Potential Of Breast Cancer Cells Research Articles

Abstract B031: Lean adipocyte-derived lipid decreases metastatic potential of breast cancer cells

Abstract Obesity rates are increasing globally, making it of critical importance to study obesity-linked diseases. Breast cancer is the most common cancer in women, and obesity is associated with both increased incidence and metastatic progression of breast cancer. Metastasis formation depends on a cancer cell leaving the primary site, migrating through the bloodstream, and establishing new growth in a distant site. Both metastasizing cell-intrinsic and metastatic site-dependent factors can help to regulate the effectiveness of this process; however, it is unknown whether obesity-linked metastasis depends on changes to the metastasizing cell and/or changes to the distant metastatic site. This study aims to characterize how the lean versus obese tumor microenvironment impacts breast cancer metastasis and identify adipocyte-derived molecules that modulate the metastatic potential of breast cancer cells. We and others have shown that obesity leads to increased metastasis formation in mouse models of orthotopic breast cancer. However, my preliminary data show that an obese metastatic site is not sufficient to increase metastasis formation in an intravenous injection model of metastasis, emphasizing the importance of the primary tumor microenvironment in programming breast cancer cells for metastasis. Cancer cell stemness helps to mediate metastasis formation and can be assessed in vitro with the mammosphere assay. Importantly, my data demonstrate that a secreted lipid from lean, but not obese, adipocytes potently inhibits the ability of breast cancer cells to form mammospheres. Furthermore, this inhibition induces a G1/S cell cycle arrest. Together, these data suggest that lean adipocytes in the breast cancer tumor microenvironment act in a paracrine manner to decrease the metastatic potential of breast cancer cells. My work demonstrates the role of adipocytes in the regulation of metastasis in breast cancer and provides important insight into the role of the tumor microenvironment in mediating metastasis in lean versus obese conditions. Citation Format: Abigail E. Jackson, Keren I Hilgendorf. Lean adipocyte-derived lipid decreases metastatic potential of breast cancer cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B031.

PFDN5 plays a dual role in breast cancer and regulates tumor immune microenvironment: Insights from integrated bioinformatics analysis and experimental validation

BackgroundAlthough the prognosis for patients with breast cancer has improved, breast cancer remains the leading cause of death for women worldwide. Prefoldin 5 (PFDN5), as a subunit of the prefoldin complex, plays a vital role in aiding the correct folding of newly synthesized proteins. However, the exact impact of PFDN5 on breast cancer development and its prognostic implications remain unclear. MethodsWe conducted bioinformatics analysis to investigate the correlation between PFDN5 and patient survival, as well as various clinicopathological characteristics in breast cancer. Additionally, various assays were employed to validate the biological functions of PFDN5 in breast cancer. Finally, RNA sequencing (RNA-seq) was utilized to investigate the molecular mechanisms associated with PFDN5. ResultsCompared to normal tissues, PFDN5 exhibited lower expression levels in breast cancer tissues, and lower expression of PFDN5 is associated with poorer prognosis. PFDN5 led to G2/M phase arrest in the cell cycle and reduced proliferative potential in breast cancer cells. However, PFDN5 also promoted migration and invasion of breast cancer cells. Also, RNA-seq analysis revealed an involvement of PFDN5 in the cell cycle and TGF-β signaling pathway. Furthermore, PFDN5 had a significant impact on tumor immune microenvironment by promoting macrophage polarization towards the M1 phenotype and exhibited a positive correlation with CD8+ T cell infiltration levels. ConclusionsPFDN5 plays a dual role in breast cancer and serves as a key factor in tumor immune microenvironment. Therefore, PFDN5 holds promise as a valuable biomarker for predicting both metastatic and prognosis in breast cancer.

NUAKs promote mTOR/c-Myc-induced glucose and glutamine reprogramming for cell growth and metastasis in breast cancer cells

Breast cancer progression and metastasis are closely connected to changes in glucose and glutamine metabolism. While Novel (nua) kinase family 1 (NUAK1) and Novel (nua) kinase family 2 (NUAK2), which are two members of the AMPK-related kinases, have been associated with breast tumorigenesis, their role in the metabolic reprogramming that occurs during breast cancer progression remains unclear. Our research uncovers that NUAKs expression is significantly higher in breast cancer tissues and cell lines, and it is positively related to glycolysis, the pentose phosphate pathway (PPP), glutamine metabolism, and a poor prognosis for breast cancer patients. We show that NUAKs significantly increase metabolic reprogramming, including aerobic glycolysis, PPP, and glutamine metabolism in triple negative breast cancer subtypes but only induce aerobic glycolysis and PPP in luminal breast cancer subtypes to meet the anabolic demands of rapidly dividing breast cancer cells. In contrast, the depletion of NUAKs has the opposite effect. Mechanistic insights reveal that NUAKs activate mammalian target of rapamycin (mTOR) signaling, which in turn upregulates the c-Myc transcription factor, a crucial regulator of glucose and glutamine metabolic gene expression. Moreover, we demonstrate that NUAKs enhance mTOR/c-Myc signaling pathways, leading to increased glucose and glutamine reprogramming, which supports rapid cell proliferation and metastatic potential in breast cancer cells. Importantly, pretreating breast cancer cells with mTOR inhibitors blocked the metabolic reprogramming and tumor-promoting effect of NUAK1/2. Therefore, targeting NUAKs may represent a novel therapeutic strategy for the treatment of breast cancer.

Microwave-assisted green synthesis of silver nanoparticles using Mitragyna parvifolia bark extract and their biological activities: an economical and environment-friendly approach

ABSTRACT Nowadays, nanotechnology is extensively employed in the medical profession. In this study, we used the aqueous extract of the bark of Mitragyna parvifolia to synthesize silver nanoparticles (AgNPs) by microwave-assisted green synthesis. The synthesis of AgNPs was confirmed by visual color change to brown color and characteristic surface plasmon resonance peak at 432 nm. The hydrodynamic diameter of the AgNPs was 171.81 nm having a zeta potential of −24.14 mV; Fourier Transmission Infrared Spectroscopy confirmed the presence of different functional groups on the NP surface; Scanning Electron Microscope and High-Resolution Transmission Electron Microscopy indicated predominantly circular shape of nanoparticle; Selected Area Electron Diffraction and X-ray Diffraction analyses determined the crystalline structure of AgNPs. Energy-Dispersive X-ray indicated the elemental composition and formation of AgNPs. The AgNPs were screened at different concentrations for antioxidant activity, antimicrobial, and anticancer potential in breast cancer cells (MCF-7 and MDA-MB-231). The AgNPs exhibited remarkable antioxidant, antimicrobial, and anticancer activities. The sedative and antinociceptive activities were also tested on Swiss albino mice, which showed mild sedative and very potent antinociceptive activity. However, detailed mechanistic studies are warranted in the future for clinical application of the AgNPs as a biologically active agent as well as a carrier for drug delivery.

A feasible strategy for fabricating pH-responsive SN-38 loaded europium metal-organic framework delivery for promising treatment for breast cancer

One of the significant challenges in cancer therapy is the development of biocompatible drug delivery systems (DDS) with enhanced penetration retention effect (EPR) and precision targeting. The current study aimed to establish a one-pot synthesis of 7-ethyl-10-hydroxy camptothecin (SN-38) loaded europium-metal-organic framework (SN-38@Eu-MOF) and evaluate its anticancer potential against breast cancer. High drug loading efficiency (85.3 ± 0.02 % in 48 h at pH 5.0) and pH-sensitive regulated release of drug SN-38 were two characteristics of the fabricated Eu-MOFs. According to material characterization, the material's surface formed a monodispersed homogeneous rod-shaped structure adorned with SN-38, with an average particle size of 72 ± 0.23 nm. Results of anticancer assays demonstrated that when compared to SN-38 alone, SN-38@Eu-MOFs showed improved anticancer potential in breast cancer cells (MCF-7 and MDA-MB-231). According to the mechanism of anticancer potential, SN-38@Eu-MOFs increased reactive oxygen species (ROS) levels and caused mitochondrial malfunction and DNA damage, which resulted in apoptosis. The study's findings indicate that SN-38@Eu-MOFs is a potentially biocompatible nanomaterial for drug delivery systems for treating breast cancer and other pharmaceutical applications.

The role of RRS1 in breast cancer cells metastasis and AEG-1/AKT/c-Myc signaling pathway.

Breast cancer is the most common malignant tumor in women. Recurrence, metastasis, and chemotherapy resistance are the main causes of death in breast cancer patients. The inhibition of breast cancer metastasis is of great significance for prolonging its survival. Ribosome biogenesis regulatory protein homolog (RRS1) is overexpressed in breast cancer tissues and is involved in regulating the carcinogenic process of breast cancer cells. However, the exact signaling pathway and molecular mechanism of RRS1 promoting breast cancer metastasis are not fully understood. Hence, the primary objective of our study is to investigate the correlation between RRS1 and breast cancer metastasis. Bioinformatic analysis was used to identify the expression levels and prognostic significance of RRS1 in breast cancer. Lenti-sh RRS1 lentivirus was constructed and employed to downregulate the RRS1 expression in MDA-MB-231 and BT549 cells, which had a high-level expression of RRS1. Subsequently, we assessed the impact of RRS1 downregulation on the proliferation, migration, and invasion of breast cancer cells using CCK-8, apoptosis, and cell cycle by flow cytometry, wound healing test, Transwell migration, and invasion experiments. Moreover, we utilized an in vivo imaging system to examine the metastatic potential of breast cancer cells after RRS1 knockdown. Picrate staining and hematoxylin-eosin staining were employed to evaluate the presence of metastatic lesions. To gain a deeper understanding of the molecular mechanism, we conducted co-immunoprecipitation and western blot. The significant overexpression of RRS1 in breast cancer indicates a worse prognosis, as determined through TCGA databases (p<0.01). Additionally, RRS1 exhibits upregulation in breast cancer (p<0.001), which is tightly linked to the occurrence of lymph node metastasis (p<0.001). Clinical breast cancer tissues and breast cancer cell lines also demonstrated a noteworthy upregulation of RRS1 (p<0.05). Loss-of-function experiment illustrated that the inhibiting of RRS1 expression reduced the rapid proliferation capacity of MDA-MB-231 and BT549 cells and hindered their migration and invasion capabilities (p<0.05). Importantly, the suppression of RRS1 significantly diminished lung metastasis in Balb/c nude mice that were injected with MDA-MB-231 cells (p<0.01). Mechanistically, RRS1 may interact with the AEG-1 to modulate the phosphorylation of AKT at T308 and S473, consequently impeding the activity of c-Myc (p<0.05). To conclude, RRS1 functions as a potential oncogene in breast cancer by leveraging the AEG-1/AKT/c-Myc signaling.

Arnicolide D Inhibits Oxidative Stress-induced Breast Cancer Cell Growth and Invasion through Apoptosis, Ferroptosis, and Parthanatos.

Breast cancer is the most common malignant tumor in women, and its pathogenesis is very complicated. More and more studies have found that Traditional Chinese Medicine plays an important role in tumor prevention. To investigate the mechanism of arnicolide D isolated from Centipeda minima in breast cancer. Cell Counting Kit-8 (CCK-8), western blot, RT-qPCR, ELISA, flow cytometry, and Transwell were used to detect the effect of arnicolide D on the biological function of breast cancer cells. Arnicolide D promoted reactive oxygen species (ROS) production and induced a decrease in mitochondrial membrane potential in breast cancer cells, thereby inhibiting cell viability and increasing lactate dehydrogenase (LDH) release. Arnicolide D activated the classical apoptosis pathway to induce cell apoptosis; it significantly promoted PARP-1 expression, enhanced the nuclear translocation of apoptosis-inducing factor (AIF), and reduced the expression of AIF in mitochondria, indicating that it can induce the occurrence of parthanatos in a ROS dependent manner. In addition, arnicolide D down-regulated glutathione peroxidase 4 (GPX4) expression and increased the accumulation of Fe2+ and malondialdehyde (MDA), thereby activating ferroptosis. Apoptosis inhibitor, ferroptosis inhibitor, PARP inhibitor, PARP-1 siRNA, AIF siRNA and GPX4 overexpression vector significantly attenuated the inhibitory effect of arnicolide D on cell viability and reduced LDH release, which indicates that arnicolide D inhibits breast cancer cell growth by inducing apoptosis, parthanatos and ferroptosis. Arnicolide D also reduced breast cancer cell invasion and inhibited the expression of matrix metallopeptidase (MMP)-2 and MMP-9. Arnicolide D can activate a variety of cell death modes by inducing oxidative stress, thereby inhibiting the growth and invasion of breast cancer cells, indicating that arnicolide D has a good anti-tumor effect.

Polyphyllin VII Promotes Apoptosis in Breast Cancer by Inhibiting MAPK/ERK Signaling Pathway through Downregulation of SOS1.

Polyphyllin VII is a biologically active herbal monomer extracted from the traditional Chinese herbal medicine Chonglou. Many studies have demonstrated the anticancer activity of polyphyllin VII against various types of cancers, such as colon, liver, and lung cancer, but its effect on breast cancer has not been elucidated. In this study, we demonstrate that polyphyllin VII inhibited proliferation, increased production of intracellular reactive oxygen species, and decreased mitochondrial membrane potential in breast cancer cells. Notably, polyphyllin VII also induced apoptosis via the mitochondrial pathway. Transcriptome sequencing was used to analyze the targets of PPVII in regulating breast cancer cells. Mechanistic studies showed that polyphyllin VII downregulated Son of Sevenless1 (SOS1) and inhibited the MAPK/ERK pathway. Furthermore, PPVII exerted strong antitumor effects in vivo in nude mice injected with breast cancer cells. Our results suggest that PPVII may promote apoptosis through regulating the SOS1/MAPK/ERK pathway, making it a possible candidate target for the treatment of breast cancer.

Abstract PO4-24-03: Adiponectin modulates cell polarity according to ERα expression in breast cancer cells

Abstract INTRODUCTION: Tumor microenvironment (TME) is a complex and heterogeneous network, consisting of stromal and immune cells, embedded with cytokines, growth factors, soluble receptors, and exosomes. Mutual and dynamic crosstalk among cancer, stromal and immune cells predispose cancer cells to metastasis. Particularly, the main component of breast TME is represented by adipocytes, which secretory activity is compromised in obesity. Indeed, hypertrophic, and hyperplastic adipocytes undergo to a modified cytokines secretory pattern, characterized by a reduced secretion of Adiponectin, now recognized as a crucial factor in the pathogenesis of breast cancer. LKB1 is one the most important effector of Adiponectin transduction pathways, and recently it has been demonstrated its role as ERα coactivator in promoting breast cancer cell growth. Moreover, LKB1 is a key regulator in controlling cell polarity along with E-cadherin and Cdc42. Cell polarity has been considered essential for mammary epithelial integrity, and disruption of individual polarity proteins is sufficient to induce tumor progression. The aim of the present study was to investigate the effects of adiponectin on the regulation and alteration of cell polarity, essential for breast cancer cell growth and invasiveness. METHODS: Immunofluorescence analyses were done to detect protein localization in breast cancer cells. Protein expression was performed by immunoblotting in the presence or absence of ERa and LKB1 siRNA. LKB1/Cdc42 and LKB1/E-cadherin interaction was detected by Proximity ligation and immunoprecipitation assay respectively. Wound healing assay was carried out to evaluate GM130 orientation. Growth and metastatic potential of breast cancer cells were assessed in vivo by orthotopic model and metastatic study respectively. RESULTS: In Adiponectin-treated MCF-7 cells LKB1 is mostly present in the nucleus, recruited as ERa-coactivator, while in BT20 cells LKB1 is localized with E-cadherin in the cytosolic compartment, suggesting its role in polarity. Our results showed that in MCF-7 cells Adiponectin increases Cdc42 level depending on LKB1 expression. Moreover, Cdc42 and LKB1 interact and colocalized in the nucleus of Adiponectin-treated MCF-7 cells. On the contrary, LKB1 and Cdc42 are located with E-cadherin in the cytosol of BT20 cells, addressing their cytosolic cooperation in maintaining cell polarity, as confirmed by an increased Cdc42 activity. Immunofluorescence analyses demonstrated a lack of Golgi realignment toward the front of the leading edge in MCF-7 cells treated with Adiponectin, not observed in BT20 cells. All this suggest a loss of cell polarity organization that predispose to cancer growth and progression, as confirmed by an increased tumor volume mass and a major number of lung metastatic events upon tail vein injection of adiponectin-treated ERα-positive cells with respect to untreated cells. CONCLUSION: In ERα-positive breast cancer cells Adiponectin modulates the expression of the main proteins involved in cell polarity, leading to increased growth, progression, and distant metastasis. Citation Format: Giuseppina Daniela Naimo, Martina Forestiero, Alessandro Paolì, Luca Gelsomino, Rocco Malivindi, Adele Elisabetta Leonetti, Francesca Giordano, Maria Luisa Panno, Loredana Mauro, Sebastiano Andò. Adiponectin modulates cell polarity according to ERα expression in breast cancer cells [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 PO4-24-03.

Abstract PO2-24-09: Prolonged Effects of Hypoxia in Luminal Breast Cancer Cells Promotes an Aggressive Phenotype

Abstract Aidan Moriarty Mentor: Dr. Min Yu Hypoxic Memory in Breast Cancer Cells Metastasis is the main cause of tumor-related mortality in breast cancer patients. Improving our understanding of the metastatic cascade is imperative to improve patient outcomes. Prior research indicates cancer cells with increased metastatic potential have acquired adaptive properties induced by factors in the tumor microenvironment (TME), including hypoxia. However, previous hypoxia research has been primarily focused on signaling changes that occur when cancer cells are in the hypoxic TME and does not account for long-term changes that may occur once cancer cells re-enter normoxia, such as when they metastasize. Our research in patient-derived circulating tumor cell (CTCs) lines and breast cancer cell lines has indicated that certain transcriptional changes are maintained after cells leave hypoxia, and that these post-hypoxic CTCs maintain a higher metastatic potential compared to cells not exposed to hypoxia. Taking an un-biased approach to define changes in hypoxia-exposed breast cancer cells upon reoxygenation, we performed bulk RNA-Seq on luminal breast cancer cell lines in various oxygen conditions over time. We found that a subset of genes that were differentially expressed in hypoxic conditions were maintained as up- or down-regulated after cells were reoxygenated, indicating a “hypoxic memory”. Additionally, a subset of genes were differentially expressed in the post-hypoxic cells compared to normoxia controls, demonstrating novel and prolonged gene expression changes induced by hypoxia exposure. We performed functional assays and observed that this post-hypoxic state is associated with a more aggressive phenotype in vitro. Current research in our lab is on-going to understand the epigenetic changes that may be responsible for this hypoxic memory to further understand the consequences of hypoxic memory in CTC biology and metastasis. Finally, we have designed a dual-reporter hypoxia-tracing system to validate our findings in vivo. This research will improve our understanding of the prolonged influence of hypoxia on metastatic potential in breast cancer cells and has the potential to inform future therapeutic strategies. Citation Format: Aidan Moriarty, Oihana Iriondo, Desirae Mecenas, Yilin Li, Yonatan Amzaleg, Remi Klotz, Min Yu. Prolonged Effects of Hypoxia in Luminal Breast Cancer Cells Promotes an Aggressive Phenotype [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 PO2-24-09.

Involvement of ferroptosis in eribulin-induced cytotoxicity in ovarian clear cell carcinoma

Ovarian clear cell carcinoma (OCCC) is a unique clinicopathological subtype of epithelial ovarian cancer that is resistant to standard chemotherapy. Eribulin, a microtubule dynamics inhibitor of halichondrin class, has unique effects in the cancer microenvironment such as induction of epithelization and reduction in metastatic potential in breast cancer cells; however, nothing is known about the effect of eribulin and the detailed mechanisms in OCCC. This study aimed to investigate the involvement of ferroptosis and its mechanism in the antitumor activity of eribulin in OCCC cells and a mouse xenograft model. We found that eribulin-induced cell death was reduced by ferroptosis inhibitors; deferoxamine, an iron chelator and ferrostatin-1, a lipid peroxidation inhibitor. Eribulin increased the levels of intracellular iron, reactive oxygen species (ROS), and lipid peroxides, and increased the mitochondrial membrane potential. Eribulin downregulated the expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), the mitochondrial enzyme dihydroorotate dehydrogenase (DHODH), and superoxide dismutase (SOD) activity. The combination of eribulin and ML210, a glutathione peroxidase 4-inhibiting ferroptosis inducer, had a synergistic effect on ferroptosis. Taken together, our findings show firstly that eribulin triggers ferroptosis in OCCC and this effect occurs via the suppression of the Nrf2–HO-1 signaling pathway, SOD activity and the promotion of lipid peroxidation. These findings suggest that eribulin-induced ferroptosis is associated with its anti-tumor effect and also could be a potential therapeutic target in OCCC.

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.

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.

A home-based lifestyle intervention program reduces the tumorigenic potential of triple-negative breast cancer cells

Translational research for the evaluation of physical activity habits and lifestyle modifications based on nutrition and exercise has recently gained attention. In this study, we evaluated the effects of serum samples obtained before and after a 12-week home-based lifestyle intervention based on nutrition and exercise in breast cancer survivors in terms of modulation of the tumorigenic potential of breast cancer cells. The home-based lifestyle intervention proposed in this work consisted of educational counselling on exercise and nutritional behaviors and in 12 weeks of structured home-based exercise. Triple-negative breast cancer cell line MDA-MB-231 was cultured in semi-solid medium (3D culture) with sera collected before (PRE) and after (POST) the lifestyle intervention program. Spheroid formation was evaluated by counting cell colonies after 3 weeks of incubation. Results show a slight but significant reduction of spheroid formation induced by serum collected POST in comparison to those obtained PRE. Moreover, statistical analyses aimed to find physiologic and metabolic parameters associated with 3D cell proliferation revealed the proliferative inducer IGF-1 as the only predictor of cell tumorigenic potential. These results highlight the importance of lifestyle changes for cancer progression control in a tertiary prevention context. Translational research could offer a useful tool to identify metabolic and physiological changes induced by exercise and nutritional behaviors associated with cancer progression and recurrence risk.

Transcriptome-based identification of key actin-binding proteins associated with high metastatic potential in breast cancer.

Actin-binding proteins (ABPs) are essential for the regulation of morphological plasticity required for tumor cells to metastasize. The aim of this study was to perform an unbiased bioinformatic approach to identify the key ABPs significantly associated with the metastatic potential of breast cancer cells. Microarray data from 181 primary breast cancer samples from our hospital were used, and all genes belonging to the Gene Ontology term actin cytoskeleton organization were obtained from QuickGO. Association with metastasis-free survival probability was tested using Cox proportional hazards regression, and pairwise co-expression was tested by Pearson correlations. Differential expression between different subgroups was analyzed using Wilcoxon tests for dichotomous traits and Kruskal-Wallis tests for categorical traits. Validation was performed using four publicly available breast cancer datasets. ARHGAP25 was significantly associated with a low metastatic potential, and CFL1, TMSB15A, and ACTL8 were significantly associated with a high metastatic potential. A significantly higher expression of CFL1, TMSB15A, and ACTL8 mRNA was found in the more aggressive Her2-positive and triple-negative subtypes as well as in ER-negative samples. Also, these genes were co-expressed in the same tumors. However, only mRNA levels of CFL1 were increased in pN1 compared to pN0 patients. External validation revealed that CFL1 and TMSB15A had significant associations with consistent hazard ratios in two breast cancer cohorts, and among these, CFL1 exhibited the highest hazard ratios. CFL1 showed the strongest correlation with the metastatic potential of breast tumors. Thus, targeted inhibition of CFL1 might be a promising approach to treat malignant breast cancer cells.

Hypoxia-induced epigenetic regulation of breast cancer progression and the tumour microenvironment.

The events that control breast cancer progression and metastasis are complex and intertwined. Hypoxia plays a key role both in oncogenic transformation and in fueling the metastatic potential of breast cancer cells. Here we review the impact of hypoxia on epigenetic regulation of breast cancer, by interfering with multiple aspects of the tumour microenvironment. The co-dependent relationship between oxygen depletion and metabolic shift to aerobic glycolysis impacts on a range of enzymes and metabolites available in the cell, promoting posttranslational modifications of histones and chromatin, and changing the gene expression landscape to facilitate tumour development. Hormone signalling, particularly through ERα, is also tightly regulated by hypoxic exposure, with HIF-1α expression being a prognostic marker for therapeutic resistance in ER+ breast cancers. This highlights the strong need to understand the hypoxia-endocrine signalling axis and exploit it as a therapeutic target. Furthermore, hypoxia has been shown to enhance metastasis in TNBC cells, as well as promoting resistance to taxanes, radiotherapy and even immunotherapy through microRNA regulation and changes in histone packaging. Finally, several other mediators of the hypoxic response are discussed. We highlight a link between ionic dysregulation and hypoxia signalling, indicating a potential connection between HIF-1α and tumoural Na+ accumulation which would be worth further exploration; we present the role of Ca2+ in mediating hypoxic adaptation via chromatin remodelling, transcription factor recruitment and changes in signalling pathways; and we briefly summarise some of the findings regarding vesicle secretion and paracrine induced epigenetic reprogramming upon hypoxic exposure in breast cancer. By summarising these observations, this article highlights the heterogeneity of breast cancers, presenting a series of pathways with potential for therapeutic applications.

Exploration of SERPINA family functions and prognostic value in breast cancer based on transcriptome and in vitro analysis.

Breast cancer poses a significant risk to women worldwide, yet specific role of SERPINA gene family in breast cancer remains unclarified. Data were collected from online databases. SERPINA family gene expression was presented, and prognosis value was evaluated. Multi-omics methods were employed to explore the SERPINA-related biological processes, followed by comprehensive analyses of their roles in breast cancer. Single-cell data were analyzed to characterize the SERPINA family gene expression in different cell clusters. We selected SERPINA5 as the target gene. Via pan-cancer analysis, SERPINA5 was also investigated in various cancers. The experimental validation was conducted in MDA-MB-231 cell line eventually. SERPINA family showed differential expression in breast cancer, which were mainly expressed in myeloid cells, epithelial cells, and dendritic cells. SERPINA5 expression was upregulated in breast cancer, which was associated with a better prognosis. Immune infiltration illustrated the positive correlativity between SERPINA5 intensity and eosinophilic recruitment. Pan-cancer analysis indicated the function of SERPINA5 as a potential biomarker in other cancers. Finally, experimental validation demonstrated that SERPINA5 contributes to lower invasion and metastatic potential of breast cancer cells. With bioinformatics analysis, the significant role SERPINA family genes functioned in breast cancer was comprehensively explored, with SERPINA5 emerging as a key gene in suppressing breast cancer progression.

Novel STAT3 Inhibitor Exerts Anti-breast Cancer Effects Both In vitro and In vivo

Background: Breast cancer is one of the most common malignant tumors. Signal transduction and activators of transcription 3 (STAT3) have been demonstrated to play important roles in breast cancer. However, no direct inhibitor of STAT3 has been approved by the FDA for clinical use. Objective: LL1 is a newly designed STAT3 inhibitor that we identified. In this study, we investigated the cytotoxic effect of LL1 on breast cancer cells and its potential mechanisms. Methods: Colony formation and CCK-8 assay were used to detect the anti-proliferation of LL1. Flow cytometry was used to evaluate mitochondrial membrane potential and apoptosis in breast cancer cells following the treatment of LL1. The expression of proteins was analyzed using western blot, and the invasion and migration of cells were analyzed by wound healing assay and transwell assay. The xenograft model was used to evaluate the anti-cancer effect of LL1 in vivo. Results: LL1 selectively inhibited the expression of p-STAT3, but had no obvious effect on total STAT3. LL1 exhibited great potential in suppressing the proliferation of breast cancer in vitro. Moreover, LL1 induces apoptosis and the decrease of mitochondrial membrane potential in breast cancer cells. LL1 can also inhibit the invasion and migration of breast cancer cells. These cell biology changes may be induced via the regulation of Bcl-2, Bax, cleaved-caspase3, Survivn, Mmp-2, Mmp-9, N-cadherin, E-cadherin, vimentin, c-myc and cyclin D1 by LL1. In addition, LL1 exhibited great antitumor activity in vivo. Conclusion: Our study suggested that LL1 can be considered a promising candidate for the treatment of breast cancer.

Heat-shock protein 90α protects NME1 against degradation and suppresses metastasis of breast cancer.

NME1 has been exploited as a potential translational target for decades. Substantial efforts have been made to upregulate the expression of NME1 and restore its anti-metastasis function in metastatic cancer. Cycloheximide (CHX) chase assay was used to measure the steady-state protein stability of NME1 and HSP90α. The NME1-associating proteins were identified by immunoprecipitation combined with mass spectrometric analysis. Gene knockdown and overexpression were employed to examine the impact of HSP90AA1 on intracellular NME1 degradation. The motility and invasiveness of breast cancer cells were examined in vitro using wound healing and transwell invasion assays. The orthotopic spontaneous metastasis and intra-venous experimental metastasis assays were used to test the formation of metastasis in vivo, respectively. HSP90α interacts with NME1 and increases NME1 lifetime by impeding its ubiquitin-proteasome-mediated degradation. HSP90α overexpression significantly inhibits the metastatic potential of breast cancer cells in vitro and in vivo. A novel cell-permeable peptide, OPT22 successfully mimics the HSP90α function and prolongs the life span of endogenous NME1, resulting in reduced metastasis of breast cancer. These results not only reveal a new mechanism of NME1 degradation but also pave the way for the development of new and effective approaches to metastatic cancer therapy.

Targeted demethylation of the CDO1 promoter based on CRISPR system inhibits the malignant potential of breast cancer cells.

Cysteine dioxygenase 1 (CDO1) is frequently methylated, and its expression is decreased in many human cancers including breast cancer (BC). However, the functional and mechanistic aspects of CDO1 inactivation in BC are poorly understood, and the diagnostic significance of serum CDO1 methylation remains unclear. We performed bioinformatics analysis of publicly available databases and employed MassARRAY EpiTYPER methylation sequencing technology to identify differentially methylated sites in the CDO1 promoter of BC tissues compared to normal adjacent tissues (NATs). Subsequently, we developed a MethyLight assay using specific primers and probes for these CpG sites to detect the percentage of methylated reference (PMR) of the CDO1 promoter. Furthermore, both LentiCRISPR/dCas9-Tet1CD-based CDO1-targeted demethylation system and CDO1 overexpression strategy were utilized to detect the function and underlying mechanism of CDO1 in BC. Finally, the early diagnostic value of CDO1 as a methylation biomarker in BC serum was evaluated. CDO1 promoter was hypermethylated in BC tissues, which was related to poor prognosis (p<.05). The CRISPR/dCas9-based targeted demethylation system significantly reduced the PMR of CDO1 promotor and increased CDO1 expression in BC cells. Consequently, this leads to suppression of cell proliferation, migration and invasion. Additionally, we found that CDO1 exerted a tumour suppressor effect by inhibiting the cell cycle, promoting cell apoptosis and ferroptosis. Furthermore, we employed the MethyLight to detect CDO1 PMR in BC serum, and we discovered that serum CDO1 methylation was an effective non-invasive biomarker for early diagnosis of BC. CDO1 is hypermethylated and acts as a tumour suppressor gene in BC. Epigenetic editing of abnormal CDO1 methylation could have a crucial role in the clinical treatment and prognosis of BC. Additionally, serum CDO1 methylation holds promise as a valuable biomarker for the early diagnosis and management of BC.