Normal Breast Epithelial Cells Research Articles

Enhancing therapeutic efficacy: sustained delivery of 5-fluorouracil (5-FU) via thiolated chitosan nanoparticles targeting CD44 in triple-negative breast cancer

Our current study reports the successful synthesis of thiolated chitosan-based nanoparticles for targeted drug delivery of 5-Fluorouracil. This process was achieved through the ionic gelation technique, aiming to improve the efficacy of the chemotherapeutic moiety by modifying the surface of the nanoparticles (NPs) with a ligand. We coated these NPs with hyaluronic acid (HA) to actively target the CD44 receptor, which is frequently overexpressed in various solid malignancies, including breast cancer. XRD, FTIR, SEM, and TEM were used for the physicochemical analysis of the NPs. These 5-Fluorouracil (5-FU) loaded NPs were evaluated on MDA-MB-231 (a triple-negative breast cell line) and MCF-10A (normal epithelial breast cells) to determine their in vitro efficacy. The developed 5-FU-loaded NPs exhibited a particle size within a favorable range (< 300 nm). The positive zeta potential of these nanoparticles facilitated their uptake by negatively charged cancer cells. Moreover, they demonstrated robust stability and achieved high encapsulation efficiency. These nanoparticles exhibited significant cytotoxicity compared to the crude drug (p < 0.05) and displayed a promising release pattern consistent with the basic diffusion model. These traits improve the pharmacokinetic profile, efficacy, and ability to precisely target these nanoparticles, offering a potentially successful anticancer treatment for breast cancer. However, additional in vivo assessments of these formulations are obligatory to confirm these findings.

Abstract PO5-27-07: A novel ROR1 inhibitor CPD86 suppresses Triple-Negative Breast Cancer cells via regulation of AKT/GSK3β pathway

Abstract Breast cancer is the second most commonly diagnosed cancer in women in the United States. The most aggressive subtype is triple negative breast cancer (TNBC), which accounts for 15-20% of new cases on average each year. Histologically, it is characterized by the absence of estrogen receptors (ER), progesterone receptors (PR) and human epidermal growth factor receptor 2 (HER2), which are typically targeted by hormone therapies such as tamoxifen, fulvestrant, or letrozole. Standard of care for TNBC includes traditional chemotherapy and radiation, which are detrimental to both cancerous and normal cells. Therefore, the lack of all hormone receptors in TNBC calls for the investigation of novel anti-cancer compounds that specifically target TNBC with minimal damage to non-cancerous tissues. Receptor orphan tyrosine kinase-like receptor 1 (ROR1) is an oncoprotein that is overexpressed in several human malignancies such as lung, breast, prostate and pancreatic cancers, but not in normal tissues. Inhibition of ROR1 signaling has shown to repress proliferation and induce apoptosis of cancer cells. Through in-silico docking and isothermal titration calorimetry, we identified a novel compound CPD86 that interacts with and inhibits ROR1. We hypothesize that CPD86 selectively targets TNBC cells by reducing the phosphorylation of AKT/GSK3β via the inhibition of ROR1. Our in vitro results suggest that CPD86 inhibits cell viability and induces intrinsic apoptosis of TNBC cells at a half-maximal inhibitory concentration of 2-5 µM, but not in normal breast epithelial cells with minimal ROR1 expression. CPD86 also represses TNBC cell migration and invasion while leaving non-malignant cells unharmed. This study highlights that ROR1 inhibitors could be developed as a potential therapeutic for TNBC and the data would serve as proof-of-concept justification for evaluation in other ROR1-upregulated cancers. Citation Format: Tram Ta, Victoria Reed, Nikhil Chandra, Nick Dwyer, Norman Fultang, Shradheya Gupta, Indrakant Singh, Bela Peethambaran. A novel ROR1 inhibitor CPD86 suppresses Triple-Negative Breast Cancer cells via regulation of AKT/GSK3β pathway [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 PO5-27-07.

Abstract PO1-06-11: Exosomal HMGB1 induce PD-L1+-tumor associated macrophages through glycometabolic reprogramming to promote lung-tropic metastasis of triple negative breast cancer

Abstract Background: Lung metastasis occurs in 30% of triple negative breast cancer (TNBC), the detailed molecular mechanism remains largely unexplored. Accumulating evidence suggests that PD-L1+-tumor associated macrophages (PD-L1+-TAMs) are closely related to immune suppression, invasion, and metastasis. Tumor-derived exosomes have been reported to remodel tumor microenvironment and accelerate metastasis via packing and delivering a variety of biologically active molecules. Our previous studies found that exosomes from breast cancer cells polarized macrophages toward TAMs within the primary tumor nest. The aim of this study was to reveal whether TNBC-derived exosomes were able to polarize lung macrophages towards PD-L1+-TAMs and generate a pre-metastatic immunosuppressive niche, therefore discovering the underlying mechanism of lung-tropic metastasis. Methods: Exosomes isolated from EO771 TNBC cell line (EO771/exo) and HC11 normal breast epithelial cell line (HC11/exo) were characterized. Uptake of exosomes by macrophages was visualized by confocal microscopy. Mass spectrometry was applied to identify the significantly highly expressed HMGB1 protein in EO771/exo, and ligands of HMGB1 on lung macrophages were detected by co-immunoprecipitation. Functions of EO771/exo and HMGB1 in inducing PD-L1+-TAMs and generating a pre-metastatic immunosuppressive niche were confirmed by both in vitro and in vivo studies. Roles of EO771/exo and HMGB1 on glycolysis and lactate production were evaluated by metabolism assays. Associations between PD-L1+-TAMs and CD3+CD8+ T lymphocytes within TNBC axillary lymph nodes and lung metastasis specimens were analyzed by immunofluorescence and immunohistochemistry. Results: Confocal microscopy showed constant internalization and absorption of EO771/exo by macrophages. EO771/exo could polarize macrophages toward PD-L1+-TAMs and generate a pre-metastatic immunosuppressive niche, with respect to HC11/exo. Mass spectrometry, clinical samples, and bioinformatics analysis revealed that HMGB1 was highly expressed in EO771/exo and was involved in cellular communication and metabolism processes. Co-immunoprecipitation and 3-dimensional modeling indicated a strong binding of HMGB1 with Tim-3 ligand on lung macrophages. Macrophages treated with EO771/exo displayed an enhanced glycolysis and lactate production. Flow cytometry demonstrated that green fluorescent protein (GFP)-EO771 tumor-bearing mice had more lung micro-metastases than brain and liver. Immunofluorescence and laser confocal microscopy confirmed that EO771/exo, with respect to HC11/exo, were able to increase lung micro-metastases and escalate PD-L1 expression on lung macrophages. Moreover, elevated PD-L1+-TAMs and reduced CD3+CD8+ T lymphocytes were found in TNBC positive axillary lymph nodes and lung metastasis specimens than TNBC negative axillary lymph nodes. Conclusions: Exosomal HMGB1 from TNBC could target Tim-3 ligand on lung macrophages and accelerate glycolysis and lactate production to induce PD-L1+-TAMs, thereby suppressing CD3+CD8+ T lymphocytes immunity and generating a pre-metastasis immune-suppressive niche. Our research would uncover a novel mechanism of lung-tropic metastasis and provide a new therapeutic approach for TNBC treatment. Keywords: breast cancer; lung metastasis; exosomes; tumor-associated macrophages; metabolic reprogramming Citation Format: Wei-Xian Chen, Jia-Hao Wu, Tian-Cheng Cheng, Sujin Yang. Exosomal HMGB1 induce PD-L1+-tumor associated macrophages through glycometabolic reprogramming to promote lung-tropic metastasis of triple negative breast cancer [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 PO1-06-11.

Drug repositioning for immunotherapy in breast cancer using single-cell analysis.

Immunomodulatory peptides, while exhibiting potential antimicrobial, antifungal, and/or antiviral properties, can play a role in stimulating or suppressing the immune system, especially in pathological conditions like breast cancer (BC). Thus, deregulation of these peptides may serve as an immunotherapeutic strategy to enhance the immune response. In this meta-analysis, we utilized single-cell RNA sequencing data and known therapeutic peptides to investigate the deregulation of these peptides in malignant versus normal human breast epithelial cells. We corroborated our findings at the chromatin level using ATAC-seq. Additionally, we assessed the protein levels in various BC cell lines. Moreover, our in-house drug repositioning approach was employed to identify potential drugs that could positively impact the relapse-free survival of BC patients. Considering significantly deregulated therapeutic peptides and their role in BC pathology, our approach aims to downregulate B2M and SLPI, while upregulating PIGR, DEFB1, LTF, CLU, S100A7, and SCGB2A1 in BC epithelial cells through our drug repositioning pipeline. Leveraging the LINCS L1000 database, we propose BRD-A06641369 for B2M downregulation and ST-4070043 and BRD-K97926541 for SLPI downregulation without negatively affecting the MHC complex as a significantly correlated pathway with these two genes. Furthermore, we have compiled a comprehensive list of drugs for the upregulation of other selected immunomodulatory peptides. Employing an immunotherapeutic approach by integrating our drug repositioning pipeline with single-cell analysis, we proposed potential drugs and drug targets to fortify the immune system against BC.

Abstract 4109: Discovery of a novel cancer-specific antigen for therapeutic targeting using the Oncotope platform

Abstract Identification of druggable cancer-specific surface antigens is hindered by the complexity of cancer's molecular diversity and the risk of normal cell cross-reactivity. We developed the Oncotope Platform that rapidly identifies multiple cancer-specific cell surface targets not expressed on normal cells (JCO 2022). In this study, we generated polyclonal antibodies to human Triple Negative Breast Cancer (TNBC; Hs578T, ATCC). Following this, we used the Oncotope Platform to filter the anti-TNBC antibody-enriched serum, to yield cancer specific polyclonal antibodies. Using a Protein Microarray, we assessed the binding affinity of both the Total Anti-TNBC Serum (before filtration) and the Oncotope Filtered Anti-TNBC Serum towards approximately 25,000 human proteins. Our results identified antigens that exhibited significantly higher affinity binding to the Oncotope Filtered Anti-TNBC Serum than the Total Anti-TNBC Serum. One of these antigens ZIP7, a zinc transporter situated in the ER and Golgi, has been shown to activate cell proliferation related signaling pathways in TNBC. Our findings revealed the aberrant cell surface expression of ZIP7 on TNBC cells and its potential as a therapeutic target. Flow cytometry analysis using rabbit polyclonal antibodies against ZIP7 revealed substantial binding to TNBC cells, while no significant binding was observed with normal breast epithelial (NBE) cells from the same patient (Hs578BsT, ATCC). Additionally, cytotoxicity assays, employing the same antibodies and a secondary anti-rabbit ADC, showed the preferential killing of TNBC cells over NBE cells (Table 1). In summary, our study marks a breakthrough in identifying cancer-specific antigens with therapeutic potential. The Oncotope Platform, in combination with protein microarrays, enables rapid identification of unique cancer-specific antigens absent in normal cells. Specifically targeting ZIP7 on TNBC cell surfaces highlights its promise for precision cancer therapy. Table 1. Cell Line Incubated with: %Binding Flow Cytometry (1:400 Ab dilution) %Cytotoxicity (4ug/ml Ab conc.) TNBC (Hs578T) Total Anti-TNBC Serum 96.65 94.5 +/- 2.5 Oncotope Filtered Anti-TNBC Serum 97.46 79.2 +/- 2.1 ZIP7 Polyclonal antibody ((Proteintech 11200-1-AP) 46.2 73.2 +/- 0.5 Normal Breast Epithelial cells (Hs578Bst) Total Anti-TNBC Serum 92.95 92.3 +/- 3.0 Oncotope Filtered Anti-TNBC Serum 20.39 17.9 +/- 4.5 ZIP7 Polyclonal antibody ((Proteintech 11200-1-AP) 6.37 14.1 +/- 1.6 Citation Format: John Sanil Manavalan, Danielle Mor, Joseph Davis, Sharanjot Saini, Ipsita Pal, David Feith, Yuichi Hikichi, Elliot Davis. Discovery of a novel cancer-specific antigen for therapeutic targeting using the Oncotope platform [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 4109.

Abstract 5680: Circular MALAT1 - the new kid on the block in breast-to-brain metastatic cancer

Abstract Introduction: Breast-to-brain metastatic (BTB met) cancer constitutes a great challenge for modern medicine and science. As the research on protein-coding genes brought only incremental progress in developing anticancer therapy, much attention is devoted to understanding the role of non-coding RNAs, including a relatively recently discovered group - of circular RNAs. This project aimed to determine the relationship between BTB met cancer cells and cells derived from a deadly brain tumor - glioblastoma stem-like cells (GSCs), considering the circular RNA signatures and selecting a reliable candidate for mechanistic study - a circular form of non-coding oncogene MALAT1 (circMALAT1). Methods: The circular RNA expression profile in patient-derived BTB met cancer cells (n=4), GSCs (n=8), and neural progenitor cells (NPC) as a non-malignant control (n=3) was evaluated by the microarray approach. Expression of circMALAT1 was also validated in non-malignant breast epithelial cells by digital PCR and data mining in the GEO database. CircMALAT1 knockdown by antisense oligonucleotides was followed by functional analysis. Results: 12,660 circular RNAs were detected using the Arraystar platform, and upon applying the cutoff of FC ≥2 and p-value &amp;lt;0.05, 547 circular RNAs were selected as significantly deregulated in all cancer groups compared to NPC. CircMALAT1 was elevated in BTB met compared to NPC and normal breast epithelial cells. Knockdown of circMALAT1 affected cancer cell viability and proliferation while promoting p21 expression, thus decelerating the cell cycle manifested by a reduction in 3D spheroids volume. Conclusions: The analysis of the circular RNA landscape showed for the first time some striking similarities between BTB mets and GSCs despite different tissue-of-origin. Of note, the circRNAome of BTB mets clustered especially closely with mesenchymal GSC - the most aggressive and therapy-resistant subtype of GSCs. These results also demonstrated the role of circMALAT1 in shaping the oncogenic traits of these cells. Therefore, circular RNAs can serve as valuable biomarkers and promising novel mechanistic candidates whose modulation may become a paradigm-shifting therapeutic approach. Citation Format: Adrian Szczepaniak, Agnieszka Bronisz, Jakub Godlewski. Circular MALAT1 - the new kid on the block in breast-to-brain metastatic cancer [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 5680.

Abstract 5687: Downregulation of DAX-1 expression by miRNA overexpression as a mechanism to potentiate breast cancer

Abstract The orphan nuclear receptor DAX-1 (Dosage Sensitive Sex Reversal, Adrenal Hypoplasia Congenita, critical region on the X chromosome, gene 1) plays a key role in sex determination and the synthesis of steroid hormones. In addition to these canonical examples, DAX-1 has been shown to play a contradictory role in cancer development. While DAX-1 is overexpressed in some types of cancer, it is completely absent or downregulated in several other cancers such as prostate and breast. One of the explanations for the paradoxical role of DAX-1 in cancer development could be microRNAs (miRNAs). Recent research has demonstrated that the dysregulated expression of these short non-coding RNAs increases the proliferative and anti-apoptotic capabilities of cancer cells. After broadly surveying over 180 miRNAs historically upregulated in MCF7 cells, we compared the activity of DAX-1 in MCF7 breast cancer cells and MCF10a normal breast epithelial cells with the presence of specific miRNA inhibitors. Our primary hypothesis remains that multiple miRNAs negatively regulate the expression of DAX-1 in human breast cancer cells and are not overexpressed in normal breast cells, providing another mechanism of lifting the repression of DAX-1 expression. Quantitative PCR and western blot analysis was performed indicating that inhibiting miRNAs 29c, 199a, and 424 significantly upregulate DAX-1 expression in MCF10a cells. We examined the invasive and metastatic properties of MCF7 cells following inhibition of these specific miRNAs. This research will allow clinicians to screen for additional miRNAs that could be elevated in breast cancer patients substantially increasing the number of people diagnosed with breast cancer early on. In addition, elucidating the pathways of how more and more miRNAs influence downstream targets will offer drug designers novel targets to improve treatment for breast cancer and other cancer patients. Citation Format: Nicholas C. DeVito, Christina Tzagarakis-Foster. Downregulation of DAX-1 expression by miRNA overexpression as a mechanism to potentiate breast cancer [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 5687.

Abstract 7660: L-Arginine metabolism is of prognostic relevance in women with breast cancer, but varies considerably between subtypes in vitro - A bedside-to-bench translational approach

Abstract Availability of the semi-essential amino acid L-arginine (L-arg) is limiting for proliferation in highly proliferative tissues. L-Arg is also a substrate for nitric oxide synthases (NOS) and arginases (ARG) 1 and 2. Methylation of L-arg residues within proteins by protein L-arg methyltransferases (PRMTs) leads to asymmetric (ADMA) and symmetric dimethylarginine (SDMA). Protein L-arg methylation is an important post-translational mechanism to regulate gene expression and cell cycle control. We measured L-arg and its metabolites in a prospective cohort of patients with primary breast cancer (BC). We further measured these metabolites and expression of genes involved in L-arg metabolic pathways in cell lines representing intrinsic BC subtypes. Plasma L-arg-related metabolite concentrations from 243 women with primary BC were associated with total mortality and disease recurrence during a median follow-up of 88 (IQR, 82-93) months. MCF-7, BT-474, SK-BR-3, and MDA-MB-468 cells representing ER-positive, HER2-positive, and triple negative BC were cultured in standard conditions and compared to MCF-12A normal breast epithelial cells. The plasma (in vivo) and intracellular concentrations (in vitro) of L-arg, L-ornithine (L-orn), L-citrulline (L-cit), ADMA, and SDMA were measured by UPLC-MS/MS. mRNA expression was quantified by qRT-PCR and protein expression was analyzed by Western Blot. In vivo, the plasma concentrations of all L-arg metabolites differed significantly between BC patients and healthy controls. L-Arg and ADMA were significantly higher in patients with recurrent disease and in those who died during follow up. ADMA was significantly associated with total mortality and recurrent disease in Luminal A patients; low L-orn/L-arg ratio was associated with survival in HER2+ BC patients, and low L-cit was associated with survival in triple negative BC. With the exception of MCF-7 cells, DDAH1 and DDAH2 mRNA concentrations in vitro were significantly higher in BC cells than in MCF-12A cells (DDAH1: 32-44 fold, DDAH2: 1.7-4.2 fold; p&amp;lt;0.05). These differences were mirrored by DDAH1 and DDAH2 protein concentrations. By contrast, MCF-7 cells were characterized by low DDAH1 and DDAH2, but high PRMT4 and PRMT6 mRNA expression and high L-arg content. BT-474 cells showed high ARG2 expression and high L-arg and L-orn concentrations, and MDA-MB-468 cells had the highest L-cit/L-arg ratio. In conclusion, regulation of L-arg metabolic pathways shows a complex and differential pattern between intrinsic BC subtypes, which is mirrored in representative cell lines in vitro. ADMA is a prognostic biomarker in Luminal A breast cancer patients; its metabolizing enzyme, DDAH, is highly overexpressed in BC cells in vitro. Thus, fingerprinting of L-arg metabolism may offer novel personalized treatment options within intrinsic BC subtypes. Citation Format: Juliane Hannemann, Leticia Oliveira Ferrer, Antonia Röglin, Fiona Kleinsang, Volkmar Müller, Isabell Witzel, Rainer Böger. L-Arginine metabolism is of prognostic relevance in women with breast cancer, but varies considerably between subtypes in vitro - A bedside-to-bench translational approach [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 7660.

Abstract 1723 Differences in barriers to immortalization in normal breast epithelial cells from donors with and without mutations in breast cancer susceptibility genes

Abstract 1723 Differences in barriers to immortalization in normal breast epithelial cells from donors with and without mutations in breast cancer susceptibility genes

Encapsulation and Delivery of Mitoxantrone Using Zirconium-Based Metal–Organic Frameworks (MOFs) and Their Cytotoxic Potential in Breast Cancer Cells

Mitoxantrone (MTX) is a drug employed in breast cancer treatment, but its application is largely limited due to side effects. A controlled delivery approach can potentially reduce the side effects. In this study, two zirconium (Zr)-based MOFs, UiO-66 and UiO-66-NH2, were studied for a more controlled delivery of MTX with a 40% and 21% loading capacity, respectively. Characterisation via powder X-ray diffraction, thermogravimetric analysis, Fourier transform infrared spectrometry, scanning electron microscopy, and dynamic light scattering confirmed the integrity of structure post-MTX loading. UV–vis spectrophotometry revealed distinctive release profiles, with UiO-66-MTX exhibiting a 25% cumulative release after 96 h in water and 120 h in PBS +10% FBS. UiO-66-NH2-MTX displayed a more sustained release, reaching 62% in water and 47% in PBS +10% FBS after 168 h. The interaction between MTX and the MOFs was also proposed based on computational modelling, suggesting a stronger interaction of UiO-66NH2 and MTX, and an optimised interaction of MTX in the tetrahedral and octahedral pores of the MOFs. The study also reports the release profile of the drug and antiproliferative activity against a panel of breast cancer cell lines (MDA-MB-231, MDA-MB-468, and MCF7) and a normal breast epithelial cell line (MCF10A). MTX-encapsulated MOFs were thoroughly characterised, and their biological activity was assessed in vitro. MTT cell viability assay indicated a higher IC50 value for MTX-loaded MOFs compared to free MTX in physiological conditions, albeit with a slower release profile. These findings suggest the potential of these MTX-loaded MOFs as an alternative avenue for formulation to mitigate side effects.

An analysis of the prognostic role of reactive oxygen species-associated genes in breast cancer.

This study aimed to type breast cancer in relation to reactive oxygen species (ROS), clinical indicators, single nucleotide variant (SNV) mutations, functional differences, immune infiltration, and predictive responses to immunotherapy or chemotherapy, and constructing a prognostic model. We used uniCox analysis, ConsensusClusterPlus, and the proportion of ambiguous clustering (PAC) to analyze The Cancer Genome Atlas (TCGA) data to determine optimal groupings and obtain differentially expressed ROS-related genes. Clinical indicators were then combined with the classification results and the Chi-square test was used to assess differences. We further examined SNV mutations, and functional differences using gene set enrichment analysis (GSEA) analysis, the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, immune cell infiltration, and response to immunotherapy and chemotherapy. A prognostic model for breast cancer was constructed using these differentially expressed genes, immunotherapy or chemotherapy responses, and survival curves. RT-qPCR was used to detect the differences in the expression of LCE3D, CA1, PIRT and SMR3A in breast cancer cell lines and normal breast epithelial cell line. We identified two distinct tumor types with significant differences in ROS-related gene expression, clinical indicators, SNV mutations, functional pathways, and immune infiltration. The response to specific chemotherapy drugs and immunotherapy treatments also documented significant differences. The prognostic model constructed with 16 genes linked to survival could efficiently divide patients into high- and low-risk groups. The high-risk group showed a poorer prognosis, higher tumor purity, distinct immune microenvironment, and lower immunotherapy response. RT-qPCR results showed that LCE3D, CA1, PIRT and SMR3A are highly expressed in breast cancer. Our methodical examination presented an enhanced insight into the molecular and immunological heterogeneity of breast cancer. It can contribute to the understanding of prognosis and offer valuable insights for personalized treatment strategies. Further, the prognostic model can potentially serve as a powerful tool for risk stratification and therapeutic decision-making in clinical settings.

Abstract A089: Role of Vardhamana Pippali Rasayana (VPR) in human breast cancer cells

Abstract Serious concerns include delayed diagnosis and adverse side-effects of conventional treatment leading to residual morbidity in Breast cancer (BC) globally. Traditional Indian medicines/herbs via combination of conventional cancer are gaining increasing acceptance worldwide, playing a pivotal role as proven by their efficacy evaluation studies. Many plants are known to reduce the cell proliferation and tumor development after their treatment. Vardhamana Pippali Rasayana (VPR), an Ayurvedic herbal preparation of pippali (Piper longum) in gradually increasing and tapering dose is used to treat many cancers without any adverse side-effects. However, its anti-cancer role in breast cancer is not known yet. The present study has explored the cytotoxic and apoptotic effects of VPR in triplicates on human breast cancer (MCF7) and normal breast epithelial (MCF10A) cell lines at different concentrations after extracting piperine in aqueous and hydro-alcoholic soxhlet extract (0.25 to 50 μg/μl). The cytotoxic activity was carried out by MTT assay and results showed that a low IC50 was observed at 3.75 μg/μl of VPR against MCF-7 as compared with normal breast epithelial cells. Detection of apoptotic cells using suitable Annexin V was done and there was a time-dependent increase in percentage of annexin V positive cells upon treatment. The above results indicate that VPR may have a strong anti-cancerous potential. But other functional assays are warranted to validate the efficacy of the drug against breast cancer which are under progress in our laboratory. Citation Format: Dr.Richa Tripathi, Shivam Singh, Subhash Gupta, VG Huddar, Tanuja M Nesari, Ashok Sharma. Role of Vardhamana Pippali Rasayana (VPR) in human breast cancer cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A089.

Abstract A037: Metabolic maneuvering of glycocalyx hypersialylation is a smart and healthy way of curbing metastatic potential in breast cancer

Abstract Metastatic breast cancer is currently the most deadly and incurable stage of breast cancer. A recent metabolic screen has identified sialic acid as a key player in progression of breast cancer metastasis. In the present study, we metabolically target the hypersialylation of metastatic breast cancer cells using appropriately designed nutritionary interventions. Our rationales are the following: (a) The aberrantly sialylated, hyperbranched glycocalyx promotes metastasis by facilitating cellular migration and immune evasion. (b) As hypersialylation relies heavily on enhanced sialic acid biosynthesis from the oncogenic glycolytic surge and preferential dietary uptake of glucose by cancer cells, it stands to reason that dietary changes can impact the extent of sialylation of the glycocalyx. Considering these points, we hypothesized that it was possible to control the degree of glycocalyx sialylation in breast cancer cells using an appropriate nutritionary regimen, which will in turn modulate the progression to invasive phenotype. The goal is to harness the metabolism of cancer cells themselves to curb their metastatic potential. Using a specific ketone-body forming metabolite called 3-hydroxybutyrate (HB) and lowering of glucose concentration, the cell culture medium was modified to mimic ketogenic diet. The response of metastatic breast cancer cells and normal breast epithelial cells was then monitored. The two main measures of cellular response were: cell surface sialylation via fluorescence microscopy and fluorescence lifetime imaging (FLIM) of intracellular NADH as a marker of metabolic index. We are currently investigating the consequences of nutritionary interventions on metastatic potential of breast cancer cells by measuring their impact on cellular proliferation, clonogenicity and migratory abilities. We have observed that the main metabolite of ketogenic diet, HB, tends to protect metastatic breast cancer cells from DOX treatment when supplemented in a low-glucose (LG) medium. However, the extent of increase in cell surface sialylation is very modest in comparison to that in normal breast epithelial cells. This differential increase in sialylation implies that a ketogenic diet might significantly promote the proliferation of healthy cells over the proliferation of metastatic breast cancer cells. FLIM measurements indicate that the chemotherapeutic, Doxorubicin tends to induce an initial preference for glycolysis in invasive breast cancer cells. While an HB rich LG media by itself promotes glycolysis in these cells, it suppresses any further Doxorubicin induced onset of glycolysis pointing to a protective mechanism against the chemotherapeutic. Through this ongoing study, we aim to decipher the right set of nutrients that would “deshield” the breast cancer cells and lower their metastatic disposition. The ultimate goal of our research is to determine a paradigm of drugs and diet that metastatic breast cancer cells are most responsive to, and that would translate into a viable remedy for metastatic breast cancer with minimal side-effects. Citation Format: Mohini Kamra, Yuan-I Chen, Paula C Delgado, Tim Yeh, Amy Brock, Sapun H Parekh. Metabolic maneuvering of glycocalyx hypersialylation is a smart and healthy way of curbing metastatic potential in breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A037.

The carcinogenic capacity of arsenic in normal epithelial breast cells and double-positive breast cancer cells.

The carcinogenic effect of arsenic is a subject of controversy in relation to breast cancer. In our current research, we aimed to simulate the effects of chronic low-level arsenic exposure on breast cells by intoxicating MCF-10A and MCF-7 cells with 1 μM Arsenic trioxide (As2O3) for 3 weeks (3w) and 6 weeks (6w), respectively. We assessed the cellular responses to As2O3 through various assays, including confocal fluorescence microscopy, flow cytometry for cell cycle analysis, Transwell invasion assay, scratch assay, and colony assay. Additionally, we analyzed the mutation burden in all the exposed cells by using the next generation sequencing technology. Our findings indicate that As2O3 has a minor carcinogenic effect in normal cells, with no definitive evidence of malignant transformation observed after 6 weeks of exposure. In the case of breast cancer cells, As2O3 exhibits a dual effect, both inhibitory and stimulatory. It leads to reduced colony formation ability at 6 weeks, while enhancing the cells' ability for invasion. The mutations triggered by As2O3 exposure are distributed across genes with both tumor-suppressive and oncogenic functions. Five mutations are common to both cell lines, involving the following genes: Kinase Insert Domain Receptor (KDR) (c.798+54G>A), Colony Stimulating Factor 1 Receptor (CSF1R) (c.*37AC>C, c.*35C>TC), SWI/SNF-Related Matrix-Associated Actin-Dependent Regulator of Chromatin Subfamily B Member 1 (SMARCB1) (c.1119-41C>T), and Fms-like Tyrosine Kinase 3 (FLT3) (c.1310-3T>C). Additionally, Human Epidermal Growth Factor Receptor 4 (ERBB4/HER4) (c.421+58A>G) and Human Epidermal Growth Factor Receptor 2 (HER2/ERBB2) (c.2307+46A>G) mutations were exclusively found in MCF-10A cells exposed to As2O3. Furthermore, MCF-7 cells exhibited unique mutations in the KIT Proto-Oncogene (KIT) (c.1594G>A) and TP53 (c.215C>G). In summary, our study reveals that a 6-weeks exposure to arsenic has a limited carcinogenic effect in normal breast cells and a dual role in breast cancer cells.

The Garlic Compound, Diallyl Trisulfide, Attenuates Benzo[a]Pyrene-Induced Precancerous Effect through Its Antioxidant Effect, AhR Inhibition, and Increased DNA Repair in Human Breast Epithelial Cells.

Exposure to B[a]P, the most characterized polycyclic aromatic hydrocarbon, significantly increases breast cancer risk. Our lab has previously reported that diallyl trisulfide (DATS), a garlic organosulfur compound (OSC) with chemopreventive and cell cycle arrest properties, reduces lipid peroxides and DNA damage in normal breast epithelial (MCF-10A) cells. In this study, we evaluated the ability of DATS to block the B[a]P-induced initiation of carcinogenesis in MCF-10A cells by examining changes in proliferation, clonogenic formation, reactive oxygen species (ROS) formation, 8-hydroxy-2-deoxyguanosine (8-OHdG) levels, and protein expression of ARNT/HIF-1β, CYP1A1, and DNA POLβ. The study results indicate that B[a]P increased proliferation, clonogenic formation, ROS formation, and 8-OHdG levels, as well as increasing the protein expression of ARNT/HIF-1β and CYP1A1 compared to the control. Conversely, DATS/B[a]P co-treatment (CoTx) inhibited cell proliferation, clonogenic formation, ROS formation, and 8-OHdG levels compared to B[a]P alone. Treatment with DATS significantly inhibited (p < 0.0001) AhR expression, implicated in the development and progression of breast cancer. The CoTx also attenuated all the above-mentioned B[a]P-induced changes in protein expression. At the same time, it increased DNA POLβ protein expression, which indicates increased DNA repair, thus causing a chemopreventive effect. These results provide evidence for the chemopreventive effects of DATS in breast cancer prevention.

The Anticancer Effects of the Garlic Organosulfide Diallyl Trisulfide through the Attenuation of B[a]P-Induced Oxidative Stress, AhR Expression, and DNA Damage in Human Premalignant Breast Epithelial (MCF-10AT1) Cells.

Benzo[a]pyrene (B[a]P) is the most characterized polycyclic aromatic hydrocarbon associated with breast cancer. Our lab previously reported that the organosulfur compound (OSC), diallyl trisulfide (DATS), chemoprevention mechanism works through the induction of cell cycle arrest and a reduction in oxidative stress and DNA damage in normal breast epithelial cells. We hypothesize that DATS will inhibit B[a]P-induced cancer initiation in premalignant breast epithelial (MCF-10AT1) cells. In this study, we evaluated the ability of DATS to attenuate B[a]P-induced neoplastic transformation in MCF-10AT1 cells by measuring biological endpoints such as proliferation, clonogenicity, reactive oxygen species (ROS) formation, and 8-hydroxy-2-deoxyguanosine (8-OHdG) DNA damage levels, as well as DNA repair and antioxidant proteins. The results indicate that B[a]P induced proliferation, clonogenic formation, ROS formation, and 8-OHdG levels, as well as increasing AhR, ARNT/HIF-1β, and CYP1A1 protein expression compared with the control in MCF-10AT1 cells. B[a]P/DATS's co-treatment (CoTx) inhibited cell proliferation, clonogenic formation, ROS formation, AhR protein expression, and 8-OHdG levels compared with B[a]P alone and attenuated all the above-mentioned B[a]P-induced changes in protein expression, causing a chemopreventive effect. This study demonstrates, for the first time, that DATS prevents premalignant breast cells from undergoing B[a]P-induced neoplastic transformation, thus providing more evidence for its chemopreventive effects in breast cancer.

A New Role for Lactate in Breast Cancer

Lactate dehydrogenase (LDH) is an enzyme that plays a key role in cellular metabolism by converting pyruvate to lactate during anaerobic glycolysis. LDH has been implicated in various cancer types, including breast cancer, where increased LDH activity is associated with tumor growth, invasion, and resistance to therapy. LDH inhibitors are being investigated as potential therapeutic agents in cancer treatment, including breast cancer. Khajah and colleagues investigated the role of LDH inhibitors in both estrogen receptor (ER)+ and ER‑ breast cancer cell lines and in normal breast epithelial cells. A significant reduction in extracellular lactate level, cell proliferation, motility, and invasion was observed after treatment with LDH inhibitors. Subsequently, changes in the levels of vimentin, E‑cadherin, p38 MAPK, ERK1/2, and AKT were also observed. Cancer cell proliferation was preferentially blocked compared with normal epithelial cell inhibition by LDH inhibitors such as quercetin and lonidamine. These results further highlight the role of lactate in breast cancer progression and indicate the practical use of various commercially available LDH inhibitors as promising therapeutic agents to oppose the processes leading to cancer progression. While LDH inhibitors show promise, challenges include achieving selectivity for cancer cells and minimizing toxicity to normal tissues. The development of specific LDH inhibitors with favorable pharmacokinetic properties is an ongoing area of research. Mol Med Rep. 2024 Jan;29(1):12. doi: 10.3892/mmr.2023.1313.

Downregulation of MALAT1 in triple-negative breast cancer cells

BackgroundMALAT1 is one of the most abundant nuclear long non-coding RNAs, which has been found to be elevated in various types of cancers. However, conflicting reports on MALAT1 in breast cancer cell lines challenge understanding of MALAT1's involvement in breast cancer progression. AimMeasurement of normalized relative quantity (NRQ) of MALAT1 transcripts in cell lines representing triple-negative breast cancer (TNBC) and luminal breast cancer. Materials and methodsThe studies were performed using cell lines representing luminal breast cancer (T47D, MCF-7), TNBC (MDA-MB-468, CAL-51, MDA-MB-231), and MCF-10A cell line of normal breast epithelial cells. Total RNA was isolated from six independent cell cultures of each line, treated with DNase I, and used to synthesize complementary DNA, which was used in quantitative real-time PCR (qPCR) assays. Four MALAT1 fragments and reference genes CCSER2, ANKRD17, PUM1, GAPDH were amplified. ResultsGeometric means of the NRQ of MALAT1 in breast cancer cell lines had the shortest 95% confidence intervals when CCSER2 was used for normalization. MALAT1 major transcript levels thus estimated in TNBC cell lines were found to be statistically significantly reduced compared to levels in both MCF-10A cells and luminal breast cancer cell lines, while MALAT1 minority splice variants were found to be increased in almost all breast cancer cell lines. ConclusionCCSER2-normalized qPCR results indicate MALAT1 downregulation in cell lines representing the more aggressive breast cancer subtype compared to both the normal breast epithelial cell line and the estrogen receptor-positive breast cancer cell lines.

The effect of lactate dehydrogenase inhibitors on proliferation, motility and invasion of breast cancer cells invitro highlights a new role for lactate.

Lactate dehydrogenase (LDH) is being increasingly recognized as a major factor in the progression of breast cancer. It was previously shown that short interfering RNA‑mediated knockdown of either LDH‑A or ‑B isoform resulted in inhibition of cell motility due to reduced lactate levels in the extracellular environment. The aim of the present study was to determine the use of pharmacological LDH inhibitors to reduce aggressive behavior of breast cancer cells. The effect of LDH inhibitors was investigated in both estrogen receptor (ER)+ and ER‑ breast cancer cell lines and in normal breast epithelial cells. Cell proliferation, motility and invasion were measured using MTT, wound healing and cultrex assays, respectively. Changes in several key mediators of mitogenic signaling important in breast cancer cells were determined using western blotting. Treatment with various inhibitors reported to block LDH activity resulted in significant reduction in extracellular lactate level, cell proliferation, motility and invasion. This was associated with changes in the levels of vimentin, E‑cadherin, p38 MAPK, ERK1/2 and AKT. A couple of these inhibitors such as quercetin and lonidamine showed preferential inhibition of cancer cell proliferation compared with normal epithelial cell inhibition. These data extend initial findings, further underlining the importance of lactate as a major factor in breast cancer progression and indicate the practical use of various commercially available LDH inhibitors as promising therapeutic agents to oppose the processes leading to cancer progression.

USP10 promotes the progression of triple-negative breast cancer by enhancing the stability of TCF4 protein

Investigating the role of ubiquitin-specific peptidase 10 (USP10) in triple-negative breast cancer (TNBC). Analyzed USP10 expression levels in tumors using public databases. Detected USP10 mRNA and protein levels in cell lines. Examined USP10 expression in tumor tissues from breast cancer patients. Conducted USP10 knockdown experiments and analyzed changes in cell proliferation and metastasis. Confirmed protein–protein interactions with USP10 through mass spectrometry, Co-IP, and fluorescence experiments. Assessed impact of USP10 on transcription factor 4 (TCF4) ubiquitination and validated TCF4′s influence on TNBC cells. We initially identified a pronounced overexpression of USP10 across multiple tumor types, including TNBC. Subsequently, we observed a conspicuous upregulation of USP10 expression levels in breast cancer cell lines compared to normal breast epithelial cells. However, upon subsequent depletion of USP10 within cellular contexts, we noted a substantial attenuation of malignant proliferation and metastatic potential in TNBC cells. In subsequent experimental analyses, we elucidated the physical interaction between USP10 and the transcription factor TCF4, whereby USP10 facilitated the deubiquitination modification of TCF4, consequently promoting its protein stability and contributing to the initiation and progression of TNBC. Collectively, this study demonstrates that USP10 facilitated the deubiquitination modification of TCF4, consequently promoting its protein stability and contributing to the initiation and progression of TNBC.