Effect of Cisplatin Treatment on Interleukin-6 Gene Expression in Breast Cancer Cells: An In Vitro Study

Development of novel approaches for quantitative analysis of gene expression in intact tumor cells should provide new means for cancer detection and for studying the response of cancer cells to biological and therapeutic reagents. We developed procedures for detecting the levels of expression of multiple genes in fixed as well as viable cells using molecular beacon imaging technology. We found that simultaneous delivery of molecular beacons targeting survivin and cyclin D1 mRNAs produced strong fluorescence in breast cancer but not in normal breast cells. Importantly, fluorescence intensity correlated well with the level of gene expression in the cells detected by real-time reverse transcription-PCR or Western blot analysis. We further show that molecular beacons can detect changes of survivin gene expression in viable cancer cells following epidermal growth factor stimulation, docetaxel treatment, and overexpression of p53 gene. Thus, molecular beacon imaging is a simple and specific method for detecting gene expression in cancer cells. It has great potential for cancer detection and drug development.

Adenoviral vectors with E1A regulated by tumor-specific promoters are selectively cytolytic for breast cancer and melanoma.

The present study addresses, by transcriptomics and quantitative stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics, the estrogen receptor α (ERα) and β (ERβ)-mediated effects on gene and protein expression in T47D breast cancer cells exposed to the phytoestrogen genistein. Using the T47D human breast cancer cell line with tetracycline-dependent ERβ expression (T47D-ERβ), the effect of a varying intracellular ERα/ERβ ratio on genistein-induced gene and protein expression was characterized. Results obtained reveal that in ERα-expressing T47D-ERβ cells with inhibited ERβ expression genistein induces transcriptomics and proteomics signatures pointing at rapid cell growth and migration by dynamic activation of cytoskeleton remodeling. The data reveal an interplay between integrins, focal adhesion kinase, CDC42, and actin cytoskeleton signaling cascades, occurring upon genistein treatment, in the T47D-ERβ breast cancer cells with low levels of ERα and no expression of ERβ. In addition, data from our study indicate that ERβ-mediated gene and protein expression counteracts ERα-mediated effects because in T47D-ERβ cells expressing ERβ and exposed to genistein transcriptomics and proteomics signatures pointing at a clear down-regulation of cell growth and induction of cell cycle arrest and apoptosis were demonstrated. These results suggest that ERβ decreases cell motility and metastatic potential as well as cell survival of the breast cancer cell line. It is concluded that the effects of genistein on proteomics and transcriptomics end points in the T47D-ERβ cell model are comparable with those reported previously for estradiol with the ultimate estrogenic effect being dependent on the relative affinity for both receptors and on the receptor phenotype (ERα/ERβ ratio) in the cells or tissue of interest.

One of the most common malignancies among men is prostate cancer. Ellagic acid (EA), a polyphenol antioxidant, has many pharmacological actions, especially anticancer effects. The purpose of this study was to evaluate the effect of EA treatment on interleukin-6 (IL-6) gene expression, cell viability, IL-6 secretion, phosphorylated STAT3, ERK, and AKT cellular signaling proteins in human prostate cancer cells (PC3). The cytotoxic effects of the EA (0-100 µM) on PC3 cells were determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay. IL-6 gene expression was down, using real-time quantitative polymerase chain reaction. The cellular concentration of phosphorylated ERK1/2, AKT, and STAT3 signaling pathways was assessed by Western blotting technic. EA treatment of PC3 cells resulted in a reduction of cell viability and phosphorylated STAT3, ERK, and AKT signaling proteins after 72 h in a dose-dependent manner. IL-6 gene expression and IL-6 levels significantly increased (P < 0.05) in a dose-dependent pattern in treated PC3 with EA. Thus, these data suggested the essential role of signaling proteins in EA-mediated anti-proliferation of PC3 cells. Our finding shows that EA can be considered as a potent agent that decreases cell proliferation through a reduction of phosphorylated STAT3, ERK, and AKT cellular signaling proteins.

Spot 14 (THRSP, S14) is a nuclear protein involved in the regulation of genes required for fatty acid synthesis in normal and malignant mammary epithelial and adipose cells. Harvatine and Bauman (1) reported that conjugated linoleic acid (CLA) inhibits S14 gene expression in bovine mammary and mouse adipose tissues and reduces milk fat production in cows. We hypothesized that CLA inhibits S14 gene expression in human breast cancer and liposarcoma cells and that this will retard their growth. Exposure of T47D breast cancer cells to a mixture of CLA isomers reduced the expression of the S14 and fatty acid synthase (FAS) genes. The mixture caused a dose-related inhibition of T47D cell growth, as did pure c9, t11 and t10, c12-CLA, but not linoleic acid. Similar effects were observed in MDA-MB-231 breast cancer cells. Provision of 8 μM palmitate fully (CLA mix, t10, c12-CLA) or partially (c9, t11-CLA) reversed the antiproliferative effect in T47D cells. CLA likewise suppressed levels of S14 and FAS mRNAs in liposarcoma cells and caused growth inhibition that was prevented by palmitic acid. CLA did not affect the growth of nonlipogenic HeLa cells or human fibroblasts. We conclude that as in bovine mammary and mouse adipose cells, CLA suppresses S14 and FAS gene expression in human breast cancer and liposarcoma cells. Rescue from the antiproliferative effect of CLA by palmitic acid indicates that reduced tumor lipogenesis is a major mechanism for the anticancer effects of CLA

The C-terminal binding protein (CtBP) is a family of dimeric nuclear proteins whose levels are increased in cancers of the colon, ovaries, prostate and breast. Elevated CtBP expression is associated with poor cancer survival and can also distinguish those node negative breast cancer patients who will show worse survival. This implicates CtBP as both a biomarker and a promising candidate for therapeutic intervention. As a dimer, CtBP provides a scaffold that couples multiple different DNA-binding transcriptional regulators with a variety of chromatin modifying protein complexes to alter the epigenetic landscape throughout the nucleus. These properties provide the rationale for pharmacological targeting of CtBP to change epigenetically regulated gene expression in cancer cells. In this study, we employ computer assisted drug design to screen for optimal quantitative structure-activity relationships (QSARs) between small molecules and CtBP to identify 24 potential CtBP inhibitors. Functional screening of these compounds identifies 4 lead compounds with low toxicity and high water solubility. Treatment of breast cancer cells at micro-molar concentrations of these small molecular inhibitors induces significant de-repression of epigenetically silenced pro-epithelial genes in the mesenchymal, triple negative breast cancer cell line, MDA-MB-231. This re-activation is associated with eviction of CtBP from the respective gene promoters, disrupted recruitment of CtBP-chromatin modifying protein complexes, increased deposition of activating epigenetic histone marks, and upregulation of both pro-epithelial gene mRNA and protein expression. In functional assays, CtBP inhibition by these small molecular inhibitors decreases cellular invasion, and improves DNA repair. FRET (Förster resonance energy transfer) analysis demonstrates that CTBP inhibition results in decreased FRET intensity, suggesting that CTBP dimerization is repressed by CTBP inhibition. In addition, pharmacological inhibition of CtBP combines with established epigenetically targeted drugs to synergistically decrease cell migration and potentiate the reactivation of silenced pro-epithelial gene expression in triple negative cancer cells. Finally, CTBP inhibition results in transcriptional repression of MDR1 expression and reduces the population of Doxorubicin resistant cells in the triple negative breast cancer cell lines. These findings implicate the possible use of this class of compounds in strategies for therapeutic intervention that may increase the efficacy and decrease the acquired resistance to targeted therapeutic intervention in breast cancer. Citation Format: Jung S. Byun, Samson Park, Dae IK Yi, Mohamed Kabbout, Genqing Liang, Kevin L. Gardner. Epigenetic re-wiring of breast cancer by pharmacological targeting of C-terminal binding protein [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1156. doi:10.1158/1538-7445.AM2017-1156

Inhibition of the Activity of Cyclophilin A Impedes Prolactin Receptor-Mediated Signaling, Mammary Tumorigenesis, and Metastases

The C‐terminal binding protein (CtBP) is a family of dimeric nuclear proteins whose levels are increased in cancers of the colon, ovaries, prostate and breast. Elevated CtBP protein expression is associated with poor cancer survival, thus implicating CtBP as both a biomarker and a promising candidate for therapeutic intervention. As a dimer, CtBP provides a scaffold that couples multiple different DNA‐binding transcriptional regulators with a variety of chromatin modifying protein complexes to alter the epigenetic landscape throughout the nucleus. These properties provide the rationale for pharmacological targeting of CtBP to alter or epigenetically change regulated gene expression in cancer cells. In this study, we employ computer assisted drug design to screen for optimal quantitative structure‐activity relationships (QSARs) between small molecules and CtBP to identify 24 potential CtBP inhibitors. Functional screening of these compounds identifies 4 lead compounds with low toxicity and high water solubility. Treatment of breast cancer cells at micro‐molar concentrations of these small molecular inhibitors induces significant de‐repression of epigenetically silenced pro‐epithelial genes in the mesenchymal, triple negative breast cancer cell line, MDA‐MB‐231. This re‐activation is associated with eviction of CtBP from the respective gene promoters, disrupted recruitment of CtBP‐chromatin modifying protein complexes, increased deposition of activating epigenetic histone marks, and upregulation of both pro‐epithelial gene mRNA and protein expression. In functional assays, CtBP inhibition by these small molecular inhibitors decreases cellular invasion, and improves DNA repair.FRET (Förster resonance energy transfer) analysis demonstrates that CTBP inhibition results in decreased FRET intensity, suggesting the CTBP dimerization interaction is repressed by CTBP inhibition. In addition, pharmacological inhibition of CtBP combines with established epigenetically targeted drugs to synergistically decrease cell migration and potentiate the reactivation of silenced pro‐epithelial gene expression in triple negative cancer cells. Finally, CTBP inhibition results in transcriptional repression of MDR1 expression and reduces the population of Doxorubicin resistant cells in triple negative breast cancer cell lines.These findings implicate the possible use of this compound in strategies for therapeutic intervention that may increase the efficacy and decrease the acquired resistance to targeted therapeutic intervention in breast cancer.

The cyclin D1/PRAD1 gene is correlated with carcinogenesis of human breast cancer. In this study, we have analyzed effects of breast cancer-related hormones on the cyclin D1 gene expression in T-47D human breast cancer cells. Estradiol (E2) and human prolactin (hPRL) equally enhanced the cyclin D1 gene expression in the cells, and 22 and 20 kDa human growth hormones (22K and 20K hGHs) showed less stimulatory effects. In the presence of E2, however, hPRL or 22K hGH showed additive stimulations of the cyclin D1 gene expression to that by E2 alone, while 20K hGH did not show any additive stimulation of the gene expression. The results suggest that the signal pathways through estrogen and hPRL receptors are important for cyclin D1 gene expression in breast cancer cells, and that 20K hGH has little effect on the cyclin D1 gene expression in these cells because of its lower affinity to PRL receptor.

The PD-1 (programmed death-1)/PD-L1 (PD-ligand 1) checkpoint is a critical regulator of activated T cell-cancer cell interactions, serving to defend tumor cells against immune destruction. PD-L1 (or B7-H1) produced by tumor cells engages PD-1 to suppress activated T cell engagement with tumor cells, but also to induce apoptosis of T cells. Overexpression of PD-L1 is observed in melanoma, pancreatic and lung cancers, among others, and may correlate with decreased survival (P. Wu et al., PLoS One 2015; 10(6):e0131403). Antibodies directed to PD-L1 have shown promise as immunotherapy for melanoma, lung and kidney cancers and hematological malignancies (D.B. Page et al., Ann Rev Med 2014; 65:185-202). Activation of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3-K) signal transducing pathways is critical to expression of the PD-L1 gene in cancer cells and interferon-γ (IFN-γ) is a potent endogenous inducer of expression of PD-L1. Nano-diamino-tetrac (NDAT; Nanotetrac) is an anticancer/anti-angiogenic agent targeted to the thyroid hormone-tetrac receptor on the extracellular domain of integrin αvβ3. The integrin is generously expressed by cancer cells and dividing endothelial cells, but not by nondividing, nonmalignant cells. NDAT is made by covalently bonding via a linker of tetrac (tetraiodothyroacetic acid), a derivative of L-thyroxine (T4), to poly(lactic-co-glycolic acid)(PLGA) to limit its action to the receptor on integrin αvβ3. NDAT inhibits the PI3-K and MAPK pathways in cancer cells and blocks expression of a panel of genes critical to cancer cell survival pathways. Tetrac is also known to block enhancement by thyroid hormone of actions of IFN-γ. We therefore examined the actions in vitro of T4 and NDAT on PD-L1 mRNA (qPCR) on human colon carcinoma (HCT116) cells and breast cancer (MDA-MB-231) cells. In HCT116 cells, T4 (10-7 M total; 10-10 M free hormone) increased PD-L1 gene expression by 100% (P &amp;lt;0.01). NDAT (10-7 M tetrac-equivalent) reduced basal level PD-L1 expression by 45% and T4-stimulated PD-L1 by 75% (each reduction, P &amp;lt;0.01). In MDA-MB-231 cells, T4-stimulated PD-L1 expression by 40% and NDAT reduced this enhancement by 50% (P &amp;lt;0.01). NDAT did not affect basal level PD-L1 gene expression in these cells. The absence of this effect is under investigation, particularly, the state of activation of αvβ3 in MDA-MB-231 cells. Thus, NDAT significantly and non-immunologically suppresses T4-induced PD-L1 gene expression in human colon and breast cancer cells. Nonmalignant cells and normal immune system surveillance will escape this novel effect of NDAT because normal cells, except for rapidly dividing endothelial cells, express little αvβ3. In the clinical setting, the gene is invariably exposed to host thyroid hormone and we speculate that T4 supports defensive PD-1/PDL1 checkpoint activity in tumor cells. Citation Format: Yu-Tang Chin, Hsuan-Yu Lai, Heng-Yuan Tang, Hung-Yun Lin, Shaker A. Mousa, Paul J. Davis. Anti-PD-L1 activity of Nano-diamino-tetrac (Nanotetrac) on cancer cells. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B37.

Parathyroid hormone-related protein (PTHrP) is an autocrine/paracrine factor produced by breast cancer cells that is speculated to play a major role in permitting breast cancer cells to grow into the bone microenvironment by stimulating the bone resorption axis. It has been previously shown that EGFR signaling induces the production of PTHrP in several primary and transformed epithelial cell types. Therefore, we investigated the relationship between EGFR and PTHrP gene expression in human breast cancer cells. Of a panel of 7 breast epithelial and cancer cell lines, the osteolytic, EGFR- positive lines (MDA-MB-231 and NS2T2A1) exhibited higher levels of PTHrP transcript expression. Amphiregulin mRNA levels in all lines were approximately 2 orders of magnitude higher than those of TGFalpha or HB-EGF. In the EGFR bearing lines, the receptor was phosphorylated at tyrosine 992 under basal conditions, and the addition of 100 nM amphiregulin did not lead to the phosphorylation of other tyrosine residues typically phosphorylated by the prototypical ligand EGF. Treatment of the EGFR positive lines with the EGFR inhibitor PD153035 and amphiregulin-neutralizing antibodies reduced PTHrP mRNA levels by 50-70%. Stable EGFR expression in the MCF7 line failed to increase basal PTHrP mRNA levels; however, treatment of this cell line with exogenous EGF or amphiregulin increased PTHrP transcription 3-fold. Transient transfection analysis suggests that the MAPK pathway and ETS transcription factors mediate EGFR coupling to PTHrP gene expression. Taken together, it appears that autocrine stimulation of EGFR signaling by amphiregulin is coupled to PTHrP gene expression via EGFR Tyr992 and MAPK, and that this pathway may contribute to PTHrP expression by breast tumor cells.

Mutant p53 accumulation has been shown to induce the multidrug resistance gene (MDR1) and ATP binding cassette (ABC)-based drug efflux in human breast cancer cells. In the present work, we have found that transcriptional activation of the oxidative stress-responsive heat shock factor 1 (HSF-1) and expression of heat shock proteins, including Hsp27, which is normally known to augment proteasomal p53 degradation, are inhibited in Adriamycin (doxorubicin)-resistant MCF-7 cells (MCF-7/adr). Such an endogenous inhibition of HSF-1 and Hsp27 in turn results in p53 mutation with gain of function in its transcriptional activity and accumulation in MCF-7/adr. Also, lack of HSF-1 enhances nuclear factor κB (NF-κB) DNA binding activity together with mutant p53 and induces MDR1 gene and P-glycoprotein (P-gp, ABCB1), resulting in a multidrug-resistant phenotype. Ectopic expression of Hsp27, however, significantly depleted both mutant p53 and NF-κB (p65), reversed the drug resistance by inhibiting MDR1/P-gp expression in MCF-7/adr cells, and induced cell death by increased G(2)/M population and apoptosis. We conclude from these results that HSF-1 inhibition and depletion of Hsp27 is a trigger, at least in part, for the accumulation of transcriptionally active mutant p53, which can either directly or NF-κB-dependently induce an MDR1/P-gp phenotype in MCF-7 cells. Upon Hsp27 overexpression, this pathway is abrogated, and the acquired multidrug resistance is significantly abolished so that MCF-7/adr cells are sensitized to Dox. Thus, clinical alteration in Hsp27 or NF-κB level will be a potential approach to circumvent drug resistance in breast cancer.

Human telomerase reverse transcriptase (hTERT) underlies cancer cell immortalization, and the expression of hTERT is regulated strictly at the gene transcription. Here, we report that transcription factor Ets2 is required for hTERT gene expression and breast cancer cell proliferation. Silencing Ets2 induces a decrease of hTERT gene expression and increase in human breast cancer cell death. Reconstitution with recombinant hTERT rescues the apoptosis induced by Ets2 depression. In vitro and in vivo analyses show that Ets2 binds to the EtsA and EtsB DNA motifs on the hTERT gene promoter. Mutation of either Ets2 binding site reduces the hTERT promoter transcriptional activity. Moreover, Ets2 forms a complex with c-Myc as demonstrated by co-immunoprecipitation and glutathione S-transferase pulldown assays. Immunological depletion of Ets2, or mutation of the EtsA DNA motif, disables c-Myc binding to the E-box, whereas removal of c-Myc or mutation of the E-box also compromises Ets2 binding to EtsA. Thus, hTERT gene expression is maintained by a mechanism involving Ets2 interactions with the c-Myc transcription factor and the hTERT gene promoter, a protein-DNA complex critical for hTERT gene expression and breast cancer cell proliferation.

KiSS-1 has been shown to function as a tumor metastasis suppressor gene and reduce the number of metastases in different cancers. The expression of KiSS-1 or KiSS1, like other tumor suppressor, is commonly reduced or completely ablated in a variety of cancers via an unknown mechanism. Here we show that the loss of KiSS-1 expression in highly metastatic breast cancer cell lines correlates directly with the expression levels of two transcription factors, activator protein-2alpha (AP-2alpha) and specificity protein 1 (Sp1), which synergistically activate the transcriptional regulation of KiSS-1 in breast cancer cells. Although the KiSS-1 promoter contains multiple AP-2alpha binding elements, AP-2alpha-mediated regulation occurs indirectly through Sp1 sites, as determined by deletion and mutation analysis. Overexpression of AP-2alpha into highly metastatic breast cell lines did not alter KiSS-1 promoter-driven luciferase gene activity. However, co-transfection of AP-2alpha wild-type or the dominant negative form of AP-2 lacking its C-terminal DNA-binding domain, AP-2B, together with Sp1, increased KiSS-1 promoter activity dramatically, suggesting that AP-2alpha regulation of KiSS-1 transcription does not require direct binding to the KiSS-1 promoter. Furthermore, we demonstrated that AP-2alpha directly interacted with Sp1 to form transcription complexes at two tandem Sp1-binding sites of the promoter to activate KiSS-1 transcription. Together, our results indicate that AP-2alpha and Sp1 are strong transcriptional regulators of KiSS-1 and that loss or decreased expression of AP-2alpha in breast cancer may account for the loss of tumor metastasis suppressor KiSS-1 expression and thus increased cancer metastasis.

There is concern that ingestion of dietary phytoestrogens may increase risk of estrogen receptor alpha (ERα)-positive breast cancer. The prenylflavone icaritin, a phytoestrogen consumed in East Asian societies for its perceived beneficial effects on bone health, stimulated the growth of breast cancer (MCF-7) cells at low concentrations. Although acting like an estrogenic ligand, icaritin exerted an unexpected suppressive effect on estrogen-stimulated breast cancer cell proliferation and gene expression at higher concentrations. Like estradiol, icaritin could dose-dependently destabilize ERα protein. However, destabilization of ERα by the estradiol/icaritin combination was profound and greater than that observed for either compound alone. Microarray gene expression analyses implicated aryl hydrocarbon receptor (AhR) signaling for this suppressive effect of icaritin. Indeed, icaritin was an AhR agonist that competitively reduced specific binding of a potent AhR agonist and increased expression of the AhR-regulated gene CYP1A1. When AhR was knocked down by small interfering RNA, the suppressive effect of icaritin on estradiol-stimulated breast cancer cell growth and gene expression was abolished, and ERα protein stability was partially restored. Similarly in an athymic nude mouse model, icaritin restricted estradiol-stimulated breast cancer xenograft growth and strongly reduced ERα protein levels. Overall, our data support the feasibility for the development of dual agonists like icaritin, which are estrogenic but yet, through activating AhR-signaling, can destabilize ERα protein to restrict ERα-positive breast cancer cell growth.