantly correlated with mRNA expression in lymphoblastoid cell lines (P=2.55×10-4 and P=8.78×10-5, respectively). Furthermore, the genotype frequency distribution varied between populations worldwide. In addition, 3 CpG islands and several transcription factor binding sites in the BRCA1 promoter region were established. The identification of such polymorphisms and CpG islands may aid in designing improved therapeutic strategies to treat patients with BRCA1-associated breast cancer.

Abstract P6-03-05: Cell-free DNA as molecular tool for monitoring disease progression and response to therapy in breast cancer patients

Abstract Background: Identification of cancer-specific genes from breast cancer cells was instrumental in the advancement of targeted breast cancer therapy. However, with genomic heterogeneity within the breast cancer and evolution of cancer over time, genomic sequencing obtained from a single biopsy site may not capture the complete genomic profile. Thus, circulating cell-free DNA (cfDNA), isolated from plasma, is potentially a non-invasive source of identifying cancer-specific genomic alterations and may provide comprehensive genomic data throughout a patient's clinical course as they undergo anti-cancer therapy. Method: We performed a retrospective chart review of 100 patients with stage 4 or high-risk stage 3 breast cancer who were tested for cfDNA genomic alterations. The most common actionable cancer specific genomic alterations were identified. In 23 patients who also had genomic analysis from tumor DNA (tDNA), an analysis using the Cohen's Kappa statistic was performed to determine the degree of agreement between genomic alterations found in tDNA and cfDNA. The proportion of patients with clinical disease progression between two cohorts determined by change in mutant allele frequency was compared using two-sided Fisher's exact test. Patients who received targeted therapy based on the identified genomic alteration were followed to determine response to therapy. Results: In cfDNA of 100 breast cancer patients, the most commonly found cancer specific genomic alterations were TP53, PIK3CA, EGFR amplification, and ERBB2 amplification, with incidence rates 27%, 22%, 9%, and 7%, respectively. In tDNA of 23 patients, incidence rates were 65%, 26%, 9%, and 13%. PIK3CA and ERBB2 amplification demonstrated robust agreement between tDNA and cfDNA (Cohen's Kappa= 0.64 and 0.77, respectively). TP53 and EGFR amplification demonstrated poor agreement between tDNA and cfDNA (Cohen's Kappa= 0.18 and 0.33, respectively). There were 22 patients who had baseline and post-therapy mutant allele frequency measurements of TP53 and PIK3CA. Directional change of mutant allele frequency was closely associated with patient's response to therapy (p=0.0017). 8 out of 8 patients (100%) who had progression of disease had increase in mutant allele frequency. 10 out of 14 patients (71%) of patients who responded to therapy had decrease in mutant allele frequency. 6 patients who were found to have ERBB2 amplification were initiated on anti-HER2 cancer therapy. 5 of 6 patients (83%) had clinical response to therapy, while one patient had progression of disease. 3 patients who were found to have EGFR amplification (2 in cfDNA, 1 in tDNA) were initiated on anti-EGFR therapy. 2 of 3 patients (67%) had clinical response to therapy, while one patient had progression of disease. Conclusion: There is no definite agreement between genomic alterations found in tDNA and those found in cfDNA. Whether this is due to tumor heterogeneity or tumor evolution over time with administration of anti-cancer treatment remains unknown. However, identification of selected cancer specific genomic alterations from cfDNA may be a non-invasive tool to monitor disease progression and response to breast cancer therapy. Citation Format: Liang DH, Patel A, Ensor JE, Patel TA, Chang JC, Rodriguez AA. Cell-free DNA as molecular tool for monitoring disease progression and response to therapy in breast cancer patients. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-03-05.

HER2 Targeted Breast Cancer Therapy with Switchable “Off/On” Multifunctional “Smart” Magnetic Polymer Core–Shell Nanocomposites

Multifunctional magnetic polymer nanocombinations are gaining importance in cancer nanotheranostics due to their safety and their potential in delivering targeted functions. Herein, we report a novel multifunctional core-shell magnetic polymer therapeutic nanocomposites (NCs) exhibiting pH dependent "Off-On" release of drug against breast cancer cells. The NCs are intact in blood circulation ("Off" state), i.e., at physiological pH, whereas activated ("On" state) at intracellular acidic pH environment of the targeted breast cancer cells. The NCs are prepared by coating the cannonball (iron nanocore) with hydrophobic nanopockets of pH-responsive poly(d,l-lactic-co-glycolic acid) (PLGA) polymer nanoshell that allows efficient loading of therapeutics. Further, the nanocore-polymer shell is stabilized by poly(vinylpyrrolidone) (PVP) and functionalized with a targeting HER2 ligand. The prepared Her-Fe3O4@PLGA-PVP nanocomposites facilitate packing of anticancer drug (Tamoxifen) without premature release in the bloodstream, recognizing the target cells through binding of Herceptin antibody to HER2, a cell surface receptor expressed by breast cancer cells to promote HER2 receptor mediated endocytosis and finally releasing the drug at the intracellular site of tumor cells ("On" state) to induce apoptosis. The therapeutic efficiency of hemo/cytocompatible NCs drug delivery system (DDS) in terms of targeted delivery and sustained release of therapeutic agent against breast cancer cells was substantiated by in vitro and in vivo studies. The multifunctional properties of Her-Tam-Fe3O4@PLGA-PVP NCs may open up new avenues in cancer therapy through overcoming the limitations of conventional cancer therapy.

Identification of p62/SQSTM1 as a component of non-canonical Wnt VANGL2-JNK signalling in breast cancer.

The non-canonical Wnt/planar cell polarity (Wnt/PCP) pathway plays a crucial role in embryonic development. Recent work has linked defects of this pathway to breast cancer aggressiveness and proposed Wnt/PCP signalling as a therapeutic target. Here we show that the archetypal Wnt/PCP protein VANGL2 is overexpressed in basal breast cancers, associated with poor prognosis and implicated in tumour growth. We identify the scaffold p62/SQSTM1 protein as a novel VANGL2-binding partner and show its key role in an evolutionarily conserved VANGL2–p62/SQSTM1–JNK pathway. This proliferative signalling cascade is upregulated in breast cancer patients with shorter survival and can be inactivated in patient-derived xenograft cells by inhibition of the JNK pathway or by disruption of the VANGL2–p62/SQSTM1 interaction. VANGL2–JNK signalling is thus a potential target for breast cancer therapy.

LKB1, A New Biomarker in Breast Cancer

Breast cancer is the most common cancer in women worldwide. Estrogen signaling pathways have been identified as efficient targets of breast cancer therapy, given their key role in promoting breast tumor growth. Agents blocking estrogen-mediated pathways are routinely used in clinical applications in patients displaying estrogen-sensitive breast cancer subtypes; however intrinsic or acquired resistance to treatment often occurs or develops, thus limiting their efficacy. This limitation has highlighted an imperative need to identify new predictive biomarkers. Recent findings have highlighted a role for the Liver Kinase B1 (LKB1) in breast cancer tumorigenesis. LKB1 is a serine/threonine kinase mutated in Peutz-Jeghers syndrome (PJS), implicated in many cellular processes including energy metabolism, cell polarization and cell cycle arrest and has also been shown to play an essential role as a tumor suppressor gene by negatively regulating the mTOR pathway. This review provides an overview of previous findings and ongoing research on LKB1, and substantiates the use of this kinase as a potential prognostic and predictive biomarker of breast cancer.

Abstract A65: A novel regulatory mechanism involving Ras-mediated activation of the zinc-finger transcription factor, SAF-1/MAZ induces EGFR/HER1 expression in breast cancer cells

Abstract Tumor microenvironment (TME) plays a critical role in tumor growth, invasion and metastasis. In TME, epidermal growth factor receptor (EGFR) family members, including HER1, HER2, HER3 and HER4, are involved in determining aggressive growth of breast cancer due to their ability to transduce the growth promoting functions of growth factors. This activity is potentiated by the over-expression of these receptor molecules in cancer cells. To reduce the activity of EGFR molecules, various inhibitors have been developed. EGFR/HER1 tyrosine kinase inhibitors (TKIs), gefitinib and erlotinib, show antitumor activity but these drugs have not meet their primary goal of improved survival in the overall patient population. A similar TKI, lapatinib, has shown some limited success in breast cancer. While these therapeutic options focus on reducing the functional activity of EGFR protein, the fundamental and major problem of over-expression of EGFR in cancer cells has not been addressed. In two-thirds of aggressive breast cancer patients, transcriptional induction of EGFR causes high EGFR/HER1 level. Therefore, the goal of this study is to identify a molecular mechanism by which EGFR is over-expressed in breast cancer cells. To understand how transcriptional induction might occur, we have explored a novel biosynthetic pathway for EGFR over-expression. Our findings reveal that EGFR promoter is significantly more active in MDA-MB-231 cells in comparison to MDA-MB-468 cells. When transcription factor SAF-1 was ectopically expressed in these cells, EGFR promoter activity was further increased in MDA-MB-231 cells. Since MDA-MB-231 cells contain a highly active form of Ras, the data suggested a possible Ras-mediated activation of SAF-1 which in turn induces EGFR expression. Consistent with these findings, inhibition of K- and H-Ras, by using both siRNA and CRISPR/Cas9-mediated knock-out systems, reduced the expression of EGFR in MDA-MB-231 cells. The effect was more profound when K-Ras was targeted for inhibition. Since SAF-1/MAZ is seen to be activated by Ras, our data implicates K-Ras - SAF-1/MAZ - EGFR axis in breast cancer cell growth. Our findings may provide new targets for breast cancer therapy. Supported by grants from College of Veterinary Medicine Faculty Research Award Grant and MU Center for Botanical Interaction Studies Pilot Project Grant. Citation Format: Alpana Ray, Brett Havis, Bimal Ray. A novel regulatory mechanism involving Ras-mediated activation of the zinc-finger transcription factor, SAF-1/MAZ induces EGFR/HER1 expression in breast cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A65.

Abstract C200: Effects of PI3K/Akt pathway inhibition on global proteome levels and phosphorylation signaling in patient-derived xenograft models of triple negative breast cancer

Abstract AIM: The aim of this study was to identify markers that have the potential to predict BKM120 (a pan-class I PI3K inhibitor) effectiveness in triple negative breast cancer. INTRODUCTION: Triple negative breast cancer is an aggressive subtype that constitutes circa 20% of all breast cancers. This subtype of breast cancer is defined by its lack of estrogen-, progesterone- and HER2/neu expression. Since these are the operative receptors for targeted breast cancer therapies, current standard treatment instead centers on conventional cytotoxic chemotherapy. Unfortunately, the majority of triple negative breast cancer patients does not have meaningful response, and new therapeutic agents are needed. The PI3K/Akt/mTOR axis is often dysregulated in breast cancer, and clinical trials are ongoing for several inhibitors targeting this pathway. Differential analysis of the global proteome and phosphoproteome of BKM120-treated responsive and resistant tumors may identify markers, patterns and pathways associated with clinical response to support precision therapy. MATERIALS AND METHODS: We collected tumor material from triple negative breast cancer xenografts propagated in the mammary fat pads of NOD SCID mice after BKM120 treatment. Long term (∼30 days) treatment was performed to assess growth rates and sensitivity to the drug. For mass spectrometry analyses, tumor material from 6 different mouse models across 5 treatment conditions was lysed and digested into peptides. Samples were labeled with TMT6-plex reagents, and analyzed together with one reference sample in a total of 6 TMT6-plex experiments. To increase proteome coverage, sample complexity was reduced using high-pH reversed-phase separation, and phospho-peptides were enriched using immobilized metal affinity chromatography. All samples were analyzed on a Q Exactive mass spectrometer. RESULTS AND DATA ANALYSES: Cumulatively, >19,000 proteins were identified, with an average of >10,000 proteins and >21,000 phosphosites per sample. The 6 mice showed a continuum of response to the drug from highly sensitive to highly resistant. Pearson correlation analysis was used to identify proteins and phosphosites that correlate to this response gradient, while moderate t-tests were used to identify commonly regulated markers. Preliminary analyses indicate several interesting proteins and phospho-sites, some pertaining to the PI3K/Akt pathway. Further canonical pathways were detected using Gene Set Enrichment Analysis on a sample-by sample basis and additional network and pathway analyses were performed using the network analysis program GeNets, as well as Ingenuity Pathway Analysis. Citation Format: Filip O. Mundt*, Cynthia Ma*, Philipp Mertins, Zhanfang Guo, Matthew Meyer, Jana Qiao, DR Mani, Karl Clauser, Shunqiang Li, Kuan Huang, Jason Held, Sherri Davies, Kelly Ruggles, Li Ding, Michael Gillette, David Fenyo, Reid Townsend, Matthew Ellis, Steven A. Carr. Effects of PI3K/Akt pathway inhibition on global proteome levels and phosphorylation signaling in patient-derived xenograft models of triple negative breast cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C200.

Breast cancer associated a2 isoform vacuolar ATPase immunomodulates neutrophils: potential role in tumor progression.

In invasive breast cancer, tumor associated neutrophils (TAN) represent a significant portion of the tumor mass and are associated with increased angiogenesis and metastasis. Identifying the regulatory factors that control TAN behavior will help in developing ideal immunotherapies. Vacuolar ATPases (V-ATPases), multi-subunit proton pumps, are highly expressed in metastatic breast cancer cells. A cleaved peptide from a2 isoform V-ATPase (a2NTD) has immunomodulatory role in tumor microenvironment. Here, we report for the first time the role of V-ATPase in neutrophils modulation. In invasive breast cancer cells, a2NTD was detected and a2V was highly expressed on the surface. Immunohistochemical analysis of invasive breast cancer tissues revealed that increased neutrophil recruitment and blood vessel density correlated with increased a2NTD levels. In order to determine the direct regulatory role of a2NTD on neutrophils, recombinant a2NTD was used for the treatment of neutrophils isolated from the peripheral blood of healthy volunteers. Neutrophils treated with a2NTD (a2Neuɸ) showed increased secretion of IL-1RA, IL-10, CCL-2 and IL-6 that are important mediators in cancer related inflammation. Moreover, a2Neuɸ exhibited an increased production of protumorigenic factors including IL-8, matrix metaloprotinase-9 and vascular endothelial growth factor. Further, functional characterization of a2Neuɸ revealed that a2Neuɸ derived products induce in vitro angiogenesis as well as increase the invasiveness of breast cancer cells. This study establishes the modulatory effect of breast cancer associated a2V on neutrophils, by the action of a2NTD, which has a positive impact on tumor progression, supporting that a2V can be a potential selective target for breast cancer therapy.

Autocrine interferon-γ may affect malignant behavior and sensitivity to tamoxifen of MCF-7 via estrogen receptor β subtype.

Mitogenic actions of estrogens are mediated by two distinct estrogen receptors (ERs), which are critical in the progression and therapeutic response of breast cancer. ER expression is a dynamic phenomenon that is regulated by numerous factors, including cytokines, in the tumor microenvironment. Recently, studies have shown that autocrine production of IL-4 promotes cancer cell growth and there is negative correlation between tumor IL-4 and hormone receptor levels, suggesting that there is crosstalk between cytokine receptors and ER. Thus, we evaluated for interaction between the two ERs and the cytokines IL-4 and IFN-γ, and if this interaction modulates malignant behavior. We identified that ERβ exerts protective activity in the progression of breast cancer cell line MCF-7, which co-expresses ERα and ERβ. IFN-γ and IL-4 have the opposite effects on malignant biological behavior. Furthermore, we found positive correlation between IFN-γ and ERβ expression in MCF-7. We also determined that autocrine IFN-γ in MCF-7 increases mRNA expression of ERβ resulting in enhanced sensitivity to tamoxifen (TAM). These results indicate that ERβ and autocrine IFN-γ represent two putative targets for breast cancer therapy.

Abstract 1838: MUC1 serves as metabolic regulator in triple negative breast cancer

Abstract Breast cancer, the second leading cause of cancer deaths in women, is the most common cancer among North American women, accounting for nearly 1 in 3 cancer cases diagnosed in the U.S. women. Triple negative breast cancer (TNBC) subtype accounts for approximately 15-25% of all breast cancer cases and has an increased incidence of metastasis, high recurrence within 1-3 years and a high mortality rate. Therefore, identifying factors that facilitate tumor growth and metastases have the potential to serve as novel molecular targets for breast cancer therapy. Mucin1 (MUC1), a glycoprotein associated with chemoresistance, is aberrantly overexpressed in TNBC and facilitates growth and metastasis of TNBC cells. This occurrence can be partially attributed to MUC1 interaction with hypoxia-inducible factor alpha (HIF1α), a key regulator of glycolysis. In the present study we examined the effect of MUC1 expression on cancer cell metabolism of TNBC cell lines under normoxic (20% Oxygen) and hypoxic (1% Oxygen) conditions. MUC1 was ectopically overexpressed in the MDA-MB-231 cell line and stably knocked down in the MDA-MB-468 cell line. Results indicate that MUC1 interacts with HIF1α and the interaction is further enhanced under hypoxic conditions. In addition, MUC1 expression enhanced glucose and glutamine uptake, enhanced lactate secretion and altered the expression of several metabolic genes. Furthermore, untargeted global metabolomic profiling identified metabolite alterations in which MUC1 expression modulates cancer cell metabolism to facilitate growth properties of TNBC cells. Thus our results support the notion that MUC1 serves as a metabolic regulator in TNBC, facilitating metabolic reprogramming that influences growth of TNBC. Citation Format: Gennifer D. Goode. MUC1 serves as metabolic regulator in triple negative breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1838. doi:10.1158/1538-7445.AM2015-1838

Abstract 155: LncRNA AK023948 promotes breast tumorigenesis by enhancing AKT phosphorylation

Abstract The human genome expresses a large number of long non-coding RNAs (lncRNAs), however, little is known about their physiological functions or their role in cancer for the vast majority of these lncRNAs. In the present study, we identified a set of lncRNAs that were differentially expressed in breast cancer tissues as compared to normal tissue. Among them is AK023948 which is up-regulated in breast cancer tissue and in breast cancer cell lines. Ectopic expression of AK023948 in breast cancer MCF-7 cells promotes proliferation and anchorage independent cell growth along with activation of AKT phosphorylation. On the other hand, knockdown or knockout of AK023948 in MCF-7 cells reduces the proliferation of cells and phosphorylation of AKT. Furthermore, there is a positive correlation between AK023948 and pAKT expression in breast cancer tissue microarray. To determine the underlying mechanism of AK023948-mediated activation of AKT, we performed RNA precipitation assays using biotin-labeled AK023948 RNA probe, followed by PAGE analysis, and detected a unique protein band by silver staining. Mass spectrometry analysis identified it as ATP dependent RNA helicase A (RHA/DHX9) which was confirmed by Western blot and RNA immunoprecipitation (IP), respectively. Co-IP indicated that DHX9 interacts with p85PI3K regulatory subunit. Further characterization revealed that p85-DHX9 interaction is essential for stability of p85α and AKT phosphorylation. Together, these results suggest that AK023948 may function as an oncogenic lncRNA in breast cancer and thus, it may serve as a novel target for breast cancer therapy. Citation Format: Pratirodh Koirala, Yin Yuan MO. LncRNA AK023948 promotes breast tumorigenesis by enhancing AKT phosphorylation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 155. doi:10.1158/1538-7445.AM2015-155

Abstract 168: MALAT1 is crucial for epithelial-mesenchymal transition of breast cancer cells in acidic microenvironment

Abstract Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT1), a long non-coding RNA (lncRNA), is found up-regulated in several tumors. However, little is known about the role of MALAT1 in breast cancer, which is one of the highly invasive tumors among women worldwide. In this study, we found that MALAT1 was highly expressed in both breast cancer cell lines and breast cancer specimens in comparison to normal specimens. We found that low extracellular pH (pHe-acidosis), a major hallmark of solid tumors, further up-regulates MALAT-1 in several breast cancer cell lines in a time-dependent manner. Furthermore, acidosis induces epithelial to mesenchymal transition (EMT)-associated vimentin and Twist 1 expression but suppresses E-cadherin levels in breast cancer cells. Moreover, breast cancer cells under low pHe adopt stem-ness property as revealed by an increase in CD44+ and ESA+ population, mammosphere formation, and expression of OCT-4, SOX-2 and Nanog. Acidosis promotes translocation of β-catenin from cytoplasm to nucleus. The antisense oligonucleotides mediated gene silencing of MALAT-1 prevents acidosis induced nuclear translocation of β-catenin. This is in consistent with our previous finding that breast cancer cells cultured under low pHe exhibit an increased invasion activity. These results suggest that acidosis-induced MALAT-1 trigger an EMT program, leading to cell invasion and metastasis. Therefore, MALAT-1 represents a potential target for breast cancer therapy. Studies are underway to delineate the mechanistic association between MALAT-1, β-catenin, and EMT in acidic microenvironment. Citation Format: Subash C. Gupta, Yin-Yuan Mo. MALAT1 is crucial for epithelial-mesenchymal transition of breast cancer cells in acidic microenvironment. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 168. doi:10.1158/1538-7445.AM2015-168

Abstract 241: A novel tumor suppressor miRNA co-regulating EMT and p53-independent cell survival in breast cancer

Abstract Breast cancer is the most common cancer and the second leading cause of cancer-related deaths among women. MicroRNAs (miRNAs) have been shown to be key regulators of cancer-related processes including cell proliferation, survival, migration and invasion. Here, we performed a small scale miRNA mimic screen to identify miRNAs leading to inhibition of cell survival and epithelial-mesenchymal transition (EMT), both of which are key parameters for tumor growth and metastasis. We identified a miRNA (miR-644) which leads to apoptosis in p53-mutated breast cancer cell lines, but not in p53-wild type ones. Furthermore, in vitro migration and invasion assays revealed that miR-644 inhibits both migration and invasion by interfering with the EMT process. Then, we combined whole genome mRNA profiling with target prediction algorithms to determine the potential targets of this miRNA, and identified transcriptional co-repressor CTBP1 as a novel direct target which is already known for limiting apoptosis by repressing Bax transcription. CTBP1 has also been reported to bind ZEB transcription factors and assists EMT by enhancing ZEB-mediated downregulation of E-Cadherin. We showed that silencing of CTBP1 phenocopies the effects of miR-644 in induction of p53-independent apoptosis and blocking EMT and cell invasion. Currently, we are testing the role of miR-644 and its target CTBP1 in tumor growth and metastasis in in vivo assays. Overall, our results suggest that miR-644 is a novel tumor suppressor miRNA co-regulating EMT and cell survival and it could be a potential target for breast cancer therapy. Note: This abstract was not presented at the meeting. Citation Format: Umar Raza, Stefan Uhlmann, Emre Yurdusev, Stefan Wiemann, Ozgur Sahin. A novel tumor suppressor miRNA co-regulating EMT and p53-independent cell survival in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 241. doi:10.1158/1538-7445.AM2015-241

Abstract 5045: Characterization of estrogen receptor alpha phosphorylation sites as a marker for tamoxifen resistant breast cancer

Abstract A major focus of breast cancer research is to understand the mechanisms responsible for disease progression and drug resistance. Most treatment options target endocrine therapy, however, the processes that modulate these signaling pathways are poorly understood. Here, we seek to gain additional understanding about the phosphorylation status of the estrogen receptor as it relates to tamoxifin drug treatment. Tamoxifen, a selective ER inhibitor, was the first targeted breast cancer therapy developed. Unfortunately, tamoxifen therapy was shown to be ineffective for certain kinds of cancers and even in affected tumors an acquired resistance was eventually seen. Previous research has shown that the estrogen receptor protein is phosphorylated at more than 20 different sites and some of these sites are linked with poor prognosis in tamoxifen treated patients. For example, phosphorylation at the S305 site may lead to an activation of ERα and to transcription of ERα-responsive gene after tamoxifen treatment, leading to tamoxifen resistance. In addition, other phosphorylation sites have also been found to correlate with poor prognosis of tamoxifen treated patients. Thus, differential phosphorylation of ERα may serve as a biomarker to identify patients unlikely to respond to tamoxifen treatment. Here, utilizing a newly developed medium throughput IHC test system we have interrogated multiple sites of ERα for phosphorylation status by screening an extensive panel of different breast cancer patient samples and other non-breast cancer tissue microarray (TMA) samples. These patient samples were classified according to their clinical grade (ER positive or negative, Her2, progesterone positive or negative, and Ki67 index) and tamoxifen treatment and then screened for their ERα phosphorylation status. The results show unique staining patterns depending upon the tumor classification and conclusions about the utilization of this information as a potential for diagnostic applications will be discussed. Citation Format: Wayne A. Speckmann, Robert Brockett, Chun Yang, Chandra Mohan, Kevin Long, Michele Hatler, Xiaoyi Xu. Characterization of estrogen receptor alpha phosphorylation sites as a marker for tamoxifen resistant breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5045. doi:10.1158/1538-7445.AM2015-5045

Targeting ceramide metabolic pathway induces apoptosis in human breast cancer cell lines

The sphingolipid ceramide is a pro apoptotic molecule of ceramide metabolic pathway and is hydrolyzed to proliferative metabolite, sphingosine 1 phosphate by the action of acid ceramidase. Being upregulated in the tumors of breast, acid ceramidase acts as a potential target for breast cancer therapy. We aimed at targeting this enzyme with a small molecule acid ceramidase inhibitor, Ceranib 2 in human breast cancer cell lines MCF 7 and MDA MB 231. Ceranib 2 effectively inhibited the growth of both the cell lines in dose and time dependant manner. Morphological apoptotic hallmarks such as chromatin condensation, fragmented chromatin were observed in AO/EtBr staining. Moreover, ladder pattern of fragmented DNA observed in DNA gel electrophoresis proved the apoptotic activity of Ceranib 2 in breast cancer cell lines. The apoptotic events were associated with significant increase in the expression of pro-apoptotic genes (Bad, Bax and Bid) and down regulation of anti-apoptotic gene (Bcl 2). Interestingly, increase in sub G1 population of cell cycle phase analysis and elevated Annexin V positive cells after Ceranib 2 treatment substantiated its apoptotic activity in MCF 7 and MDA MB 231 cell lines. Thus, we report Ceranib 2 as a potent therapeutic agent against both ER+ and ER− breast cancer cell lines.

Intercellular interactions and progression of hormonal resistance of breast cancer cells

The main goal of the study is the analysis of the role of cell-cell interactions in the formation of the tumor cell resistance to hormonal drugs. About 70 % of breast tumors contain estrogen receptor (ER), a key molecular target for hormone (endocrine) therapy. However, the efficiency of endocrine therapy of breast cancer is limited by the development of hormone resistance which leads to progression of tumor cells to hormone-independent phenotype, increase in tumor malignancy and worse prognosis. Hormonal independence may be accompanied with the loss of the receptors, as well as with the another mechanisms including ligand-independent receptor activation, disbalance between receptor activators and repressors, stimulation of hormone-independent pathways. It is less known about the role of the intercellular interactions in the progression of hormonal resistance. Several studies demonstrate the involvement of cell junctions in the mediating of cell response to (anti) estrogens, however the significance of cell-cell contacts in the formation of hormonal resistance still not clear. Here we have hypothesized that the formation of the hormone resistance of tumors may be based, at least in part, on the transferring of the resistant phenotype from the resistant to hormone-sensitive cells – as a result of the secretion of the specific factors acting in the paracrine manner or via the direct cell-cell contacts. Using the estrogen-dependent breast cancer cells MCF-7 and the resistant subline MCF-7 / T developed by long-term cultivation of MCF-7 cells in the presence of antiestrogen tamoxifen, we investigated the possible changes in the hormonal sensitivity of these cells caused by the co-cultivation in vitro. To discern the cell cultures, the MCF-7 / T cells were previously transfected with the plasmid containing the gene of the green fluorescent protein (GFP), and GFP-positive hormone-resistant subline MCF-7 / T / GFP+ was developed. We showed that the co-cultivation of the parent and resistant cells lead to increase in the resistance of the parent cell to tamoxifen. To further explore the mechanism of such resistance, the analysis of the biological activity of exosomes prepared from the estrogen-sensitive and resistant cells was performed. The exosome preparations isolated from the resistant MCF-7 / T cells were found to induce the similar resistance in the recipient MCF-7 cells – in contrast to the control exosomes isolated from the MCF-7 cells. The subsequent cloning of these newly formed resistant cells showed that the cells retain the resistant phenotype for at least 80 days of cultivation. Totally, the results presented demonstrate the important role of cell-cell interactions in the progression of hormonal resistance, opening a new perspectives in the development of probable targets for breast cancer therapy.

Development of RNA aptamers as molecular probes for HER2(+) breast cancer study using cell-SELEX.

Development of molecules that specifically recognize cancer cells is one of the major areas in cancer research. Human epidermal growth factor receptor 2 (HER2) is specifically expressed on the surface of breast cancer cells. HER2 is associated with an aggressive phenotype and poor prognosis. In this study we aimed to isolate RNA aptamers that specifically bind to HER2 overexpressing TUBO cell line. Panel of aptamers was selected using cell-based systematic evolution of ligands by exponential enrichment (cell-SELEX). Binding studies showed that selected aptamers can identify TUBO cell line with high affinity and selectivity. Our preliminary investigation of the target of aptamers suggested that aptamers bind with HER2 proteins on the surface of TUBO cells. We believe the selected aptamers could be useful ligands for targeted breast cancer therapy.

Targeting CD151 by lentivirus-mediated RNA interference inhibits luminal and basal-like breast cancer cell growth and invasion.

CD151 is a member of the tetraspanin family, which is involved in diverse cellular processes, including proliferation, motility, and invasion. However, the role of CD151 in breast cancer especially luminal and basal-like subtype breast cancer remains obscure. Here, we report the role of CD151 in the biological behaviors of luminal and basal-like subtype cell lines and the underlying molecular mechanism. A eukaryotic expression vector expressing both CD151 shRNA and GFP was transfected into MCF-7 and MDA-MB-468 cells. The CD151 gene-silencing effect is authenticated by real-time PCR and Western blot. Our data show that the capacity for proliferation, migration, and invasion of two kinds of cells is diminished after Knockdown of CD151 via lentivirus-mediated CD151 specific shRNA. Tumor cells are arrested in G0/G1 phase. Apoptosis is increased. Moreover, we also demonstrate that the expressions of mmp26 and CD147 are inhibited by knockdown of CD151. But the inhibition depends on the cell type. We can conclude that silencing gene CD151 inhibits expression of properties that are associated with the malignant phenotype of MCF-7 and MDA-MB-468 cells. It may become a potential target in breast cancer therapy especially for luminal and basal subtypes.