T47D Cell Lines Research Articles

ERα status of invasive ductal breast carcinoma as a result of regulatory interactions between lysine deacetylases KAT6A and KAT6B

Breast cancer (BC) is the leading cause of death among cancer patients worldwide. In 2020, almost 12% of all cancers were diagnosed with BC. Therefore, it is important to search for new potential markers of cancer progression that could be helpful in cancer diagnostics and successful anti-cancer therapies. In this study, we investigated the potential role of the lysine acetyltransferases KAT6A and KAT6B in the outcome of patients with invasive breast carcinoma. The expression profiles of KAT6A/B in 495 cases of IDC and 38 cases of mastopathy (FBD) were examined by immunohistochemistry. KAT6A/B expression was also determined in the breast cancer cell lines MCF-7, BT-474, SK-BR-3, T47D, MDA-MB-231, and MDA-MB-231/BO2, as well as in the human epithelial mammary gland cell line hTERT-HME1 – ME16C, both at the mRNA and protein level. Statistical analysis of the results showed that the nuclear expression of KAT6A/B correlates with the estrogen receptor status: KAT6ANUC vs. ER r = 0.2373 and KAT6BNUC vs. ER r = 0.1496. Statistical analysis clearly showed that KAT6A cytoplasmic and nuclear expression levels were significantly higher in IDC samples than in FBD samples (IRS 5.297 ± 2.884 vs. 2.004 ± 1.072, p < 0.0001; IRS 5.133 ± 4.221 vs. 0.1665 ± 0.4024, p < 0.0001, respectively). Moreover, we noticed strong correlations between ER and PR status and the nuclear expression of KAT6A and KAT6B (nucKAT6A vs. ER, p = 0.0048; nucKAT6A vs. PR p = 0.0416; nucKAT6B vs. ER p = 0.0306; nucKAT6B vs. PR p = 0.0213). Significantly higher KAT6A and KAT6B expression was found in the ER-positive cell lines T-47D and BT-474, whereas significantly lower expression was observed in the triple-negative cell lines MDA-MB-231 and MDA-MB-231/BO2. The outcomes of small interfering RNA (siRNA)-mediated suppression of KAT6A/B genes revealed that within estrogen receptor (ER) positive and negative cell lines, MCF-7 and MDA-MB-231, attenuation of KAT6A led to concurrent attenuation of KAT6A, whereas suppression of KAT6B resulted in simultaneous attenuation of KAT6A. Furthermore, inhibition of KAT6A/B genes resulted in a reduction in estrogen receptor (ER) mRNA and protein expression levels in MCF-7 and MDA-MMB-231 cell lines. Based on our findings, the lysine acetyltransferases KAT6A and KAT6B may be involved in the progression of invasive ductal breast cancer. Further research on other types of cancer may show that KAT6A and KAT6B could serve as diagnostic and prognostic markers for these types of malignancies.

Novel benzenesulfonamides as dual VEGFR2/FGFR1 inhibitors targeting breast cancer: Design, synthesis, anticancer activity and in silico studies

In the current study, a new series of benzenesulfonamides 6a-r was designed and synthesized as dual VEGFR-2 and FGFR1 kinase inhibitors with anti-cancer activity. The 4-trifluoromethyl benzenesulfonamide 6l exhibited the highest dual VEGFR-2/FGFR1 inhibitory activity with IC50 values of 0.025 and 0.026 µM, respectively. It showed a higher activity than sorafenib and staurosporine by 1.8- and 1.3-fold, respectively. Furthermore, compound 6l was further tested on EGFR and PDGFR-β kinases showing IC50 values of 0.106 and 0.077 µM, respectively. The target compounds were tested for their anticancer activity against NCI-60 panel of cancer cell lines at 10 µM concentration, where compound 6l displayed the highest mean growth inhibition percent % (GI%) of 60.38%. Compounds 6a, 6b, 6e, 6f, 6h-l, and 6n-r revealed promising GI% on breast cancer cell lines (MCF-7, T-47D, and MDA-MB-231), and were subjected to IC50 determination on these cell lines. The tested compounds showed a higher activity on T-47D and MCF-7 cell lines over MDA-MB-231 cell line compared to the used reference standard; sorafenib. Compounds 6e, 6h-j, 6l and 6o revealed IC50 values ≤ 20 µM against T-47D cell line, furthermore, they were found to be non-cytotoxic on Vero normal cell line. Furthermore, the effect of the most active compounds 6i, and 6l in T-47D cells on cell cycle analysis progression, cell apoptosis, and apoptosis markers was investigated. Both compounds arrested cell cycle progression at G1 phase, furthermore, they enhanced early and late apoptosis, as well as necrosis. The capability of compounds 6i, and 6l to induce apoptosis was further confirmed by their ability to raise BAX/BCl-2 ratio and caspase-3 level in the treated cells. Cell migration assay revealed that both compounds 6i and 6l have anti-migratory effects compared to control T-47D cells after 24, and 48 h. Molecular docking studies for compounds 6a-r on VEGFR-2 and FGFR1 binding sites showed that they exhibit an analogous binding mode in both target kinases which agrees with that of type II kinase inhibitors.

Investigation of a tamoxifen-RACK7/KDM5C-IFN-I axis for ER+ breast cancer immunomodulation.

9 Background: ER+ breast cancer patients generally have good prognosis. However, significant relapse rates (i.e., hormone drugs, Tamoxifen/TMX) and the poor response to immune checkpoint blockade (ICB) remain critical issues to be addressed. Thus, exploration of anti-ICB resistant mechanisms and alternative targets is needed to improve therapeutic efficacy. Methods: To investigate potential cross-resistant mechanisms between hormone and ICB therapy, ER+ cell lines T47D and MCF7 cells were chronically-treated with TMX for 6 months, and subjected to RNA-Seq and pathway enrichment analysis. Dysregulation of identified factors were validated via western blotting, RT-qPCR, and flow cytometry. Target factors were then inhibited or depleted by shRNA knockdown (KD) or small-compound inhibition. Identified ICB resistance mechanisms were further elaborated by in vitro co-culture assays, syngeneic mouse models, and immune-profiling of tumor microenvironment (TME). Results: Chronic exposure to TMX activates Type I interferon (IFN-I) signaling, induces IFN I-stimulated gene (ISG) expression, and downregulates the H3K4 demethylase and ISG repressor complex known as RACK7/KDM5C. Chronic TMX also upregulates CEACAM1, a well-known ligand for the immune checkpoint receptor TIM3. Results prompt us to evaluate whether loss of RACK7 combined with TMX treatment may modulate a lymphocyte-attractive (via IFN-I), but T-cell exhaustive (via CEACAM1-TIM3) TME. Indeed, in vitro treatment of TMX under RACK7-KD conditions triggers STING upregulation, TBK1 hyperphosphorylation, and a more pronounced activation of ISGs and CEACAM1. In vivo, TMX combined with RACK7-KD enhances tumor growth in the TS/A (ER+) syngeneic mouse model, while immune-profiling reveal that this combination promotes lymphocyte infiltration with a higher population of terminally exhausted CD8+ T-cells in the TME. Conclusions: Our data demonstrate that the TMX-induced activation of cGAS/STING pathway and RACK7 downregulation coordinately shape the IFN-I and immune checkpoint signaling axes in ER+ breast cancer. This TMX–RACK7–IFN-I regulatory network promotes a lymphocyte-attractive TME via IFN-I signaling activation, whereas induction of inhibitory ligands renders T-cells to be terminally-exhausted and unable to kill tumors. These findings highlight the potential use of TMX’s STING-agonistic effect in re-shaping the “cold” breast cancer TME, and the use of combined TMX and anti-TIM3 or anti-CEACAM1 blockade to improve ICB therapy.

Investigation of the effect of macrophage and epithelial cell interaction in the progression of different subtypes of breast cancer.

10 Background: In the realm of tumor progression, the immunosuppressive role of macrophages has been well-established. However, a significant lacuna persists concerning the mechanisms through which epithelial cells regulate macrophages. Furthermore, the precise manner in which macrophages modulate breast cancer cells remains unclear. Hence, the primary objective of the current investigation is to delve into the influence of macrophages on the progression of distinct subtypes of breast cancer. Methods: Human monocyte cell-derived macrophages (M0) were cultivated in the presence of conditioned media (CM) from various subtypes of breast cancer cells. Conversely, human luminal breast cancer cell line T47D, and basal cell line MDA MB 231 were cultured in the presence of CM from macrophages. Results: When macrophages were incubated with CM of T47D, they exhibited a spindle-shaped morphology with pseudopodia. Conversely, exposure to CM of MDA MB 231 induced macrophages to adopt a foamy, round shape with numerous filopodia. Flow cytometry analysis revealed that T47D-CM promoted a higher proportion of CD80+ cells, while MDA MB 231-CM led to an increase in both CD206+ and CD80+ cells. Moreover, both T47D and MDA MB 231-CM escalated the production of various cytokines including IL-1β, IL-2, IL-4, IL-5, IL-7, IL-12(p70), IL-13, IL-17, G-CSF, IFN-γ, and MIP-1β. Intriguingly, MDA MB 231-CM induced a significant increase in IL-6, GM-CSF, and TNF-α. Subsequent cultivation of cancer cells in the presence of macrophage-CM resulted in a marked proliferation of MDA MB 231 cells, while T47D cell proliferation decreased significantly. Correspondingly, there was a notable increase in apoptosis among T47D cells. Scratch wound healing assays revealed heightened migration of MDA MB 231 cells following treatment with macrophage-CM, whereas there was negligible alteration in T47D cell migration. Proteomic analysis unveiled dysregulation of 247 and 596 proteins in MDA MB 231 and T47D cells, respectively. Particularly noteworthy were three proteins—MDHM, H1.5, and LEG8—abundant in MDA MB 231 cells, yet scarce in T47D cells. These proteins have been implicated in promoting tumor growth across various cancer types. Conclusions: This study first time elucidates subtype specific role of macrophage in breast tumour. In triple-negative breast cancer, infiltrating macrophages induce an immunosuppressive effect, altering cytokine expression and fostering the expression of tumor-promoting factors in epithelial cells, thereby facilitating cellular proliferation and metastasis. Conversely, in luminal breast cancer, infiltrating macrophages exhibit a predominantly pro-inflammatory profile, resulting in minimal tumor-promoting effects. Key Words: Breast Cancer, Macrophage polarization, Conditioned Media, Cytokines, Proteomics. Acknowledgements, DBT – Govt of India, NIBMG, Kalyani.

Oestrogen Represses Noggin Expression by Interfering With BMP/Smad Signalling in ER Positive Breast Cancer.

As an antagonist of bone morphogenetic protein (BMP), Noggin facilitates osteolytic bone metastases from breast cancer. The present study aimed to further dissect its role in oestrogen receptor (ER) positive breast cancer. Noggin expression in ER positive breast cancer cell lines (MCF-7 and T-47D) was determined under conditions of oestrogen deprivation and treatment with 17-β-oestradiol (E2). Activation of Smad1/5/8 in the oestrogen-regulated Noggin was examined using recombinant human BMP7 (rhBMP7) and a BMP receptor inhibitor (LDN-193189). The influence of Noggin on cellular functions was evaluated in MCF-7 and T-47D cell lines. Responses to tamoxifen and chemotherapy drugs were determined in MCF-7 and T-47D cells with Noggin over-expression using MTT assay. Noggin expression was negatively correlated with ERα in breast cancers. Noggin was up-regulated upon oestrogen deprivation, an effect that was eliminated by E2 Furthermore, increased levels of phosphorylated Smad1/5/8 were observed in the oestrogen-deprived MCF-7 and T-47D cells, which was prevented by E2 and LDN-193189, respectively. BMP7-induced Noggin expression and activation of Smad1/5/8 was also prevented by E2 and LDN-193189. Noggin over-expression resulted in an increase in the proliferation of both MCF-7 and T-47D cells. MCF-7 and T-47D cells over-expressing Noggin exhibited a good tolerance to tamoxifen (TAM), DTX, and 5-FU, but the percentage of viable cells was higher compared with the controls. Noggin expression can be repressed by oestrogen through inference with the BMP/Smad signalling. Over-expression of Noggin promotes the proliferation of MCF-7 and T-47D cells, contributing to drug resistance.

Improving Tamoxifen Performance in Inducing Apoptosis and Hepatoprotection by Loading on a Dual Nanomagnetic Targeting System.

Although tamoxifen (TMX) belongs to selective estrogen receptor modulators (SERMs) and selectively binds to estrogen receptors, it affects other estrogen-producing tissues due to passive diffusion and non-differentiation of normal and cancerous cells and leads to side effects. The problems expressed about tamoxifen (TMX) encouraged us to design a new drug delivery system based on magnetic nanoparticles (MNPs) to simultaneously target two receptors on cancer cells through folic acid (FA) and hyaluronic acid (HA) groups. The mediator of binding of two targeting agents to MNPs is a polymer linker, including dopamine, polyethylene glycol, and terminal amine (DPN). Zeta potential, dynamic light scattering (DLS), and Field emission scanning electron microscopy (FESEM) methods confirmed that MNPs-DPN-HA-FA has a suitable size of ~105 nm and a surface charge of -41 mV, and therefore, it can be a suitable option for carrying TMX and increasing its solubility. The cytotoxic test showed that the highest concentration of MNPs-DPN-HA-FA-TMX decreased cell viability to about 11% after 72 h of exposure compared to the control. While the protective effect of modified MNPs on normal cells was evident, unlike tamoxifen, the survival rate of liver cells, even after 180 min of treatment, was not significantly different from the control group. The protective effect of MNPs was also confirmed by examining the amount of malondialdehyde, and no significant difference was observed in the amount of lipid peroxidation caused by modified MNPs compared to the control. Flow cytometry proved that TMX loaded onto modified MNPs can induce apoptosis by targeting the overexpressed receptors on cancer cells. Real-time PCR showed that the modified MNPs activated the intrinsic and extrinsic mitochondrial pathways of apoptosis, so the Bak1/Bclx ratio for MNPs-DPN-HAFA- TMX and free TMX was 70.82 and 0.38, respectively. Also, the expression of the caspase-3 gene increased 430 times compared to the control. On the other hand, only TNF gene expression, which is responsible for metastasis in some tumors, was decreased by both free TMX and MNPs-DPN-HA-FA-TMX. Finally, molecular docking proved that MNPs-DPN-HA-FA-TMX could provide a very stable interaction with both CD44 and folate receptors, induce apoptosis in cancer cells, and reduce hepatotoxicity. All the results showed that MNPs-DPN-HA-FA-TMX can show good affinity to cancer cells using targeting agents and induce apoptosis in metastatic breast ductal carcinoma T-47D cell lines. Also, the protective effects of MNPs on hepatocytes are quite evident, and they can reduce the side effects of TMX.

Estrogen receptor alpha (ER-α) antagonistic activity of phytoconstituents from Potentilla atrosanguinea and Potentilla fulgens in breast cancer

The Potentilla genus has long been used traditionally as food and a folklore medicine. In the present study, aerial parts of two Potentilla species, Potentilla fulgens and Potentilla atrosanguinea, of western Himalayan origin, were studied for their anti-breast cancer activity. Ethyl acetate (PAA-EA, PFA-EA), methanolic (PAA-ME, PFA-ME) and hydro-methanolic extract (PAA-HM, PFA-HM) of the plants were tested for their antiproliferative activities against MCF-7 and T-47D breast cancer cell lines. The extracts showed good antiproliferative activity against ER-α dominant breast cancer cell line T-47D, having IC50 values 6.19 ± 0.01 to 33.23 ± 0.04 μg/ml. Eight compounds were isolated, characterized, and quantified from ethyl acetate and methanolic extracts by column chromatography, 1D, 2D-NMR, HRMS and TLC densitometric analysis. Two compounds (4 and 6) have shown better antiproliferative activity than standard bazedoxifene and were further evaluated for their ER-α binding affinity via—fluorescence polarization-based competitive binding assay. The antiestrogenic properties of both compounds were assessed using western blotting. Compounds 4 and 6 were found to have significant affinity for the ER-α and managed to decrease its expression by 38 and 54% respectively. Compounds 4 and 6 also had good stability and reactivity as measured by minimal fluctuations in molecular dynamic simulation analysis, a good dock score in molecular docking, and a respectable HOMO-LUMO energy gap in DFT calculations. Compounds 4 and 6 have shown reliable results and can be used in the development of natural product-based anti-breast cancer agents.

The Composition of Triterpene Glycosides in the Sea Cucumber Psolus peronii: Anticancer Activity of the Glycosides against Three Human Breast Cancer Cell Lines and Quantitative Structure-Activity Relationships (QSAR).

Eight sulfated triterpene glycosides, peronioside A (1) and psolusosides A (2), B (3), G (4), I (5), L (6), N (7) and P (8), were isolated from the sea cucumber Psolus peronii. Peronioside A (1) is a new glycoside, while compounds 2-8 were found previously in Psolus fabricii, indicating the phylogenetic and systematic closeness of these species of sea cucumbers. The activity of 1-8 against human erythrocytes and their cytotoxicity against the breast cancer cell lines MCF-7, T-47D and triple-negative MDA-MB-231 were tested. The most active against cancer cell compounds, psolusosides A (2) and L (6), which were not cytotoxic to the non-transformed cells of the mammary gland, were chosen to study the inhibition of the migration, formation and growth of colonies of the cancer cell lines. Glycoside 2 effectively inhibited the growth of colonies and the migration of the MDA-MB-231 cell line. Compound 6 blocked the growth of colonies of T-47D cells and showed a pronounced antimigration effect on MDA-MB-231 cells. The quantitative structure-activity relationships (QSAR) indicated the strong impact on the activity of the form and size of the molecules, which is connected to the length and architecture of the carbohydrate chain, the distribution of charge on the molecules' surface and various aspects of hydrogen bond formation, depending on the quantity and positions of the sulfate groups. The QSAR calculations were in good accordance with the observed SAR tendencies.

Novel naphthoquinones as potent aromatase inhibitors: Synthesis, anticancer, and in silico studies

A set of 26 naphthoquinone derivatives (3-28) were synthesized by nucleophilic substitution or Michael addition of various amines with 1,4-naphthoquinones and were investigated for their aromatase inhibitory and anticancer activities. The 1,4-naphthoquinone derivatives with amino substituents (14-16 and 24) and the N-alkylated products (25-28) showed promising aromatase inhibitory activity (IC50 = 0.006–2.6 µM). Interestingly, 2-((4-aminophenyl)amino)-3-chloronaphthalene-1,4-dione 14 was noted as a highly potent aromatase inhibitor (IC50 = 6 nM) possessing preferable selective anticancer effect against the breast cancer T47D cell line (IC50: cytotoxic T47D = 24.3 µM, non-cytotoxic to MRC-5 normal cell line, selective index > 6.9). Findings from molecular docking study also suggested that the hydrogen bond formation with Asp309 as well as the pi-sulfur interaction with Met374 residues may be essential for a striking inhibitory effect of the most potent compound 14. Moreover, the in silico drug-likeness prediction indicated that all active naphthoquinone-based compounds are drug-like molecules with potential to be further developed as dual-action anticancer agents for breast cancer management.

Cytotoxic Effects of Plant Bioactive Compounds on T47d Breast Cancer Cells Line: A Review

The continuous cell line known as T47D cells originates from the isolation of epithelial cells from the ductal breast tissue of a 54-year-old woman with metastatic breast cancer. Numerous plant species have been employed in alternative anti-breast cancer therapy, showing advancements in recent years. The aim of this systematic review is to assess the potential bioactive compounds from herbal plant extracts and analyze the effects of herbal plants on T47D breast cancer cells. This research employs a systematic approach to gather articles, encompassing related research questions, chosen databases, keyword selection, inclusion and exclusion criteria, as well as outcomes and conclusions regarding the collected articles. From the reviewed articles, several plants exhibit anti-breast cancer activity on T47D cell lines, namely Calamintha incana, Sterculia quadrifida, Psidium guajava, Uncaria nervosa, Syzygium aqueum, Capsicum annum, Bauhinia scandens, Lansium domesticum, and Garcinia cowa due to their content of bioactive compounds such as triterpenoids, alkaloids, phenolics, flavonoids, saponins, and tannins. These bioactive compounds from the plants exert significant influence on cell cycle, proliferation, fatty acid synthesis, and apoptosis of cancer cells.

Synthesis, X-ray, Hirshfeld combined with DFT, anticancer effects with molecular docking confirmation of (E)-4-cyanobenzaldehyde oxime and (Z)–N-methyl-C-4-substituted phenyl aldonitrones

(E)-4-cyanobenzaldehyde oxime 1 and (Z)–N-methyl-C-4-substituted phenyl aldonitrones 2 and 3 were synthesised and fully characterised. With the help of Hirshfeld calculations, the molecular packing of 1 is found to be controlled by short N…H contacts (28.3%) in addition to the aromatic π-π stacking where the%C…C interactions is calculated to be 9.7%. In this case, the second most abundant interaction is H…H contact (28.1%). For 2, the N…H (24.4%) and O…H (13.8%) contacts are the most important while the most abundant interaction is H…H contacts (36.0%). DFT calculations were utilised to explore the structural and electronic parameters of both compounds at their optimised geometries which were found in good agreement with the X-ray structures. The structure of 2 (6.9829 Debye) is more polar than 1 (4.5282 Debye). The compounds 1–3 reduced the growth of MCF-7 and T47D. Compound 1 which has the lowest IC50 values against the MCF-7 and T47D cell lines, is thought to be the most promising applicant as an anticancer drug. The in-silico combination of compounds 1–3 with proliferative proteins PIK3 and CDK5, autophagic proteins mTOR and beclin, antiapoptotic proteins BCL2, and apoptotic proteins caspase 3 was examined. Compound 3 binds to PIK3, CDK5, mTOR, Beclin, BCL2 and caspase 3, and displays higher free energy bindings of −5.12, −5.45, −4.923, −5.72, −4.45 and −4.73 kcal/mol, which suggested that these proteins had the strongest inhibitory or enhanced effects. The findings show that compound 3 was the most well-docked chemical with the greatest IC50. Compound 3 impacted the autophagy, apoptotic and mitotic proteins of cell growth.

Abstract GS03-10: Allosteric PI3K-alpha inhibition overcomes on-target resistance to orthosteric inhibitors mediated by secondary PIK3CA mutations

Abstract Background: PIK3CA mutations occur in ~40% of HR-positive breast cancers, where alpelisib, an orthosteric PI3Kα inhibitor, is FDA-approved in combination with fulvestrant. Although prior studies have identified potential resistance mechanisms, such as PTEN loss, clinical acquired resistance to orthosteric PI3Kα inhibitors and the role of next-generation allosteric PI3Kα inhibitors remain poorly understood. Methods: To identify on-target and off-target alterations potentially mediating resistance to PI3Kα inhibitors, we used a targeted next-generation sequencing assay (Guardant360; Guardant Health) to analyze ctDNA in serially collected plasma samples from 32 patients with PIK3CA-mutated advanced HR-positive, HER2-negative breast cancer treated with alpelisib and inavolisib. In addition, we performed whole exome sequencing (WES) of 100 tissue samples collected from 8 autopsy series from patients with metastatic, PIK3CA-mutant HR-positive, HER2-negative breast cancer previously treated with PI3Kα inhibitors. Acquired alterations were prioritized through a combination of structural modeling and free-energy perturbation simulation and validated in genomically engineered PIK3CA mutant breast cancer cell lines T47D (PIK3CA H1047R-mutant) or MCF7 (PIK3CA E545K-mutant). Results: We observed that 50% of patients acquire genomic alterations within the PI3K-pathway, including PTEN loss and activating AKT1 mutations. Notably, while secondary PIK3CA mutations were previously reported to increase sensitivity to PI3Ka-inhibitors, we identified emergent secondary resistance mutations in PIK3CA that alter the inhibitor binding pocket including PIK3CA Q859K and PIK3CA W780R. Some mutations had differential effects on PI3Ka-selective vs. pan-PI3K inhibitors, but resistance induced by all mutations could be overcome by the novel allosteric pan-mutant-selective PI3Ka-inhibitor RLY-2608. Conclusion: In one of the largest patient cohorts analyzed to date, this study defines the clinical landscape of acquired resistance to PI3Ka inhibitors. Genomic alterations within the PI3K pathway represent a major mode of resistance and identify a novel class of secondary PIK3CA resistance mutations that can be overcome by an allosteric PI3Ka inhibitor. Together, these findings provide insights to guide strategies to overcome resistance in PIK3CA-mutated cancers. Citation Format: Andreas Varkaris, Ferran Fece de la Cruz, Elizabeth Martin, Bryanna Nordern, Nicholas Chevalier, Ignaty Leshchiner, Anastasia Stavridi, Janice Kim, Alkistis Papatheodoridi, Hakan Gunaydin, Brian Danysh, Laxmi Parida, Ioannis Sanidas, Yongli Ji, Kayao Lau, Gerburg Wulf, Aditya Bardia, Laura Spring, Steven Isakoff, Jochen Lennerz, Levi Pierce, Ermira Pazolli, Gad Getz, Ryan Corcoran, Dejan Juric. Allosteric PI3K-alpha inhibition overcomes on-target resistance to orthosteric inhibitors mediated by secondary PIK3CA mutations [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr GS03-10.

Abstract PO4-23-11: Resistance Mechanisms to CDK4/6 Inhibitors and/or Tamoxifen Using Comprehensive Thermostable Group II Intron Reverse Transcriptase Sequencing

Abstract Background Understanding the mechanisms of resistance to CDK4/6 inhibitors (CDK4/6i) and endocrine therapy (ET) is pivotal in exploring new therapeutic strategies for hormone receptor-positive (HR+), HER2-negative metastatic breast cancer. To decipher these resistance mechanisms, we analyzed the alterations in comprehensive RNA-seq (coding and non-coding RNAs) of HR+ HER2-negative BC cell lines via thermostable group II intron reverse transcriptase sequencing (TGIRT-seq). Methods We established Tamoxifen-resistant (TMR), Abemaciclib-resistant (ACR), Palbociclib-resistant (PCR), Tamoxifen/Abemaciclib double-resistant (TMR-ACR), and Tamoxifen/Palbociclib double-resistant (TMR-PCR) BC cell lines from MCF7 and T47D HR+/HER2- BC cell lines through stepwise dose-escalation continuous drug exposure. We performed a TGIRT-seq transcriptomic analysis using a protocol that allows the sequencing of both long and short non-coding and protein-coding RNAs in a single library. All libraries were sequenced using paired-end 150 bp on the Novaseq platform, resulting in an average of 50 million reads per library. For analysis, raw reads underwent adapter trimming, small RNA mapping, whole genome mapping, and the generation of annotated genes' read count. We used raw counts to detect differentially expressed genes (DEGs) with DESeq2 in R using the cut-off (log2 [fold change] &amp;gt; 1, FDR &amp;lt; 1e-3). We then used the selected DEGs for Gene Set Enrichment Analysis (GSEA) and Kaplan-Meier survival analysis from the TCGA database. Results The TGIRT-seq analysis identified 1171 to 3472 DEGs in different drug resistance and cell line combinations. Most DEGs in resistant cells were consistently downregulated compared to parent cells. Seventy-five percent of DEGs were protein-coding genes, with the rest being non-coding RNAs, such as small nucleolar RNAs, microRNAs, long non-coding RNAs, and transposable element RNAs, which conventional RNA-seq poorly detects. In principal component analysis (PCA) plots, replicates per drug resistance clustered clearly in both cell line backgrounds. PCA also indicated distinctive pathways for acquiring drug resistance in MCF7 and T47D cells. The estrogen receptor (ER) expression decreased, while HER2 increased in resistant cell lines. DEGs identified were enriched in ESR1-related pathways in all MCF7 and T47D cell lines resistant models. Several ER-regulating genes like IL-1R1 and RET were upregulated, while ADCY1 was consistently downregulated across different resistance types. A significant overlap with single-resistance DEGs was observed among the DEGs in double-resistance cell lines, but 20-50% of up/down-regulated DEGs in double-resistance cell lines were uniquely altered. Several previously identified DEGs (e.g., SALL4, TOP2A) and 21 novel candidate genes correlated with poor survival outcomes. Conclusion The analysis identified unique DEGs in double resistance cell lines, suggesting that double resistance mechanisms may not merely be a cumulative effect of single resistance mechanisms, necessitating further investigation and validation. CDK4/6i and/or ET-resistant BC cell lines displayed significant transcriptome reprogramming during the development of drug resistance. In previously published research, ESR1-related pathway alterations were proposed in tamoxifen resistance cell lines. Here we find that CDK4/6i-resistant cells also modify these ESR1-related pathways. Additionally, we identified several targetable genes, such as IL-1R1 and RET, involved in ESR1-related pathways that could pave the way for developing new treatment strategies. Citation Format: Toshiaki Iwase, Hengyi Xu, Nakyung Oh, Jangsoon Lee, Alan M Lambowitz, Naoto Ueno. Resistance Mechanisms to CDK4/6 Inhibitors and/or Tamoxifen Using Comprehensive Thermostable Group II Intron Reverse Transcriptase Sequencing [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-23-11.

Oleate Epoxides Derived from Palm Oil as New Anticancer Agents: Synthesis, Cytotoxicity Evaluation, and Molecular Docking Studies Against FASN Protein

AbstractPalm oil contains an abundant concentration of oleic acid which is known to have low toxicity to human health. The unsaturated double‐bond structure in oleic acid can be modified to synthesize oleate epoxides. The potential benefit of epoxide derivatives is their high biological activity that can be directed as new anticancer agents. In this work, the synthesis of epoxides from purified fatty acid methyl ester (EPFAME) and epoxides from purified fatty acid ethyl ester (EPFAEE) from palm oil in addition to cytotoxicity evaluations as new anticancer agents were investigated by in vitro and in silico studies. Both EPFAME and EPFAEE derivatives were successfully obtained in 82.80 % and 85.80 % yield, respectively. The MTT assay results revealed that EPFAME and EPFAEE significantly decreased the cell viability of WiDr cancer cells with IC50 of 2.70 and 14.23 μg/mL, respectively. They also showed moderate potency against T47D and HeLa cancer cell lines with IC50≥20 μg/mL. The results suggested that the addition of a methylene group from EPFAME to EPFAEE lowers cytotoxicity. A molecular docking study revealed that EPFAME and EPFAEE bind to the fatty acid synthase (FASN) protein interacting with high stability. This is represented by the binding affinity values of EPFAME and EPFAEE to FASN protein which are −7.3 and −8.1 kcal/mol, respectively. Using epoxide compounds derived from palm oil, fatty acid esters were shown to have potential medical benefits due to their low cytotoxicity and anticancer agency against several cell lines by inhibiting proliferation and inducing apoptosis of cancer cells.

Novel Adamantane Derivatives: Synthesis, Cytotoxicity and Antimicrobial Properties

Seventeen adamantane derivatives were synthesized according to facile condensation reaction protocols. Spectral analysis (1H NMR and 13C NMR) was applied to confirm the chemical structure of the obtained substances. The synthesized compounds were tested for in vitro antimicrobial activity against a panel of Gram-positive and Gram-negative bacterial strains and towards fungi from Candida spp. Among them, four derivatives numbered 9, 14, 15 and 19 showed the highest antibacterial potential with MIC = 62.5–1000 µg/mL with respect to all Gram-positive bacteria. S. epidermidis ATCC 12228 was the most susceptible among the tested bacterial strains and C. albicans ATCC 10231 among fungi. Additionally, the cytotoxicity for three derivatives was measured with the use of the MTT test on A549, T47D, L929 and HeLa cell lines. Our cytotoxicity studies confirmed that the tested substances did not cause statistically significant changes in cell proliferation within the range of the tested doses.

Abstract LB127: A first-in-class bispecific ADC targeting FORL1 and MUC1 exhibits promising anti-tumor activity in a FOLR1low xenograft model

Abstract Folate receptor alpha (FOLR1) is specifically overexpressed in malignant tumors of epithelial origin, including ovarian, lung and breast cancers. While antibody-drug conjugate (ADC) therapies targeting FOLR1 have shown promise in clinical trials, there is still a large proportion of patients, particularly those with low FOLR1 expression, who do not respond, indicating new therapeutic strategies are needed. MUC1 is another tumor-associated antigen that is likewise overexpressed in epithelial ovarian cancer, breast cancer, and NSCLC, among other cancers; analysis of ovarian PDX tumors by immunohistochemistry (IHC) suggests that FOLR1 and MUC1 are both highly expressed in this cancer type. We therefore hypothesized that dual targeting of FOLR1 and MUC1 could provide therapeutic benefit in ovarian cancer as well as a broad range of tumors. We generated a FOLR1 x MUC1 bispecific antibody (bsAb) from anti-FOLR1 and anti-MUC1 monoclonal antibodies that were produced using RenLite® fully human common light chain mice. These parental monoclonal antibodies recognize both human and cynomolgus monkey antigens. Anti-FOLR1 antibodies were highly specific for FOLR1, as they did not recognize other FOLR family members. The anti-MUC1 antibody was previously confirmed to target MUC1-C, the juxtamembrane domain of MUC1, ensuring tumor cell recognition regardless of MUC1 cleavage. Assessment of the novel FORL1 x MUC1 bsAb indicates binding to several ovarian, breast and NSCLC cell lines, and shows enhanced internalization in MDA-MB-468 and T47D cell lines when compared to parental antibodies. Subsequently, the FORL1 x MUC1 bsAb was conjugated to vcMMAE with a DAR of 4, to generate a bispecific ADC, BCG023. BCG023 exhibited superior tumor growth inhibition compared to benchmark ADCs conjugated with the same payload and DAR in a low FOLR1-expressing NSCLC PDX model. These data suggest that targeting FOLR1 and MUC1 with a bispecific ADC is a promising therapeutic strategy for the treatment of ovarian and other tumors. Citation Format: Yifu Zhang, Chengzhang Shang, Chaoshe Guo, Yi Yang. A first-in-class bispecific ADC targeting FORL1 and MUC1 exhibits promising anti-tumor activity in a FOLR1low xenograft model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB127.

Reliability aspects in repulsive steric hindrance approach: Selectivity and sensitivity investigations

Dual metal regulated bottom gate top contact structured Organic Thin Film Transistor based biosensor (Bio-OTFT) for recognition of breast cancer cell lines along with the non-tumorigenic epithelial cell line is presented here. A feasible fabrication scheme for Bio-OTFT has been demonstrated. Repulsive steric hindrance (RSH) consideration inside the cavity zone directs more practical and realistic sensing mechanisms. The insightful electrostatic analysis regarding electric field, energy band diagram, transfer characteristics (IDS-VGS), and transconductance (gm) has been described. Alteration of the dielectric constant (k) of these cancerous and healthy cell lines stated the recognition scheme in this reported Bio-OTFT. The efficiency of the recommended model has been assessed thoroughly with the reported OTFT literatures. The sensitivity is examined respecting drain current (IDS) in (A/μm), gm in (μS/μm), threshold voltage (VTH) in Voltage and sub-threshold swing (SS) in (mV/dec) and reaches to the maximum value of 2.3 × 108, 1.85 × 1010, 0.68, and 0.08 for T-47D cell line at VGS = −1.25 V. The recommended model thus suggests an enormous reliability and possibility for biosensing purposes.

Abstract 3334: Combined treatment with PG3 and Nelfinavir induced synergistic apoptosis though sustained activation of integrated stress response

Abstract Integrated stress response (ISR) activation has been linked to many human diseases, including cancer and neurological diseases. The ISR controls protein synthesis and proteostasis. ISR main effector ATF4 transcriptional factor regulates the balance between survival and cell death. Under acute ISR, ATF4 promotes cell adaptation and survival. Sustained ISR leads to ATF4-mediated apoptosis. In many cancers, mutations or overexpression of oncogenes, results in enhanced cell proliferation and increased protein synthesis, which activate the ISR. Therefore, protein translation is reduced to maintain proteostasis. In other words, many cancers are primed by the ISR. Therefore, additional push by small molecule ISR-inducers will disrupt the balance and force cancer cells to enter apoptosis. Nelfinavir activates ATF4 through inhibition of eIF2α specific phosphatases CReP and GADD34. PG3 upregulates ATF4 through the HRI/eIF2α/ATF4 pathway. We hypothesize that combined treatment of PG3 and Nelfinavir will sustain upregulation of ATF4, and lead to ATF4-mediated cell apoptosis. Cell viability assays indicated that the combined treatment potently and synergistically inhibited cell viability in colorectal cancer cell lines HT29, SW480 and HCT116 p53−/−, osteosarcoma cell line U2OS and breast cancer cell lines MCF7 and T47D. The combined treatment sustained the induction of ATF4 and enhanced the expression of its proapoptotic target genes, CHOP and PUMA, and cell apoptosis. We confirmed that the enhanced induction of ATF4, CHOP and PUMA is through the ISR because ISRIB (ISR inhibitor) blocked the combined treatment-induced upregulation of ATF4, CHOP and PUMA. Citation Format: Xiaobing Tian, Wafik S. El-Deiry. Combined treatment with PG3 and Nelfinavir induced synergistic apoptosis though sustained activation of integrated stress response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3334.

Nutraceuticals known to promote hair growth do not interfere with the inhibitory action of tamoxifen in MCF7, T47D and BT483 breast cancer cell lines.

Hair loss/thinning is a common side effect of tamoxifen in estrogen receptor (ER) positive breast cancer therapy. Some nutraceuticals known to promote hair growth are avoided during breast cancer therapy for fear of phytoestrogenic activity. However, not all botanical ingredients have similarities to estrogens, and in fact, no information exists as to the true interaction of these ingredients with tamoxifen. Therefore, this study sought to ascertain the effect of nutraceuticals (+/- estrogen/tamoxifen), on proliferation of breast cancer cells and the relative expression of ERα/β. Kelp, Astaxanthin, Saw Palmetto, Tocotrienols, Maca, Horsetail, Resveratrol, Curcumin and Ashwagandha were assessed on proliferation of MCF7, T47D and BT483 breast cancer cell lines +/- 17β-estradiol and tamoxifen. Each extract was analysed by high performance liquid chromatography (HPLC) prior to use. Cellular ERα and ERβ expression was assessed by qRT-PCR and western blot. Changes in the cellular localisation of ERα:ERβ and their ratio following incubation with the nutraceuticals was confirmed by immunocytochemistry. Estradiol stimulated DNA synthesis in three different breast cancer cell lines: MCF7, T47D and BT483, which was inhibited by tamoxifen; this was mirrored by a specific ERa agonist in T47D and BT483 cells. Overall, nutraceuticals did not interfere with tamoxifen inhibition of estrogen; some even induced further inhibition when combined with tamoxifen. The ERα:ERβ ratio was higher at mRNA and protein level in all cell lines. However, incubation with nutraceuticals induced a shift to higher ERβ expression and a localization of ERs around the nuclear periphery. As ERα is the key driver of estrogen-dependent breast cancer, if nutraceuticals have a higher affinity for ERβ they may offer a protective effect, particularly if they synergize and augment the actions of tamoxifen. Since ERβ is the predominant ER in the hair follicle, further studies confirming whether nutraceuticals can shift the ratio towards ERβ in hair follicle cells would support a role for them in hair growth. Although more research is needed to assess safety and efficacy, this promising data suggests the potential of nutraceuticals as adjuvant therapy for hair loss in breast cancer patients receiving endocrine therapy.

CYTOTOXIC ACTIVITY OF TAXUS SUMATRANA (MIQ.) DE LAUB. BARK, LEAVES, AND SHOOTS ON HELA, T47D, AND MCF-7/HER2 CELL LINES

Objective: Taxus sumatrana (Miq.) de Laub. (cemara Sumatra) is one of the plants found in Indonesia and other countries known as a medicine plant. Taxus's bark, leaves, and shoots are used traditionally and massively for some diseases (cancer, etc.), so recently it has become a rare plant. The chemical constituents of T. sumatrana are alkaloids, steroids, tannins, and flavonoids. This study aimed to investigate the potential anticancer properties of T. sumatrana bark, leaves, and shoot extracts.&#x0D; Methods: The cytotoxic activity against the HELA, T47D, and MCF-7/HER2 cell lines was determined using the MTT assay. Each cell was cultured on 96 well plates treated with extract of T. sumatrana with concentrations of 100, 10, 1, and 0,1 µg/ml. Cells were incubated for 48 h at 37 °C, 5% CO2 and then given 100 µl MTT solution 0.5 mg/ml in PBS (Phosphate Buffer Saline) for 4 h. The results of the measurements were processed with the GraphPad Prism Program.&#x0D; Results: The bark, leaves, and shoots extracts have strong cytotoxic activity based on IC50 parameters. The mean IC50 of bark, leaves, and shoots on the HELA cell line consecutively 8.94; 5.93; and 4.08 μg/ml; on the T47D cell line 5.80, 4.86, and 4,11 μg/ml; and on MCF-7/HER2 cell line 7.46, 10.60, and 13.74 μg/ml).&#x0D; Conclusion: T. sumatrana bark, leaves, and shoots have potential anti-cancer properties.