T47D Human Breast Cancer Cells Research Articles

Kuwanon C inhibits proliferation and induction of apoptosis via the intrinsic pathway in MDA-MB231 and T47D breast cancer cells

Breast cancer ranks as the most prevalent malignancy, presenting persistent therapeutic challenges encompassing issues such as drug resistance, recurrent occurrences, and metastatic progression. Therefore, there is a need for targeted drugs that are less toxic and more effective against breast cancer. Kuwanon C, an isoamylated flavonoid derived from mulberry resources, has shown promise as a potential candidate due to its strong cytotoxicity against cancer cells. The present study focused on investigating the anticancer activity of kuwanon C in two human breast cancer cell lines, MDA-MB231 and T47D cells. MTS assay results indicated a decrease in cell proliferation with increasing concentrations of kuwanon C. Furthermore, kuwanon C upregulated the expression levels of the cyclin-dependent kinase inhibitor p21 and effectively inhibited cell DNA replication and induced DNA damage. Flow cytometry confirmed that kuwanon C induced cell apoptosis and upregulated the expression levels of pro-apoptotic proteins (Bax and c-caspase3). Additionally, it stimulated the production of reactive oxygen species (ROS) in the cells. Transmission electron microscopy and Fluo-4 AM-calcium ion staining experiments provided insights into the endoplasmic reticulum (ER), revealing that kuwanon C induced ER stress. Kuwanon C upregulated the expression levels of unfolded protein response-related proteins (ATF4, GADD34, HSPA5, and DDIT3). Overall, the present findings suggested that kuwanon C exerts a potent inhibitory effect on breast cancer cell proliferation through modulating of the p21, induction of mitochondrial-mediated apoptosis, activation of ER stress and induction of DNA damage. These results position kuwanon C as a potential targeted therapeutic agent for breast cancer.

Synthesis and computational study of metal complex of α-mangostin as an anticancer candidate

Context: The number of breast cancer patients from year to year is increasing, accompanied by an increase in global mortality; the position in Indonesia has reached 60-70% with end-stage conditions. ERα antiproliferative activity exists in α-mangostin compounds, but this compound still has several physicochemical problems, including its solubility. Therefore, efforts are needed to increase the activity of α-mangostin and its affinity as an alpha estrogen receptor antagonist through a structure-based design method. Aims: To obtain new compounds derived from α-mangostin in the form of a metal complex with cobalt (AM-Co), platinum (AM-Pt), and iron (AM-Fe) as medicinal ingredients that can be used as breast cancer therapeutic agents. Methods: The research methods include in silico studies (pharmacokinetic and toxicity prediction, molecular docking, and molecular dynamics), semi-synthesis to form a complex compound, and testing cytotoxic activity against MCF-7 and T-47D human breast cancer cells. Results: From the in vitro test results, it can be seen that the AM-Pt compound showed the best anticancer activity on MCF-7 cells compared to others from IC50 and SI values; this was supported by the results of in silico studies either through molecular docking or molecular dynamics, where this compound was more effective. Conclusions: Furthermore, it can be used as an anticancer candidate; particularly, its activity is predicted to be more potent as an ER antagonist than -mangostin and cisplatin.

Estrogen-induced reactive oxygen species, through epigenetic reprogramming, causes increased growth in breast cancer cells

Despite the progress made in cancer diagnosis and treatment, breast cancer remains the second leading cause of cancer-related death among the women. Exposure to elevated levels of endogenous estrogen or environmental estrogenic chemicals is an important risk factor for breast cancer. Estrogen metabolites and ROS generated during estrogen metabolism are known to play a critical role in estrogen carcinogenesis. However, the molecular mechanisms through which estrogen-induced ROS regulate gene expression is not clear. Epigenetic changes of DNA methylation and histone modifications are known to regulate genes expression. Therefore, the objective of this study was to evaluate whether estrogen-induced ROS, through aberrant expression of epigenetic regulatory genes and epigenetic reprogramming, causes growth of breast cancer cells. Estrogen responsive MCF-7 and T47D human breast cancer cells were exposed to natural estrogen 17 beta-estradiol (E2) and synthetic estrogen Diethylstilbestrol (DES) both alone and in combination with antioxidant N-acetyl cysteine. Effects of NAC-mediated scavenging of estrogen-induced ROS on cell growth, gene expression, and histone modifications were measured. The result of MTT and cell cycle analysis revealed significant abrogation of E2 and DES-induced growth by scavenging ROS through NAC. E2 and DES caused significant changes in expression of epigenetic regulatory genes for DNA methylation and histone modifications as well as changes in both gene activating and repressive marks in the Histone H3. NAC restored the expression of epigenetic regulatory genes and changes in histone marks. Novel findings of this study suggest that estrogen can induce growth of breast cancer cells through ROS-dependent regulation of epigenetic regulatory genes and epigenetic reprogramming of histone marks.

Constitutive knock-in of MnSODK68R causes dilated cardiomyopathy and senescence in mice

The mitochondrial superoxide dismutase (SOD2 or MnSOD) is an essential enzyme for managing oxidative stress, particularly by removing the highly reactive superoxide generated during the normal functioning of the electron transport chain. The activity of MnSOD is regulated post-translationally by acetylation, specifically at lysine 68. The deacetylated form of MnSOD is catalytically active, and it has been shown that the deacetylation mimic, MnSOD K68R in which lysine 68 has been replaced with arginine, has upregulated dismutase activity in MCF7 and T47D human breast cancer cells. Overexpression of MnSOD has been shown to increase lifespan and protect against oxidative stress in the aging heart. We investigated a MnSOD K68R knock-in mouse with ubiquitous expression of the mutant enzyme and expected similar effects as the MnSOD overexpressing mice. However, we have found that MnSOD K68R homozygous knock-in mice develop dilated cardiomyopathy and have significantly decreased ejection fraction as evidenced by echocardiography at a young age. These mice also exhibit lower blood pressure compared to wild-type mice. Strikingly, these mice exhibit higher body weight, with increased lean mass and food consumption. Mitochondria isolated from MnSOD K68R knock-in mouse hearts showed higher respiratory capacity than WT counterparts. Biochemical analysis revealed increased lipid peroxidation, oxidized peroxiredoxin and senescence markers, possibly indicative of a premature aging phenotype. We conclude that MnSOD constitutive function upregulation may produce a sustained increase in hydrogen peroxide levels that could cause senescence and lead to dilated cardiomyopathy. This research was funded by NCI grant R01CA152601 and, R01CA214025, and the Cancer Prevention and Research Institute of Texas grant number RR20012. JS is supported by T32 AG 021890. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract P4-07-50: mRNA expression and delivery efficacy of lipid nanoparticles in the cells breast tumor microenvironment: in vitro and in vivo evaluation

Abstract During the COVID-19 pandemics we have all witnessed the clinical importance of mRNA as current vaccines and future therapeutics. mRNA therapies have a potential to revolutionize cancer treatment. Delivery of mRNA requires lipid nanoparticles (LNP) to protect the cargo from degradation. mRNA has a negative charge and depends on positively charged lipids to be encapsulated in LNP. These lipids can be either ionizable at certain pH or constantly cationic. Even though previous studies had evaluated the formulation properties of ionizable and cationic LNP systems, there is the need to understand their specificity in terms of mRNA delivery and protein expression in breast cancer tumor microenvironment. The objective of this work was to assess the kinetics of LNP cellular uptake and mRNA expression inv breast cancer (BC) cells and fibroblasts, the most frequent cell type in the tumor microenvironment cells, while studying the mechanisms involved in differential behaviors of LNP formulated with cationic and ionizable lipids. To achieve this goal mRNA-LNP containing ionizable lipids (LNP-A) and cationic lipids (LNP-B) were designed and formulated using Nanoassemblr® Benchtop microfluidics mixer (Precision NanoSystems). mRNA-LNP were characterized for size, zeta potential using dynamic light scattering (DLS) and mRNA encapsulation efficiency using RiboGreen assay. LNP were tagged with rhodamine lipid to investigate the uptake kinetic and a reporter GFP mRNA to evaluate mRNA expression in murine 4T1 and human MCF7, MDA-231, SUM-159 and T-47D breast cancer cells and BJ fibroblasts. Live fluorescence microscopy imaging, IncuCyte S3®, was used to determine the LNP uptake and GFP mRNA expression. In vitro biocompatibility was assessed with WST-1 assay. Additionally, expression of mRNA delivered from LNP in tumor microenvironment was evaluated in vivo in a syngeneic 4T1 breast cancer model using mRNA luciferase and IVIS imaging. mRNA-LNPs possessed an average diameter of 77 - 107 nm, narrow size distribution, neutral zeta potential and high mRNA encapsulation efficiency (>94%). Our results demonstrated that mRNA expression was higher in breast cancer cells when delivered from LNP-A formulation and in BJ fibroblasts when delivered from LNP-B. LNP-A, the ionizable LNP, was tested in the breast cancer cells to confirm the efficacy of the delivery. The highest transfection efficacy, from high to low, T-47D, MCF7, SUM-159, 4T1 and MDA-231.We have further investigated the cellular uptake mechanisms of LNP using uptake pathway inhibitors for caveolae endocytosis, clathrin endocytosis, and phagocytosis. Our data confirm that there are differences in mechanisms that govern the uptake of mRNA LNP in breast cancer cells and fibroblasts. Clathrin-mediated endocytosis was active in 4T1 breast cancer cells for ionizable and cationic LNP. Interestingly, despite in vitro differences in uptake and mRNA expression, in vivo results show that both formulations efficiently delivered luciferase-mRNA in the tumor microenvironment. Histology results demonstrated similar luciferase expression for both LNP in tumors. Additionally, we were able to confirm the prominent presence of fibroblast and similar distribution in the 4T1 subcutaneous model which could explain the similar efficacy of cationic and ionizable LNP. Understanding uptake and mRNA expression of different LNP formulations in the tumor microenvironment can help in achieving the necessary protein expression for breast cancer therapies. Furthermore, determining the most efficient carrier in early stages may reduce the time required for clinical translation. Acknowledgement: This research was supported in part by CPRIT Core for RNA Therapeutics and Research. Citation Format: Karem A. Court, Anjana Tiwari, Eric Chau, John Cooke, Biana Godin. mRNA expression and delivery efficacy of lipid nanoparticles in the cells breast tumor microenvironment: in vitro and in vivo evaluation [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-07-50.

Abstract 1149: Decitabine inhibits breast cancer cell proliferation through inhibition of endogenous retroviruses in vitro

Abstract The purpose of the study is to elucidate the antineoplastic mechanisms of decitabine, a DNA methyltransferase inhibitor. Previous literature suggests that decitabine activates human endogenous retroviruses which subsequently enhances expression of interferons, thereby suppressing tumor cell proliferation. Contrary to this hypothesis, our in vitro study with human and mouse breast cancer cell lines showed a positive correlation between the antiproliferative effect of decitabine and its suppressive effect on two endogenous retroviruses, the human endogenous retrovirus type K (HERV-K) and the mouse mammary tumor virus (MMTV). Decitabine stimulated proliferation of the T47D human breast cancer cell line at lower doses but inhibited cell proliferation at higher doses. Correspondingly, HERV-K transcripts (env and pol) increased at lower doses and decreased at higher doses. Interestingly, expression of the E-cadherin gene, an indicator of epithelial differentiation, followed an opposite trend. Decitabine inhibited proliferation of the mouse 4T1 breast cancer cell line and its expression of MMTV in a dose-dependent manner, while enhancing expression of the mouse E-cadherin. 4T1 cells overexpressing MMTV env became more resistant to decitabine, so was a human breast cancer cell line (BT-20) overexpressing HERV-K env, while T47D cells with HERV-K knockdown became more susceptible to the drug. Meanwhile, in vivo studies showed that decitabine successfully suppressed tumor development in BALB/c mice inoculated with 4T1 cells. Surprisingly, expression of MMTV env was elevated in tumors of mice treated with decitabine, along with enhanced E-cadherin expression. These data suggest decitabine inhibits cancer cell proliferation (at least partially) through inhibition of endogenous retroviruses in vitro, but its mechanisms of action in vivo are different. Further studies of the interaction between decitabine and endogenous retroviruses using engineered 4T1 cells in BALB/c mice are currently underway. Citation Format: Jiayi Li, John Lin, Leo Andrada, Bryce Grohol, Serratt Nong, Yingguang Liu. Decitabine inhibits breast cancer cell proliferation through inhibition of endogenous retroviruses in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1149.

Comparative Study of Antiproliferative Activity in Different Plant Parts of Phaleria macrocarpa and the Underlying Mechanism of Action.

Phaleria macrocarpa is a medicinal plant widely used in Indonesian folk medicine to treat several diseases, including cancer. However, the comparative evaluation of various plant parts of P. macrocarpa has not been studied for their anticancer properties on breast cancer. The study aimed to assess the antiproliferative activity of the ethanol extract of various parts of Phaleria macrocarpa against T47D human breast cancer cell lines. Several parts of P. macrocarpa, including pericarp, mesocarp, seed, and leaf, were used to determine the most potent plant part to inhibit the growth of T47D cells. The cytotoxic effects of each plant part were evaluated by WST-1 assay. The apoptotic level of T47D cells was determined by annexin V-FITC-PI and DNA fragmentation assay. Propidium iodide staining and the CFSE assay were used to examine the effect of each extract on cell cycle distribution and cell division, respectively. The relative number of caspase-3, Bax, and Bcl-2 was analyzed by flow cytometry technique. WST-1 assay revealed that P. macrocarpa leaves exhibited the most potent antiproliferative activity (p < 0.05) compared to other plant parts with selectivity only to T47D cells. P. macrocarpa leaves extract effectively induced apoptosis, inhibited proliferation, and arrested the cell cycle of T47D cells. The relative number of caspase-3 was significantly (p < 0.05) increased after being treated with P. macrocarpa leaf extract. P. macrocarpa leaf extract also leads to the dose-dependent accumulation in the Bax/Bcl-2 ratio due to upregulation of Bax and downregulation of Bcl-2. The overall results indicated that P. macrocarpa leaves could inhibit the proliferation of T47D cells and trigger apoptosis through caspase-3 activation and Bax/Bcl regulation. Therefore, P. macrocarpa leaves can be used for breast cancer therapy.

A novel LncRNA PTH-AS upregulates interferon-related DNA damage resistance signature genes and promotes metastasis in human breast cancer xenografts

Long noncoding RNAs (lncRNAs) are important tissue-specific regulators of gene expression, and their dysregulation can induce aberrant gene expression leading to various pathological conditions, including cancer. Although many lncRNAs have been discovered by computational analysis, most of these are as yet unannotated. Herein, we describe the nature and function of a novel lncRNA detected downstream of the human parathyroid hormone (PTH) gene in both extremely rare ectopic PTH-producing retroperitoneal malignant fibrous histiocytoma and parathyroid tumors with PTH overproduction. This novel lncRNA, which we have named “PTH-AS,” has never been registered in a public database, and here, we investigated for the first time its exact locus, length, transcription direction, polyadenylation, and nuclear localization. Microarray and Gene Ontology analyses demonstrated that forced expression of PTH-AS in PTH–nonexpressing human breast cancer T47D cells did not induce the ectopic expression of the nearby PTH gene but did significantly upregulate Janus kinase–signal transducer and activator of transcription pathway–related genes such as cancer-promoting interferon-related DNA damage resistance signature (IRDS) genes. Importantly, we show that PTH-AS expression not only enhanced T47D cell invasion and resistance to the DNA-damaging drug doxorubicin but also promoted lung metastasis rather than tumor growth in a mouse xenograft model. In addition, PTH-AS–expressing T47D tumors showed increased macrophage infiltration that promoted angiogenesis, similar to IRDS-associated cancer characteristics. Although the detailed molecular mechanism remains imperfectly understood, we conclude that PTH-AS may contribute to tumor development, possibly through IRDS gene upregulation.

Transcriptome profiling of curcumin-treated T47D human breast cancer cells by a system-based approach

A systemic approach provides a comprehensive scheme from the molecular regulatory mechanism of drugs in living systems. This study investigated the regulatory mechanism of curcumin in breast cancer cells by analyzing the transcriptome alterations in T47D breast cancer cells. For this purpose, the cytotoxic effect of curcumin was evaluated by MTT assay after treating T47D cells with 0–50 μM concentration of curcumin. Furthermore, transcriptome analysis was performed using RNA sequencing to find differentially expressed genes following curcumin treatment in T47D cells. Transcription factor and microRNA profiles related to curcumin gene signature were retrieved from related databases. A system-based network analysis was performed to deepen our understanding of the curcumin mechanism of action in T47D cells. In an independent analysis, a publicly available microarray dataset including gene expression and clinical data was used to evaluate the prognostic power of curcumin signature and its relation with patients' outcomes by the Cox model. Our results suggest that curcumin triggers cell death-related pathways and regulates the metabolism of lipids and apoptotic processes in T47D cells. Curcumin signature had significant prognostic power. Regulatory network analyses found specific proteins, microRNAs and transcription factors as the hub nodes, which corresponded to the effects of curcumin treatment. This work provides a picture of the cytotoxicity mechanism of curcumin and potential therapeutic targets for treatment optimization of breast cancer.

Studying Chemotactic Migration in Dunn Chamber: An Example Applied to Adherent Cancer Cells.

Cell migration is a vital process in the development of multicellular organisms. When deregulated, it is involved in many diseases such as inflammation and cancer metastisation. Some cancer cells could be stimulated using chemoattractant molecules, such as growth factor Heregulin β1. They respond to the attractant or repellent gradients through a process known as chemotaxis. Indeed, chemotactic cell motility is crucial in tumour cell dissemination and invasion of distant organs. Due to the complexity of this phenomenon, the majority of available in vitro methods to study the chemotactic motility process have limitations and are mainly based on endpoint assays, such as the Boyden chamber assay. Nevertheless, in vitro time-lapse microscopy represents an interesting opportunity to study cell motility in a chemoattracting gradient, since it generates large volume image-based information, allowing the analysis of cancer cell behaviours. Here, we describe a detailed time-lapse imaging protocol, designed for tracking T47D human breast cancer cell line motility, toward a gradient of Heregulin β1 in a Dunn chemotaxis chamber assay. The protocol described here is readily adapted to study the motility of any adherent cell line, under various conditions of chemoattractant gradients and of pharmacological drug treatments. Moreover, this protocol could be suitable to study changes in cell morphology, and in cell polarity.

4-Flourophenyl-substituted 5H-indeno[1,2-b]pyridinols with enhanced topoisomerase IIα inhibitory activity: Synthesis, biological evaluation, and structure–activity relationships

A series of fluorinated and hydroxylated 2,4-diphenyl indenopyridinols were designed and synthesized using l-proline-catalyzed and microwave-assisted synthetic methods for the development of new anticancer agents. Adriamycin and etoposide were used as reference compounds for the evaluation of topo IIα inhibitory and anti-proliferative activity of the synthesized compounds. Exploring the structure–activity relationships of 36 prepared compounds and biological results, most of the compounds with ortho- and para-fluorophenyl at 4-position of indenopyridinol ring displayed strong topo IIα inhibition. In addition, the majority of the ortho- and meta-fluorophenyl substituted compounds 1–24 displayed strong anti-proliferative activity against DU145 prostate cancer cell line compared to the positive controls. Interestingly, compound 4 possessing ortho-phenolic and ortho-fluorophenyl group at 2- and 4-position, respectively of the central pyridine ring showed high anti-proliferative activity (IC50 = 0.82 μM) against T47D human breast cancer cell line, while para-phenolic and para-fluorophenyl substituted compound 36 exhibited potent topo IIα inhibitory activity with 94.7% and 88.6% inhibition at 100 μM and 20 μM concentration, respectively. A systematic comparison between the results of this study and the previous study indicated that minor changes in the position of functional groups in the structure affect the topo IIα inhibitory activity and anti-proliferative activity of the compounds. The findings from this study will provide valuable information to the researchers working on the medicinal chemistry of topoisomerase IIα-targeted anticancer agents.

Pd(II) complexes with chelating N-(1-alkylpyridin-4(1H)-ylidene)amide (PYA) ligands: Synthesis, characterization and evaluation of anticancer activity

The bidentate N-(1-Alkylpyridin-4(1H)-ylidene)amide (PYA) pro-ligands [H2LBn][Cl]2 (2), and [H2LMe][TfO]2 (3) were prepared by simple alkylation reactions of the known compound, N,N-di(pyridin-4-yl)oxalamide (H2L, 1). The Pd(II) complexes, [Pd(LBn)2][Cl]2 (4), [Pd(LMe)2][Cl][TfO] (5), Pd(LBn)Cl2 (6) and Pd(LMe)Cl2 (7) were synthesized through reactions between these pro-ligands and suitable Pd(II) substrates in the presence of base. The molecular structures of 3 and 6 were obtained by single crystal X-ray structure determinations. Studies of the experimental and computational DNA binding interactions of the compounds 1–7 revealed that overall 4 and 6 have the largest values for the binding parameters Kb and ΔGbo. The results showed a good correlation with the steric and electronic parameters obtained by quantitative structure activity relationship (QSAR) studies. In-vitro cytotoxicity studies against four different cell lines showed that the human breast cancer cell lines MCF-7, T47D and cervical cancer cell line HeLa had either higher or similar sensitivities towards 4, 6 and 2, respectively, compared to cisplatin. In general, the cytotoxicity of the compounds, represented by IC50 values, decreased in the order 4 > 6 > 2 > 5 > 3 > 1 > 7 in cancer cell lines. Apoptosis contributed significantly to the cytotoxic effects of these anticancer agents as evaluated by apoptosis studies.

MICA-G129R: A bifunctional fusion protein increases PRLR-positive breast cancer cell death in co-culture with natural killer cells.

Breast cancer cells were reported to up-regulate human prolactin receptor (PRLR) to assist their growth through the utilization of prolactin (PRL) as the growth factor, which makes PRLR a potential therapeutic target for breast cancer. On the other hand, advanced cancer cells tend to down-regulate or shed off stress signal proteins to evade immune surveillance and elimination. In this report, we created a fusion protein consisting of the extracellular domain of MHC class I chain-related protein (MICA), a stress signal protein and ligand of the activating receptor NKG2D of natural killer (NK) cells, and G129R, an antagonistic variant of PRL. We hypothesize that the MICA portion of the fusion protein binds to NKG2D to activate NK cells and the G129R portion binds to PRLR on breast cancer cells, so that the activated NK cells will kill the PRLR-positive breast cancer cells. We demonstrated that the MICA-G129R fusion protein not only binds to human natural killer NK-92 cells and PRLR-positive human breast cancer T-47D cells, but also promotes NK cells to release granzyme B and IFN-γ and enhances the cytotoxicity of NK cells specifically on PRLR-positive cells. The fusion protein, therefore, represents a new approach for the development of breast cancer specific immunotherapy.

Comparison of hormone-induced mRNA and protein biomarker expression changes in breast cancer cells.

Protein biomarkers estrogen receptor (ER), progesterone receptor (PR), and marker of proliferation (Ki67) are routinely assessed by immunohistochemistry to guide treatment decisions for breast cancer. Now, quantification of mRNA encoding these proteins is being adopted in the clinic. However, mRNA and protein biomarkers may be differentially regulated by fluctuations in estrogen and progesterone that occur across the menstrual cycle in premenopausal breast cancer patients. This study aimed to compare how estrogen and progesterone affect mRNA and protein biomarker expression in hormone-responsive breast cancer cells. Hormone-responsive ZR-75-1 and T-47D human breast cancer cell lines were xenografted into the mammary fat pad of BALB/c nude mice supplemented with estrogen. Progesterone or vehicle was administered prior to dissection of tumors. Protein expression of ER, PR and Ki67 was quantified by immunohistochemistry, and mRNA encoding these proteins, ESR1, PGR and KI67, respectively, was quantified by real-time PCR. mRNA expression was also quantified in breast cancer cell lines treated with estrogen and progesterone in vitro. In T-47D-xenografted tumors, estrogen and progesterone treatment reduced PGR and KI67 mRNA expression, and reduced PR and Ki67 protein positivity, compared to estrogen treatment alone. In ZR-75-1 xenografted tumors, no significant differences in protein or mRNA biomarker expression were observed. In vitro, estrogen and progesterone co-treatment significantly reduced ESR1 and PGR mRNA expression in both T-47D and ZR-75-1 cell lines. Estrogen and progesterone similarly affect mRNA and protein biomarker expression in hormone-responsive breast cancer xenografts. Further research is needed to investigate concordance between protein and mRNA biomarkers in premenopausal breast cancer.

A Targeted-Covalent Inhibitor of 17β-HSD1 Blocks Two Estrogen-Biosynthesis Pathways: In Vitro (Metabolism) and In Vivo (Xenograft) Studies in T-47D Breast Cancer Models.

Simple Summary17β-Hydroxysteroid dehydrogenase type 1 (17β-HSD1) is responsible for the production of estrogens estradiol (E2) and 5-androsten-3β,17β-diol (5-diol). This enzyme is therefore a target of choice for the treatment of estrogen-dependent diseases such as breast cancer and endometriosis, by blocking estrogen biosynthesis. After we developed the first irreversible and non-estrogenic 17β-HSD1 inhibitor, a molecule named PBRM, our goal was to demonstrate its therapeutic potential. PBRM was able to block the formation of E2 and 5-diol in T-47D human breast cancer cells. When given orally to mice, PBRM was also able to block the tumor growth without any observed toxic effects. Thanks to its irreversible type of inhibition, PBRM retained its anti-tumor growth effect, even after reducing its frequency of administration to only once a week, a clear advantage over reversible inhibitors. These results strongly support the use of PBRM as a new approach in the treatment of breast cancer.17β-Hydroxysteroid dehydrogenase type 1 (17β-HSD1) plays an important role in estrogen-dependent breast tumor growth. In addition to being involved in the production of estradiol (E2), the most potent estrogen in women, 17β-HSD1 is also responsible for the production of 5-androsten-3β,17β-diol (5-diol), a weaker estrogen than E2, but whose importance increases after menopause. 17β-HSD1 is therefore a target of choice for the treatment of estrogen-dependent diseases such as breast cancer and endometriosis. After we developed the first targeted-covalent (irreversible) and non-estrogenic inhibitor of 17β-HSD1, a molecule named PBRM, our goal was to demonstrate its therapeutic potential. Enzymatic assays demonstrated that estrone (E1) and dehydroepiandrosterone (DHEA) were transformed into E2 and 5-diol in T-47D human breast cancer cells, and that PBRM was able to block these transformations. Thereafter, we tested PBRM in a mouse tumor model (cell-derived T-47D xenografts). After treatment of ovariectomized (OVX) mice receiving E1 or DHEA, PBRM given orally was able to reduce the tumor growth at the control (OVX) level without any observed toxic effects. Thanks to its irreversible type of inhibition, PBRM retained its anti-tumor growth effect, even after reducing its frequency of administration to only once a week, a clear advantage over reversible inhibitors.

Nectin-4 and p95-ErbB2 cooperatively regulate Hippo signaling-dependent SOX2 gene expression, enhancing anchorage-independent T47D cell proliferation

Nectin-4, upregulated in various cancer cells, cis-interacts with ErbB2 and its trastuzumab-resistant splice variants, p95-ErbB2 and ErbB2∆Ex16, enhancing DNA synthesis through the PI3K-AKT signaling in human breast cancer T47D cells in an adherent culture. We found here that nectin-4 and p95-ErbB2, but not nectin-4 and either ErbB2 or ErbB2∆Ex16, cooperatively enhanced SOX2 gene expression and cell proliferation in a suspension culture. This enhancement of T47D cell proliferation in a suspension culture by nectin-4 and p95-ErbB2 was dependent on the SOX2 gene expression. In T47D cells, nectin-4 and any one of p95-ErbB2, ErbB2, or ErbB2∆Ex16 cooperatively activated the PI3K-AKT signaling, known to induce the SOX2 gene expression, to similar extents. However, only a combination of nectin-4 and p95-ErbB2, but not that of nectin-4 and either ErbB2 or ErbB2∆Ex16, cooperatively enhanced the SOX2 gene expression. Detailed studies revealed that only nectin-4 and p95-ErbB2 cooperatively activated the Hippo signaling. YAP inhibited the SOX2 gene expression in this cell line and thus the MST1/2-LATS1/2 signaling-mediated YAP inactivation increased the SOX2 gene expression. These results indicate that only the combination of nectin-4 and p95-ErbB2, but not that of nectin-4 and either ErbB2 or ErbB2∆Ex16, cooperatively regulates the Hippo signaling-dependent SOX2 gene expression, enhancing anchorage-independent T47D cell proliferation.

Cowanin, a Cytotoxic Xanthone from Asam Kandis (Garcinia cowa, Roxb.) Reduced Cell Migration and Induced Cell Cycle Arrest on T47D Human Cancer Cell

This study aims to investigate and evaluate the mechanism action of cowanin, a cytotoxic xanthone isolated from ethanolic extract of the stem bark of asam kandis (Garcinia cowa Roxb). This compound was isolated after successive column and radial chromatography to give a yellow needless crystal, m.p. 121-124 oC. Based on ultraviolet, infrared, mass and nuclear magnetic resonance spectroscopic data and comparison with those of the literature, this compound was elucidated as cowanin. Since it had activity against T47D human breast cancer cell lines, further investigation of its mechanism activity was performed to explore the effects of cowanin on cell viability, migration of cells, and the cell cycle activities against T47D breast cancer cells. Viability of cell was carried out by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, migration of cells by scratch migration assay, and the cell cycle analysis by flow cytometry method. As a result of this investigation, it can be seen that cowanin could inhibited T47D cells' growth at concentration 0.1, 1, 10, and 100 µg/ml at 48 hours with the IC50 value of 11.11 µg/ml. Cowanin exhibited inhibiting the cell migration T47D cells at concentration 11.1 μg/mL treated cells at 48 h was 0.32 fold compared to control, suggesting the potent inhibitory effect of it. Cowanin is caused in significant detention of T47D cells at the G0-G1 phase of the cell cycle. Based on these data, it can be concluded that cowanin is a potential candidate to be developed as an anticancer drug.

Alterations in Progesterone Receptor Isoform Balance in Normal and Neoplastic Breast Cells Modulates the Stem Cell Population.

To investigate the role of PR isoforms on the homeostasis of stem cells in the normal and neoplastic mammary gland, we used PRA and PRB transgenic mice and the T47D human breast cancer cell line and its derivatives, T47D YA and YB (manipulated to express only PRA or PRB, respectively). Flow cytometry and mammosphere assays revealed that in murine breast, overexpression of PRB leads to an increase in luminal and basal progenitor/stem cells. Ovariectomy had a negative impact on the luminal compartment and induced an increase in mammosphere-forming capacity in cells derived from WT and PRA mice only. Treatment with ICI 182,780 augmented the mammosphere-forming capacity of cells isolated from WT and PRA mice, whilst those from PRB remained unaltered. T47D YB cells showed an increase in the CD44+/CD24Low/− subpopulation; however, the number of tumorspheres did not vary relative to T47D and YA, even though they were larger, more irregular, and had increased clonogenic capacity. T47D and YA tumorspheres were modulated by estrogen/antiestrogens, whereas YB spheres remained unchanged in size and number. Our results show that alterations in PR isoform balance have an impact on normal and tumorigenic breast progenitor/stem cells and suggest a key role for the B isoform, with implications in response to antiestrogens.

Ubiquitin-specific protease 7 downregulation suppresses breast cancer in vitro.

Because breast cancer is complicated at the pathological, histological, clinical, and molecular levels, identification of new genetic targets against carcinogenic pathways is required to generate clinically relevant treatment options. In the current study, ubiquitin-specific protease 7 (USP7), which regulates various cellular pathways including Mdm2, p53, and NF–κB, was selected as a potential gene editing strategy for breast cancer in vitro. Anticancer activity of USP7 gene suppression has been evaluated through cell proliferation, gene expression, cell cycle, sphere dissemination, and cell migration analysis. Here, siRNA and shRNA strategies and an allosteric small-molecule inhibitor of USP7 were used to define potential anticancer activity against MCF7 and T47D human breast cancer cell lines. Both blockage of deubiquitination by p5091 and knockdown of USP7 reduced cell proliferation, cell migration, colony formation, and sphere dissemination for both MCF7 and T47D breast cancer cell lines. Restriction of USP7 activity strongly enhanced apoptotic gene expression and reduced metastatic ability of breast cancer cell lines. This study describes one potential molecular target for the suppression of breast cancer proliferation and metastasis. Identification of USP7 as a promising gene editing candidate might open up the possibility of new molecular drug research in targeting the ubiquitination pathway in cancer.

Abstract 5828: Effect of Roscovitine and mifepristone in luminal breast cancer cells

Abstract The cyclin dependent kinase (CDK)4/6 inhibitors have been recently approved in combination with endocrine therapy for luminal breast cancer treatment. On the other hand, the use of CDK2 inhibitors, such as Roscovitine, appears as a therapeutic alternative to overcome the acquisition of resistance to CDK4/6 inhibitors. However, there are few clinical trials using CDK2 inhibitors for breast cancer treatment. Most of the reports on the effects of Roscovitine were performed in MCF-7 or MDA-MB-231 cells. The aim of this work was to evaluate the effect of Roscovitine on cell proliferation in T47D human breast cancer cells which express both progesterone receptor (PR) isoforms PRA and PRB, and in the T47D-YA and T47D-YB cell lines expressing only PRA or PRB, respectively. We have previously demonstrated that cells overexpressing PRB showed higher levels of cyclin A as compared with those overexpressing PRA, thus we hypothesized that they would be more sensitive to CDK2 inhibitors. Roscovitine inhibited cell proliferation in a dose dependent manner similarly in both MCF-7 cells (used as positive control) and in T47D cells, with IC50 of 11.99 µM and 11.93 µM respectively. In addition, Roscovitine inhibited cell proliferation induced by estrogen treatment in both cell lines (p&amp;lt;0.01); however the percentage of inhibition was higher in MCF-7 compared with T47D cells (70% versus 50%, respectively; p&amp;lt;0.05). Roscovitine also inhibited cell proliferation induced by the fibroblast growth factor FGF2 in T47D cells (Roscovitine 2 µM p&amp;lt;0.001 and 5 µM p&amp;lt;0.05). Whereas both T47D-YA and -YB cells were similarly inhibited by Roscovitine, only in T47D-YA cells the combination of the antiprogestin mifepristone (MFP; 10 nM) and Roscovitine 1 µM in the presence of FGF2 induced a higher inhibitory effect than MFP or Roscovitine single treatments (p&amp;lt;0.01). We conclude that CDK2 inhibitors may be an option for luminal breast cancer treatment independent of the levels of cyclin A, and that they might be used in combination with antiprogestins only in breast cancer cells overexpressing PRA isoform. Citation Format: Lucía Vottero, Claudia Lanari, Victoria T. Fabris. Effect of Roscovitine and mifepristone in luminal breast cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5828.