Positive Breast Cancer Cell Lines Research Articles

Insight on pyrimido[5,4-g]indolizine and pyrimido[4,5-c]pyrrolo[1,2-a]azepine systems as promising photosensitizers on malignant cells

Searching for new small molecules as photosensitizing agents, we have developed a class of twenty-five pyrimido[5,4-g]indolizine and pyrimido[4,5-c]pyrrolo[1,2-a]azepines with a good substitution pattern defining a versatile synthetic pathway to approach the title ring system. All compounds were evaluated for their photocytotoxicity on a triple negative human breast cancer cell line (MDA-MB-231) in the dark and under UVA light (2.0 J/cm2). The most effective compounds exhibited a photoantiproliferative activity with IC50 values up to nanomolar ranges. Interestingly, these new developed compounds showed high selectivity towards cancerous cells with respect to non-cancerous ones. Moreover, four representative derivatives demonstrated to be phototoxic also against an additional human HER2 positive breast cancer cell line (HCC1954), and against the HER2 positive vesical cancer cell line (T24) harboring Hras mutation. Mechanistic studies performed in triple negative MDA-MB-231 cancer cells revealed the ability of the compounds to increase reactive oxygen species (ROS) production and to induce a thiol redox stress, thus triggering cancer cell death through apoptosis. Apoptotic cell death was also induced in highly aggressive and metastatic HER2 positive Hras mutated T24-treated bladder cancer cells. Overall, our data confirm that these new small photosensitizing agents may represent very promising candidates for phototherapy application against highly aggressive and resistant cancers.

Effect of combining CBD with standard breast cancer therapeutics

Breast cancer is the most common malignancy in women worldwide. Sixty-five percent of breast cancers are estrogen and/or progesterone receptor positive. Estrogen receptor expression is a prognostic and predictive biomarker of response to endocrine therapy, which consists of the selective estrogen receptor modulator tamoxifen, aromatase inhibitors, and the selective estrogen receptor degrader fulvestrant. Cannabidiol is a phytocannabinoid that is emerging as a potential therapeutic agent. The aim of this study was to investigate the effect of cannabidiol on estrogen receptor-positive and estrogen receptor-negative representative breast cancer cell lines in combination with standard therapeutic agents used in clinical practice. To compare the effects of cannabidiol on breast cancer cell viability, cancer cell lines were exposed to increasing concentrations of cannabidiol. The effects of cannabidiol in combination with the endocrine therapeutics tamoxifen, fulvestrant, and the cyclin-dependent kinase inhibitor palbociclib on breast cancer cell viability were examined. We demonstrated that cannabidiol dose-dependently decreased the viability of all breast cancer cell lines independent of estrogen receptor expression. The addition of cannabidiol to tamoxifen had an additive negative effect on cell viability in ER+ ​in estrogen receptor positive T-47D line. Cannabidiol did not attenuate the effect of standard treatment of hormone receptor-positive breast cancer with fulvestrant and palbociclib. In addition, cannabidiol did not attenuate the effect of standard treatment of triple-negative breast cancer and human epidermal growth factor receptor 2 positive breast cancer cell lines with trastuzumab and cisplatin.

Estrogens and Progestins Cooperatively Shift Breast Cancer Cell Metabolism.

Simple SummaryBreast cancers are largely controlled by hormones, expressing abundant receptors for estrogen (ER) and progesterone (PR). Reprogramming of energy metabolism is a common feature of tumors, yet how estrogen and progestins (synthetic progesterone drugs) control breast cancer cell metabolism, particularly in combination, is understudied. Here we evaluated the impact of estrogens and progestins, alone or together, on breast cancer cell metabolism. Our results show that hormones significantly impact metabolism, especially in combination. Estrogens tend to target tumor-promoting genes that alter glucose metabolism while progestins target fat storage. Combined hormone treatment increases both glucose metabolism and fat storage, features advantageous for tumor progression. These results may explain, in part, why estrogen-progestin combinations increase breast cancer incidence in post-menopausal women. Targeting hormone-regulated metabolism is a potential novel therapeutic strategy for ER+PR+ breast cancer.Metabolic reprogramming remains largely understudied in relation to hormones in estrogen receptor (ER) and progesterone receptor (PR) positive breast cancer. In this study, we investigated how estrogens, progestins, or the combination, impact metabolism in three ER and PR positive breast cancer cell lines. We measured metabolites in the treated cells using ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS). Top metabolic processes upregulated with each treatment involved glucose metabolism, including Warburg effect/glycolysis, gluconeogenesis, and the pentose phosphate pathway. RNA-sequencing and pathway analysis on two of the cell lines treated with the same hormones, found estrogens target oncogenes, such as MYC and PI3K/AKT/mTOR that control tumor metabolism, while progestins increased genes associated with fatty acid metabolism, and the estrogen/progestin combination additionally increased glycolysis. Phenotypic analysis of cell energy metabolism found that glycolysis was the primary hormonal target, particularly for the progestin and estrogen-progestin combination. Transmission electron microscopy found that, compared to vehicle, estrogens elongated mitochondria, which was reversed by co-treatment with progestins. Progestins promoted lipid storage both alone and in combination with estrogen. These findings highlight the shift in breast cancer cell metabolism to a more glycolytic and lipogenic phenotype in response to combination hormone treatment, which may contribute to a more metabolically adaptive state for cell survival.

In Vitro Anti-cancer Effect of Crataegus oxyacantha Berry Extract on Hormone Receptor Positive and Triple Negative Breast Cancers via Regulation of Canonical Wnt Signaling Pathway.

Breast cancer treatment strategy depends mainly on the receptor status. Our aim was to identify a herbal preparation, effective against breast cancer, irrespective of hormone sensitivity, and to understand its molecular mechanism. The rich antioxidant composition of hawthorn (Crataegus oxyacantha) makes it a promising anti-cancer drug candidate. Polyphenol-rich methanolic extract of C. oxyacantha berry (M.Co) was found to be cytotoxic on hormone receptor positive (MCF-7) and triple negative (MDA-MB-231) breast cancer cell lines, at a dose (75μg/ml) safe on normal cells. It could effectively inhibit tumor cell proliferation and arrest cell cycle at G1/S transition in both cell lines. Molecular targets were selected from different levels of canonical Wnt signaling pathway (such as autocrine and antagonistic ligands, receptor, effector, cytoplasmic components, downstream targets, and pathway antagonist), since they are frequently found dysregulated in all breast cancers and their aberrant activation is associated with cancer stem cell expansion. M.Co could significantly downregulate the expression of Wnt pathway agonists and upregulate that of Wnt antagonists at transcriptional and translational levels, in both cell lines. To conclude, C. oxyacantha berry extract is effective against breast cancer irrespective of its hormone dependency, and cancer growth inhibition at stem cell level can be expected.

Abstract P2-13-28: Inhibiting retinoic acid receptor signalling enhances the effect of neratinib in HER2 positive breast cancer cell lines

Abstract Introduction: Treatment strategies for HER2+ breast cancer (BC) involves first line treatment with the monoclonal antibody trastuzumab, however resistance to treatment can occur. The pan-HER tyrosine kinase inhibitor neratinib is an approved adjuvant treatment for early stage HER2+ BC after one year of treatment with trastuzumab. Recent studies have highlighted the potential benefits of using retinoic acid as an adjuvant treatment for BC. The retinoic acid receptor (RAR) family of nuclear transcription factors consists of RARα, RARβ and RARγ. Interestingly ~30% of HER2+ BC have a co-amplification of the RARA gene, which codes for RARα. We examined the effect of RARα agonists (Fenretinide & AM580) and antagonist (AGN194310) in combination with neratinib in two HER2+ cell lines: SKBR3, has co-amplification of ERBB2 and RARA, and HCC1569 has amplified ERBB2 but are not RARA-amplified. Methods: SKBR3 and HCC1569 cell lines were cultured in RPMI/10% FCS at 370C/5% CO2. 10mM stock concentrations of fenretinide (H7779-Sigma), AM580 (A8843-Sigma), AGN194310 (SML2665-Sigma) and neratinib (Puma Biotechnology, Inc) were prepared in DMSO. For proliferation assays, cells were seeded in 96 well plates at a density of 3 X 104 cells per well for 24h. Cells were treated with a drug alone (2X concentration) or combination of drugs (4X concentration) in 100μL of medium. Proliferation was measured using an acid phosphatase-based assay after 5 days as percentage growth versus DMSO control. The half maximal inhibitory concentration (IC50) was calculated for each drug, using CalcuSyn. The combination assays were performed using fixed ratios. The combination index (CI) values were calculated at the effective dose that inhibits 50% growth (ED50), using CalcuSyn. Values < 1 represent a synergistic effect, a value of 1 is additive and values > 1 represent an antagonistic effect. All data presented as the mean of biological triplicate experiments ± standard deviation. Results: This research has found that the synthetic retinoic acid fenretinide, a pan-RAR (α, β and γ) activator, was more potent in the HCC1569 cells compared to the SKBR3 cell line with an IC50 of 0.21 ± 0.03μM compared to 4.55 ± 0.87 μM, respectively. However, when combined with neratinib there was a strong antagonistic effect observed in the HCC1569 cell line (CI value: 15.63 ± 9.5). Conversely, fenretinide enhanced the effect of neratinib in the SKBR3 cell line (CI value: 0.83 ± 0.4). The RARα-specific activator AM580 had an IC50 >10μM for both cell lines. When combined with neratinib there was an additive effect observed in the RARα negative HCC1569 cells (CI value: 0.97 ± 0.4), and a synergistic effect observed in the RARα upregulated SKBR3 cell line (CI value: 0.78 ± 0.08). Next we wanted to determine if inhibiting RAR activity would have any effect on the proliferation of these cell lines. The IC50 for the pan-RAR (α, β and γ) antagonist AGN194310 was >10μM for each cell line. However, when combined with neratinib treatment there was a strong synergistic effect observed in the HCC1569 cell line (CI value: 0.52 ± 0.17) and the SKBR3 cell line (CI value: 0.66 ± 0.19). Conclusions: This study suggests a differential effect of RAR agonists when combined with neratinib in RARα amplified versus non-amplified HER2+ BC. Targeting RAR signalling, particularly with a RAR antagonist, in combination with the pan-HER inhibitor warrants further investigation in HER2+ BC. Citation Format: Debbie O'Reilly, Nicola Gaynor, Neil Conlon, Irmina Diala, Lisa D Eli, John Crown, Denis M Collins. Inhibiting retinoic acid receptor signalling enhances the effect of neratinib in HER2 positive breast cancer cell lines [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-13-28.

Abstract P4-02-01: Targeting insulin receptor in estrogen receptor positive breast cancer

Abstract Obesity and metabolic dysfunction are on the rise in the United States. Hyperinsulinemia, frequently seen in these conditions, correlates with an increased risk of development and mortality from estrogen receptor (ER) positive breast cancer. Expression of components of the insulin and insulin-like growth factor (IGF) family of proteins promote cancer cell growth in vitro, link to shorter progression-free survival, and is associates with an increased risk of metastatic disease and death in breast cancer patients. Previously our lab found that development of resistance to the commonly used ER-targeting drug tamoxifen (Tam) in vitro is associated with a loss of the type I IGF receptor expression with an increased dependence on the homologous insulin receptor (IR). These data argue for targeting IR in breast cancer, but concern has been raised due to the potential of disrupting glucose homeostasis in patients with or without existing metabolic disease. To address this concern, a novel insulin-Fc fusion protein (AKS-130) was developed to lower blood glucose levels without causing clinical hypoglycemia with a potential to target tumor IR. AKS-130 was found to downregulate IR expression in models of colorectal cancer (HCT-116) and malignant melanoma (WM266.4). Further, AKS-130 suppressed xenograft growth in fed and fasted states. We examined if AKS-130 had similar effects on ER positive breast cancer cells. To test the hypothesis that resistance to endocrine therapy increases the reliance of ER positive breast cancer cells on IR signaling, we utilized the ER positive breast cancer cell lines (MCF-7L, T47D) and their Tam resistant derivatives (MCF-7L TamR, T47D TamR). Similar to in HCT-116 and WM266.4 cells, we found that AKS-130 is a modest agonist of IR in ER+ breast cancer cells. Exposure to AKS-130 led to a marked reduction in IR expression in both parental and TamR cell lines, and was associated with partially suppressed downstream signaling to Akt. Interestingly, AKS130 induced IR downregulation was inhibited by pretreatment of BMS 536924, a dual IGF1R/IR kinase inhibitor. In vivo, AKS-130 treatment by itself scaled down MCF7L parental xenograft growth as compared to vehicle group, and AKS-130 in combination with Tam trended toward extending time to a tumor size of 500mm3. In MCF7L TamR xenografts, addition of AKS-130 to Tam treatment also trended toward delaying xenograft growth, though it was not statistically significant. Importantly, prolonged treatment with AKS-130 did not lead to significant changes in weight or blood glucose. Taken together these data show that novel agents, such as AKS-130, may serve as way to disrupt IR signaling in cancer cells without affecting host glucose metabolism. Addition of AKS-130 to Tam treatment trended toward delaying xenograft growth though further studies are needed, especially animal model systems of insulin resistance. These data indicate that IR signaling may represent a target to overcome or delay endocrine therapy resistance in breast cancer. Citation Format: Jingran Cao, Lynsey M. Fettig, Kelly LaPara, Xihong Zhang, Sylaja Murikipudi, Andrea R. Delpero, Thomas M. Lancaster, Todd C. Zion, Douglas Yee. Targeting insulin receptor in estrogen receptor positive breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-02-01.

Abstract P5-04-05: Loss of REST in breast cancer promotes tumor progression through estrogen sensitization, MMP24 and CEMIP overexpression

Abstract Breast cancer is the most common malignancy in women and is the second leading cause of cancer-related deaths. Breast cancer is both pathologically and genetically heterogeneous, making early detection and treatment difficult. A subset of breast cancers expresses normal levels of REST (Repressor Element 1 Silencing Transcription factor) mRNA but lacks functional REST protein. Loss of REST function is seen in ~20% of breast cancers and is associated with a more aggressive phenotype and poor prognosis. Despite the frequent loss of REST, little is known about the role of REST in the molecular pathogenesis of breast cancer. TCGA data was analyzed for the expression of REST and its target genes in breast cancer patient samples. MCF7, an estrogen and progesterone receptor positive breast cancer cell line, and MDA-MB-231, a triple-negative breast cancer cell line were used for in vitro studies to understand the function of REST. To evaluate the role of REST in the regulation of hormone receptor pathways, RNA sequencing was carried out on total RNA from MCF7 cells after siRNA knockdown of REST followed by treatment of hormones estrogen and progesterone. Chromatin Immunoprecipitation and PCR were performed on REST knockdown cells to evaluate gene regulation by REST and understand its tumor suppressor role in breast cancer. We show that REST directly regulates CEMIP (Cell Migration-Inducing and Hyaluronan-Binding Protein, KIAA1199) and MMP24 (Matrix Metallopeptidase 24), genes known to have roles in invasion and metastasis. REST knockdown in breast cancer cell lines leads to significant upregulation of CEMIP and MMP24. In addition, we found that REST binds to RE-1 sites (Repressor Element-1) within the genes and influences their transcriptional regulation. Aberrant expression of CEMIP and MMP24 results in CD44 dependent cell signaling that promotes cell proliferation and survival. We also found that the estrogen receptor signaling pathway is activated in the absence of REST regardless of hormone treatment. Our studies show that REST plays a significant role in altering the estrogen signaling pathway in hormone-sensitive breast cancers. REST transcriptionally regulates genes known to affect cell invasion and metastasis. Importantly, we demonstrate that loss of REST promotes aggressive breast cancer and see REST as an attractive therapeutic target. Citation Format: Aditya M. Vargheese, Ashley S. Cloud, Sumedha Gunewardena, Raeann M. Shimak, Sornakala Ganeshkumar, Easwari Kumaraswamy, Roy A. Jensen, Vargheese M. Chennathukuzhi. Loss of REST in breast cancer promotes tumor progression through estrogen sensitization, MMP24 and CEMIP overexpression [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-04-05.

Abstract P5-11-03: Altered adipose-derived mesenchymal stem/stromal cells induced by obesity in the breast cancer microenvironment

Abstract It is well documented that obesity, characterized by an increased body mass index (BMI≥30), is an important risk factor for the development of aggressive breast cancer (BC), which is associated with a reduced overall survival rate and decreased treatment response. The tumor microenvironment (TME), a cellular network majorly constructed by adipocytes, adipose-derived mesenchymal stem/stromal cells (bASCs) and immune cells, impacts progression of breast cancer and treatment response of patients. This study aims to examine how obesity influences the functional characteristics of bASCs in comparison to lean bASCs in the TME. To address this issue, various molecular, biological and cellular methods are applied including transcriptomic profiling and functional assays to characterize at first the features of bASCs in the TME (adjacent to tumor; -aT/distant to tumor; -dT) in obese (ob-) and lean (ln-) bASCs (n=5, for each group) patients with breast cancer. Second, based on the transcriptomic data, we investigated the interaction of bASCs with triple negative as well as triple positive BC cell lines in 3D-spheroids and in standard 2D-cell culture conditions.Indicated by the gene signature the TME shifts bASCs towards a cancer supporting phenotype with a significant deregulated protein expression, cytokine/chemokine secretion, and reduced differentiation capacity. These changes affect critically the cell-cell interaction of BC cells with bASCs. bASCs isolated adjacent to the tumor increase the malignancy of BCs by stimulating the proliferation, migration, invasion and chemoresistance of these cells significantly. These observed effects are further enhanced by ob-aT bASCs. In conclusion, this study identifies over 900 genes deregulated in bASCs adjacent to breast cancer. Many of these genes are associated with pathways known for the development of malignant cells. Moreover, it clarifies differences in obese versus lean bASCs and their impacts on progression of breast cancer cells. Citation Format: Andreas Ritter, Susanne Roth, Alexandra Friemel, Nina-Naomi Kreis, Frank Louwen, Christine Solbach, Juping Yuan. Altered adipose-derived mesenchymal stem/stromal cells induced by obesity in the breast cancer microenvironment [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-11-03.

Abstract P5-04-04: Identification of AR driven tumors within TNBC using a novel gene signature

Abstract Background The androgen receptor (AR) is an emerging biomarker and favourable therapeutic target in breast cancer (BC), especially in Triple negative BC (TNBC) which lacks a targeted treatment and shows high molecular heterogeneity. Proportion of AR positive tumours are known to vary (10 to 43%) within TNBC and recent multi-institutional study on AR positive TNBC, showed AR protein status alone is not a reliable prognostic marker for therapeutic response. A deeper examination of AR regulated pathways is necessary to derive AR regulated gene signatures which could identify TNBC tumours truly driven by AR and amenable to anti androgen therapies. In this study, we attempted to develop AR regulated gene signature using gene expression data derived from cell lines under the influence of androgens using systematic bioinformatic approach and tested the ability of the signature to identify AR regulated tumours within TNBC. Methods Microarray data of AR positive BC cell lines (GSE61368 & GSE28305) was used to derive AR regulated genes by a bioinformatic pipeline, using integrated method by incorporating protein interaction networks, topological parameters, and semantic similarity to arrive at final set of 16 genes. Correlation of these genes with AR protein status by reverse phase protein array (RPPA) was evaluated in 447 tumors of TCGA dataset. A 3rd quartile cut-off in the AR expression (by RPPA) was used to classify them as AR positive and negative and a logistic regression model was constructed using positive AR as determinant to generate AR probability score with minimum set of genes. 3rd quartile cut-off in the probability score was used to classify tumors as AR high and low. Distribution of AR probability score was examined in 144 and 187 TNBC tumors in TCGA and METABRIC, respectively. Comparisons between AR high and low tumors were done for clinical parameters, mutational spectrum, expression of ER regulated genes (FOXA1, GATA3 and TFF1) and EMT gene signatures (Byers et al., 2013, Tan et al., 2014). Luminal androgen receptor (LAR) subtype identification by TNBCtype (Lehmann et. al) was done in both datasets to examine the overlap with our method of identification of AR driven tumors within TNBC. Results Logistic regression model identified 5 (SOCS2, ID1,PIP,GADD45 and ZNF689) of the 16 AR regulated genes as predictors for AR probability score which ranged from 0.009 to 0.68 in 144 TNBC tumors within TCGA data. 41/144 (29%) TNBC tumors were categorized as having AR high at cut off of 0.13. The AR probability score ranged from 0.04 to 0.64 in 187 TNBC tumors within METABRIC dataset and 47/187 (25%) were classified as AR driven tumors at cut off of 0.22. Though differences in the clinical variables like age , tumor size and lymph node(LN) status were not different between the AR high and low groups in the TCGA dataset, a significant increase in age, LN positive tumors was observed in the AR high groups in METABRIC. A significant higher PIK3CA mutations was observed in the AR high tumors both in the TCGA and METABRIC (p=0.012 and p<0.0001 respectively) with a proportionate lower p53 mutations in the AR high tumors in both data sets (p=0.03 and p=0.35 respectively). Further, AR high tumors showed significant higher expression of ER regulated genes (TCGA-p<0.05 & METABRIC, p<0.05), were more epithelial by EMT score (TCGA-p<0.05 & METABRIC, p<0.05). Analysis by TNBCtype showed that 30% of AR high tumors in TCGA and 40% of AR high tumors in METABRIC were identified as belonging to LAR subgroup (Lehmann et al). Conclusion Identification of AR driven tumors within TNBC has both prognostic and predictive utility. Methods using limited number of AR regulated genes could be easily applied to larger BC cohorts to identify TNBC tumours driven by AR signalling and may respond well to anti-androgen therapies. Citation Format: Savitha Rajarajan, Snijesh V.P, Maalavika Pillai, Mohit Kumar Jolly, Jyothi S Prabhu. Identification of AR driven tumors within TNBC using a novel gene signature [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-04-04.

Metastatic profiling of HER2-positive breast cancer cell lines in xenograft models.

Most studies on breast cancer metastasis have been performed using triple-negative breast cancer cells; thus, subtype-dependent metastatic ability of breast cancer is poorly understood. In this research, we performed intravenous injection (IVI) and intra-caudal arterial injections using nine human epidermal growth factor receptor-2 (HER2)-positive breast cancer cell lines for evaluating their metastatic abilities. Our results showed that MDA-MB-453, UACC-893, and HCC-202 had strong bone metastatic abilities, whereas HCC-2218 and HCC-1419 did not show bone metastasis. HER2-positive cell lines could hardly metastasize to the lung through IVI. From the genomic analysis, gene signatures were extracted according to the breast cancer subtypes and their metastatic preferences. The UACC-893 cell line was identified as a useful model for the metastasis study of HER2-positive breast cancer. Combined with our previous result on brain metastasis ability, we provide a characteristic metastasis profile of HER2-positive breast cancer cell lines in this study.

Anti-oxidant, anti-diabetic and anti-lipidemic activities of root bark extracts from Rhus tripartitum and cytotoxicity evaluation of isolated compounds

This study was undertaken to investigate anti-oxidant, anti-diabetic and anti-lipidemic activities of three extracts from Rhus tripartitum root bark prepared by solvents with increasing polarity (hexane, ethyl acetate and n-butanol).Our result demonstrated that the n-butanol extract is endowed with the highest phenolic content 872.503 ± 0.788 mg (GAE)/g Extract, flavonoid content 325.19±0.294 mg(QE)/g Extract and tannin content 698.99± 0.605 mg(CE)/g Extract. In addition this extract was found to exhibit the highest anti-oxidant activity using DPPH radical-scavenging (IC50 = 0.008 mg/ml), ferric-reducing power (IC50=0.844 mg/ml), total antioxidant capacity concentration (756.758±0.940 mg (GAE)/g Extract) and nitric oxide radical scavenging (IC50 = 0.01 mg/ml) assays. Mean while, the ethyl acetate extract present the best anti-diabetic activity (30.11%) and anti-lipidemic activity (49.79%). Three compounds were isolated for the first time from the ethyl acetate extract of Rhus tripartitum and identified using extensive spectral techniques (MS, NMR 1D and 2D). The in vitro cytotoxic activity of isolated compounds on MCF-7 cells positive breast cancer cell line was investiguated. One of the isolated compounds present the interesting growth inhibitory effect on MCF-7 cell.

Proteomic Analysis Revealed Underlying Biological Pathways Associated with Hormetic Response of Hormone Positive Breast Cancer Cell Line Exposed to Low-Dose Flavonoid Mixture

Objective: A considerable level of evidence has accumulated about the breast cancer risk-reducing effect of consuming specific flavonoids, through the increasing amount of research and epidemiologic studies. Different flavonoids may have different cellular bioavailability and favor, i.e., the occurrence of a hormetic effect, thus it is important to evaluate breast cancer cells’ response to different doses of flavonoids. This study aims to investigate the alterations of the biological pathways in a hormone-positive (HR+) breast cancer cell line as a resemblance for the most common breast cancer subtype, related to the low-dose exposure of the flavonoids. Materials and Methods: Different levels of doses were applied to MCF-7 breast cancer cells. In order to determine cellular proliferation, WST-1 analysis was conducted. The highest proliferation was observed with cell lines exposed to a low-dose flavonoid mixture and these were selected for further analysis. Intracellular protein expression were investigated by peptide analysis on a nano LC-MS/MS platform. A protein-protein interaction network and pathway analysis were conducted for the proteins expressed differently between the groups. Results: A total of 214 proteins were identified and 36 proteins with significant alterations (≥1.2-fold change, p≤0.05) were detected. Significant changes were observed in the pathways related to carbon metabolism, amino acid biosynthesis, splicing mechanism, mitochondrial protein import and translation elongation pathways. Conclusion: Our study demonstrated that flavonoids can have a hormetic effect which can initially alter metabolic pathways vital for cell proliferation and survival. These pathways may include potential targets for enhancing the anticancer activity of the flavonoids.

Abstract P131: The role of UDP-6 glucose dehydrogenase (UGDH) in estrogen-mediated phenotypes in both estrogen receptor positive and estrogen receptor negative breast cancer

Abstract A better understanding of pathologic mechanisms underlying breast cancer progression and metastasis is critical to improve current treatment modalities. UDP-6- glucose dehydrogenase (UGDH) is an enzyme that has become a recent oncologic target of interest across various cancer subtypes for its role in increasing the aggressiveness and migratory capacity of tumor cells both in vitro and in vivo. UGDH is ubiquitously expressed and plays a critical role in forming the extracellular matrix (ECM), producing nucleotide sugars, and processing hormones through glucuronidation. For these reasons, investigating the effect of UGDH on breast cancer in the setting of hormonal stimulation is important. We found that knocking down UGDH in estrogen receptor positive (ER+) breast cancer cell lines (MCF7 marco and T47D) and an estrogen receptor negative (ER-) breast cancer cell line (MDA-MB-231) could abrogate estrogen induced migration-specific phenotypes in vitro. Furthermore, we found that knocking down UGDH mitigated estrogen stimulated primary tumor growth in vivo for both MCF7 macro and MDA-MB-231 mammary fat pad tumor models in mice. Thus, we found that UGDH regulates estrogen stimulated migratory phenotypes in both ER+ and ER- breast cancers in vitro and primary tumor growth in vivo. For this reason, UGDH and its associated pathways are promising targets for future drug development and small molecular targets in the treatment of both metastatic ER positive and ER negative breast cancers. Citation Format: Meghan Price, Catherine Lavau, Cesar Baeta, Jovita Byemerwa, Suzanne Wardell, Olivia Brueckner, Debarati Mukherjee, Corinne Haynes, Annee Nguyen, Chingyi Chang, Donald McDonnell, C. Rory Goodwin. The role of UDP-6 glucose dehydrogenase (UGDH) in estrogen-mediated phenotypes in both estrogen receptor positive and estrogen receptor negative breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P131.

PGRMC1 Promotes Progestin-Dependent Proliferation of Breast Cancer Cells by Binding Prohibitins Resulting in Activation of ERα Signaling.

Simple SummaryCombined menopausal hormone therapy is associated with increased breast cancer risk in postmenopausal women. In our previous studies, progesterone receptor membrane component 1 (PGRMC1) was shown to play a role in progestins’ elicitation of enhanced proliferation of breast cancer cells. Here we describe a potential mechanism by which PGRMC1 contributes to breast cancer progression via interaction with prohibitins, inhibiting their function as transcriptional repressors. This facilitates estrogen receptor alpha (ERα) transcriptional activity and enhances oncogenic signaling upon treatment with certain progestins, including norethisterone and dydrogesterone. Our data underline the contribution of PGRMC1 to especially hormone receptor positive breast cancer pathogenesis and demonstrate the need for further studies to understand its role in cancer.In previous studies, we reported that progesterone receptor membrane component 1 (PGRMC1) is implicated in progestin signaling and possibly associated with increased breast cancer risk upon combined hormone replacement therapy. To gain mechanistic insight, we searched for potential PGRMC1 interaction partners upon progestin treatment by co-immunoprecipitation and mass spectrometry. The interactions with the identified partners were further characterized with respect to PGRMC1 phosphorylation status and with emphasis on the crosstalk between PGRMC1 and estrogen receptor α (ERα). We report that PGRMC1 overexpression resulted in increased proliferation of hormone receptor positive breast cancer cell lines upon treatment with a subgroup of progestins including norethisterone and dydrogesterone that promote PGRMC1-phosphorylation on S181. The ERα modulators prohibitin-1 (PHB1) and prohibitin-2 (PHB2) interact with PGRMC1 in dependency on S181-phosphorylation upon treatment with the same progestins. Moreover, increased interaction between PGRMC1 and PHBs correlated with decreased binding of PHBs to ERα and subsequent ERα activation. Inhibition of either PGRMC1 or ERα abolished this effect. In summary, we provide strong evidence that activated PGRMC1 associates with PHBs, competitively removing them from ERα, which then can develop its transcriptional activities on target genes. This study emphasizes the role of PGRMC1 in a key breast cancer signaling pathway which may provide a new avenue to target hormone-dependent breast cancer.

Elucidation of xenoestrogen metabolism by non-targeted, stable isotope-assisted mass spectrometry in breast cancer cells

Environmental exposure to xenoestrogens, i.e., chemicals that imitate the hormone 17β-estradiol, has the potential to influence hormone homeostasis and action. Detailed knowledge of xenobiotic biotransformation processes in cell models is key when transferring knowledge learned from in vitro models to in vivo relevance. This study elucidated the metabolism of two naturally-occurring phyto- and mycoestrogens; namely genistein and zearalenone, in an estrogen receptor positive breast cancer cell line (MCF-7) with the aid of stable isotope-assisted metabolomics and the bioinformatic tool MetExtract II. Metabolism was studied in a time course experiment after 2 h, 6 h and 24 h incubation. Twelve and six biotransformation products of zearalenone and genistein were detected, respectively, clearly demonstrating the abundant xenobiotic biotransformation capability of the cells. Zearalenone underwent extensive phase-I metabolism resulting in α-zearalenol (α-ZEL), a molecule known to possess a significantly higher estrogenicity, and several phase-II metabolites (sulfo- and glycoconjugates) of the native compound and the major phase I metabolite α-ZEL. Moreover, potential adducts of zearalenone with a vitamin and several hydroxylated metabolites were annotated. Genistein metabolism resulted in sulfation, combined sulfation and hydroxylation, acetylation, glucuronidation and unexpectedly adduct formation with pentose- and hexose sugars. Kinetics of metabolite formation and subsequent excretion into the extracellular medium revealed a time-dependent increase in most biotransformation products. The untargeted elucidation of biotransformation products formed during cell culture experiments enables an improved and more meaningful interpretation of toxicological assays and has the potential to identify unexpected or unknown metabolites.

Delphinidin induces cell cycle arrest and apoptosis in HER-2 positive breast cancer cell lines by regulating the NF-κB and MAPK signaling pathways.

Delphinidin is an anthocyanidin monomer, commonly found in vegetables and fruits, and has demonstrated antitumor effects in the HER-2-positive MDA-MB-453 breast cancer cell line, with low cytotoxicity on normal breast cells. However, the direct functional mechanisms underlying the effect of delphinidin on HER-2-positive breast cancer cells has not been fully characterized. In the present study, it was found that delphinidin could induce G2/M phase cell cycle arrest by inhibiting the protein expression level of cyclin B1 and Cdk1 in HER-2-positive breast cancer cell lines. In addition, delphinidin promoted the mitochondrial apoptosis pathway by inhibiting the ERK and NF-κB signaling pathway and activating the JNK signaling pathway. Therefore, delphinidin markedly suppressed the viability of the HER-2-positive breast cancer cell lines by modulating the cell cycle and inducing apoptosis. Overall, the findings from the present study demonstrated that delphinidin treatment could induce the mitochondrial apoptosis pathway in human HER-2-positive breast cancer cell lines, providing an experimental basis for the prevention and treatment of HER-2-positive breast cancer by flavonoids.

Synthesis and Biological Evaluation of (S)-2-(Substituted arylmethyl)-1-oxo-1,2,3,4-tetrahydropyrazino[1,2-a]indole-3-carboxamide Analogs and Their Synergistic Effect against PTEN-Deficient MDA-MB-468 Cells.

A series of twenty-six compounds of furfuryl or benzyl tetrahydropyrazino[1,2-a]indole analogs were synthesized and evaluated for cytotoxic activity against the estrogen receptor (ER)-positive breast cancer cell line (MCF-7) and the epidermal growth factor receptor (EGFR) over-expressed triple-negative breast cancer cell line (MDA-MB-468). Among them, compounds 2b, 2f and 2i showed more potent activity and selectivity against MDA-MB-468 cells than gefitinib, as an EGFR- tyrosine kinase inhibitor. In addition, it was confirmed by means of isobologram analysis of combinational treatment with gefitinib that they have a synergistic effect, especially compounds 2b and 2f, which inhibit Akt T308 phosphorylation. Moreover, it was confirmed that 2-benzyl-1-oxo-1,2,3,4-tetrahydropyrazino[1,2-a]indole-3-carboxamide analogs (2b, 2f, and Ref 2) tend to selectively inhibit PI3Kβ, which is involved in the phosphorylation of Akt.

Pharmacological Inhibition of MCT4 Reduces 4-Hydroxytamoxifen Sensitivity by Increasing HIF-1α Protein Expression in ER-Positive MCF-7 Breast Cancer Cells.

The rate of glycolysis in cancer cells is higher than that of normal cells owing to high energy demands, which results in the production of excess lactate. Monocarboxylate transporters (MCTs), especially MCT1 and MCT4, play a critical role in maintaining an appropriate pH environment through lactate transport, and their high expression is associated with poor prognosis in breast cancer. Thus, we hypothesized that inhibition of MCTs is a promising therapeutic target for adjuvant breast cancer treatment. We investigated the effect of MCT inhibition in combination with 4-hydroxytamoxifen (4-OHT), an active metabolite of tamoxifen, using two estrogen receptor (ER)-positive breast cancer cell lines, MCF-7 and T47D. Lactate transport was investigated in cellular uptake studies. The cytotoxicity of 4-OHT was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In both cell lines evaluated, MCT1 and MCT4 were constitutively expressed at the mRNA and protein levels. [14C]-L-lactate uptake by both cells was significantly inhibited by bindarit, a selective MCT4 inhibitor, but weakly affected by 5-oxoploline (5-OP), a selective MCT1 inhibitor. The results of the MTT assay showed that combination with bindarit, but not 5-OP, decreased 4-OHT sensitivity. Bindarit significantly increased the levels of hypoxia-inducible factor-1α (HIF-1α) in MCF-7 cells. Moreover, HIF-1α knockdown significantly increased 4-OHT sensitivity, whereas induction of HIF-1α by hypoxia decreased 4-OHT sensitivity in MCF-7 cells. In conclusion, pharmacological MCT4 inhibition confers resistance to 4-OHT rather than sensitivity, by increasing HIF-1α protein levels. In addition, HIF-1α inhibition represents a potential therapeutic strategy for enhancing 4-OHT sensitivity.

218P Differential effects of estrogen exposure on steroid metabolizing CYP enzyme expression in estrogen receptor alpha versus beta positive breast cancer cell lines

Estrogen receptors (ER), ERα and ERβ, might serve as extra-hepatic regulators of CYP enzymes involved in steroid hormone metabolism such as CYP1B1, 1A1, 2B6, and CYP3A4. The aim of this project was, to study the responsiveness of either ERα negative and ERα positive cell lines on estrogen induced steroid metabolizing CYP enzyme expression, and changes in ERα or ERβ expression.

Cytotoxic Effects of Hellebrigenin and Arenobufagin Against Human Breast Cancer Cells.

Development of new therapeutic strategies for breast cancer is urgently needed due to the sustained emergence of drug resistance, tumor recurrence and metastasis. To gain a novel insight into therapeutic approaches to fight against breast cancer, the cytocidal effects of hellebrigenin (Helle) and arenobufagin (Areno) were investigated in human estrogen receptor (ER)-positive breast cancer cell line MCF-7 and triple-negative breast cancer cell line MDA-MB-231. Helle exhibited more potent cytotoxicity than Areno in both cancer cells, and MCF-7 cells were more susceptible to both drugs in comparison with MDA-MB-231 cells. Apoptotic-like morphological characteristics, along with the downregulation of the expression level of Bcl-2 and Bcl-xL and the upregulation of the expression level of Bad, were observed in Helle-treated MCF-7 cells. Helle also caused the activation of caspase-8, caspase-9, along with the cleavage of poly(ADP-ribose) polymerase in MCF-7 cells. Helle-mediated necrosis-like phenotype, as evidenced by the increased propidium iodide (PI)-positive cells was further observed. G2/M cell cycle arrest was also induced by Helle in the cells. Upregulation of the expression level of p21 and downregulation of the expression level of cyclin D1, cyclin E1, cdc25C and survivin were observed in MCF-7 cells treated with Helle and occurred in parallel with G2/M arrest. Autophagy was triggered in MCF-7 cells and the addition of wortmannin or 3-MA, two well-known autophagy inhibitors, slightly but significantly rescued the cells. Furthermore, similar alterations of some key molecules associated with the aforementioned biological phenomena were observed in MDA-MB-231 cells. Intriguingly, the numbers of PI-positive cells in Helle-treated MCF-7 cells were significantly reduced by wortmannin and 3-MA, respectively. In addition, Helle-triggered G2/M arrest was significantly corrected by wortmannin, suggesting autophagy induction contributed to Helle-induced cytotoxicity of breast cancer cells by modulating necrosis and cell cycle arrest. Collectively, our results suggested potential usefulness of both Helle and Areno in developing therapeutic strategies to treat patients with different types of breast cancer, especially ER-positive breast cancer.