Breast Cancer Stem Cells Research Articles

Breast cancer stem cells origins and the memory stem cells: clinical significance of biomarkers and the active therapeutic approaches

Despite recent improvements in diagnosis and treatment, breast cancer (BC) remains one of the leading causes of cancer-related deaths among women. In this complex disease, breast cancer stem cells (BCSCs) are a small but significant subset of different cancer cells with the ability to proliferate and self-renew. According to an increasing amount of studies, BCSCs are essential for breast cancer metastasis, drug resistance, and recurrence. Due to its diverse nature, BC includes numerous subtypes, each of which displays unique BCSC types and concentrations that are connected to different therapy outcomes and outcomes. Despite significant advancements in the treatment of early-stage breast cancer, there are still few effective therapy approaches for metastatic BC. The development, progression, and dissemination of BC are largely attributed to cancer stem-like cells (CSCs), which are characterized by their exceptional adaptability and self-renewal ability. An overview of the development of BCSCs, their biomarkers, clinical significance, and the mechanisms behind their behavior is the goal of the current study. The active therapy strategies being employed to address BCSCs will also be examined.

NSDHL contributes to breast cancer stem-like cell maintenance and tumor-initiating capacity through TGF-β/Smad signaling pathway in MCF-7 tumor spheroid

BackgroundNAD(P)-dependent steroid dehydrogenase-like protein (NSDHL), which is involved in breast tumor growth and metastasis, has been implicated in the maintenance of cancer stem cells. However, its role in regulating breast cancer stem-like cells (BCSCs) remains unclear. We have previously reported the clinical significance of NSDHL in patients with estrogen receptor-positive (ER +) breast cancer. This study aimed to elucidate the molecular mechanisms by which NSDHL regulates the capacity of BCSCs in the ER + human breast cancer cell line, MCF-7.MethodsNSDHL knockdown suppressed tumor spheroid formation in MCF-7 human breast cancer cells grown on ultralow-attachment plates. RNA sequencing revealed that NSDHL knockdown induced widespread transcriptional changes in the MCF-7 spheroids. TGF-β signaling pathway was the most significantly enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway (fold change ≥ 2, P ≤ 0.05) identified in NSDHL-knockdown MCF-7 spheroids compared with the control. In orthotopic tumor models injected with NSDHL-knockdown MCF-7 spheroids, tumor initiation and growth were strongly suppressed compared with those in the control.ResultsBCSC populations with CD44+/CD24- and CD49f+/EpCAM + phenotypes and high ALDH activity were decreased in NSDHL-knockdown MCF-7 spheroids and xenograft tumors relative to controls, along with decreased secretion of TGF-β1 and 3, phosphorylation of Smad2/3, and expression of SOX2. In RNA-sequencing data from The (TCGA) database, a positive correlation between the expression of NSDHL and SOX2 was found in luminal-type breast cancer specimens (n = 998). Our findings revealed that NSDHL plays an important role in maintaining the BCSC population and tumor-initiating capacity of ER-positive MCF-7 spheroids, suggesting that NSDHL is an attractive therapeutic target for eliminating BCSCs, thus preventing breast cancer initiation and progression.ConclusionsOur findings suggest that NSDHL regulates the BCSC/tumor-initiating cell population in MCF-7 spheroids and xenograft tumors.

ZNF480 Accelerates Chemotherapy Resistance in Breast Cancer by Competing With TRIM28 and Stabilizing LSD1 to Upregulate the AKT-GSK3β-Snail Pathway.

Zinc finger protein 480 (ZNF480) may interact with lysine-specific demethylase 1 (LSD1), which is highly expressed in many malignant tumors; however, ZNF480 expression has not previously been investigated in breast cancer. Therefore, we explored the expression and molecular mechanisms of ZNF480 in breast cancer. According to public databases and immunohistochemical staining analysis, ZNF480 is highly expressed in the tissue of patients with breast cancer, and ZNF480 expression is positively correlated with advanced TNM stage (p = 0.036), lymph node metastasis (p = 0.012), and poor prognosis (p = 0.005). ZNF480 overexpression enhances breast cancer cell proliferation, migration, and stemness by activating AKT-GSK3β-Snail signaling both in vitro and in vivo. Moreover, ZNF480 binds to LSD1 through its KRAB domain, thereby activating AKT signaling. Mass spectrometry and co-immunoprecipitation revealed that ZNF480 abrogates ubiquitination degradation and subsequently stabilizes LSD1 through competitive binding with TRIM28. Ipragliflozin was identified as a small-molecule inhibitor of ZNF480 and LSD1 interaction that may block breast cancer progression. Moreover, ZNF480 expression was significantly higher in treatment-resistant patients than in treatment-sensitive patients. Thus, ipragliflozin may neutralize neoadjuvant chemotherapy resistance induced by ZNF480 overexpression. Overall, elevated ZNF480 expression is positively associated with poor patient outcomes. Mechanistically, ZNF480 accelerates proliferation and neoadjuvant chemotherapy resistance in breast cancer cells via the AKT-GSK3β-Snail pathway by interacting with and stabilizing LSD1 in a competitive manner within TRIM28. This research has implications for developing targeted drugs against chemotherapy resistance in breast cancer.

GD2 in Breast Cancer: A Potential Biomarker and Therapeutic Target.

Expression of disialoganglioside GD2 in normal tissues is primarily limited to the central nervous system, peripheral sensory nerve fibers, dermal melanocytes, lymphocytes, and mesenchymal stem cells. Its widespread overexpression in various cancer types allows it to be classified as a tumor-associated antigen with potential diagnostic and therapeutic implications. This article reviews the synthesis pathways of GD2 and its role in cancer cell adhesion, proliferation, and metastasis with a focus on breast cancer. GD2 appears to be overexpressed on the outer membrane of most breast cancer cells and breast cancer stem cells (BCSCs) and is closely linked to epithelial-mesenchymal transition (EMT). GD3 synthase (GD3S) is considered to be the rate-determining step in GD2 synthesis. Clinical studies indicate that GD2 expression is increased in 35-70% of breast cancer samples, with higher levels in triple-negative breast cancer (TNBC). This overexpression correlates with more aggressive tumor features and worse prognosis. Therapeutic targeting of GD2 with monoclonal antibodies (moABs) like dinutuximab and naxitamab has demonstrated anti-cancer activity in preclinical cancer models and human clinical trials against high-risk neuroblastoma reducing tumor growth and enhancing survival. GD2-specific chimeric antigen receptor (CAR) T-cell therapy and GD3S inhibition present other promising therapeutic strategies to improve clinical outcomes. Furthermore, GD2-targeted vaccines are currently being investigated in cancer therapy. This narrative review article underscores the critical role of GD2 in breast cancer pathogenesis and highlights the promising therapeutic opportunities it offers. It advocates for the initiation of clinical trials to further explore the potential of GD2-targeted treatment in combination with standard breast cancer therapies.

Navigating the therapeutic landscape for breast cancer: targeting breast cancer stem cells.

Breast cancer is a common and deadly malignancy that affects women globally, and breast cancer stem cells (BCSCs) play an important role in tumorigenesis, development, metastasis, and recurrence. Traditional therapies often fail to eliminate BCSCs, leading to treatment resistance and relapse. This review explores the therapeutic strategies which are designed to target BCSCs, including inhibition of key signaling pathway and targeting receptor. This paper also explores the approaches to targeting BCSCs including chemotherapy, phytomedicines, and nanotechnology. Nanotechnology has gained a lot of importance in cancer therapy because of its ability to deliver therapeutic agents with more precision and minimal side effects. Various chemotherapeutic drugs, siRNAs, or gene editing tools are delivered efficiently with the use of nanocarriers which target pathways, receptors, and proteins associated with BCSCs. Over the past few years, stimuli-responsive and receptor-targeted nanocarriers have been explored for better therapeutic effects. In recent times, strategies such as chimeric antigen receptor (CAR) T-cell therapy, ablation therapy, and cell-free therapies are explored for targeting these stem cells. This review provides a recent developmental overview of strategies to attack BCSCs from conventional chemotherapeutic agents to nanotechnological platforms such as polymeric, lipidic, and metal-based nanoparticles and advanced technologies like CAR T cell therapies.

Unveiling the Potential of Hyptis Capitata and Hyptis Brevipes Compounds in Overcoming Apoptosis Resistance and Inducing Targeted Cell Cycle Arrest in Breast Cancer Stem Cells

Breast cancer stem cells (CSC), as a kind of tumor cells, are able to regenerate themselves, leading to apoptosis resistance and cancer relapse. In this study, we investigated the potential of Hyptis Capitata and Hyptis Brevipes compounds in preventing apoptosis resistance and inducing targeted cell cycle arrest in CSC. The cytotoxic effects of the compounds were evaluated using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay and the cell cycle distribution and apoptosis induction were assessed through flow cytometry analysis. The phytochemical characterization of Hyptis bravipes extract (HBE) and Hyptis capitata extract (HCE) conducted under thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC) analysis. The results revealed that Hyptis Brevipes extract (HBE) exhibited strong cytotoxicity towards CSCs, with an IC50 value of 89 µg/mL. In contrast, Hyptis Capitata extract (HCE) showed weak growth inhibition, with an IC50 value greater than 500 µg/mL. Subsequent analysis demonstrated that HBE induced cell cycle arrest in the G2/M and S phases and significantly increased apoptosis in the CSC population. Furthermore, phytochemical characterization using TLC and HPLC revealed that HBE and HCE share similar constituent compounds, with HBE exhibiting a more diverse compound content and stronger cytotoxic effects. These findings provide valuable insights into the potential of Hyptis Capitata and Hyptis Brevipes compounds as targeted agents against breast cancer stem cells and contribute to understanding their chemical profiles and pharmacological properties. HIGHLIGHTS Hyptis brevipes extract induced cell cycle arrest in G2/M and S phases. Cytotoxicity of Hyptis brevipes leading to apoptosis induction on CSC Hyptis brevipes significantly targeted Breast Cancer Stem Cells GRAPHICAL ABSTRACT

Double-Edged Sword Effect of Diet and Nutrition on Carcinogenic Molecular Pathways in Breast Cancer.

Environmental exposure to a mixture of chemical xenobiotics acts as a double-edged sword, promoting or suppressing tumorigenesis and the development of breast cancer (BC). Before anything else, we are what we eat. In this review, we highlight both "the good" and "the bad" sides of the daily human diet and dietary patterns that could influence BC risk (BCR) and incidence. Thus, regularly eating new, diversified, colorful, clean, nutrient-rich, energy-boosting, and raw food, increases apoptosis and autophagy, antioxidation, cell cycle arrest, anti-inflammation, and the immune response against BC cells. Moreover, a healthy diet could lead to a reduction in or the inhibition of genomic instability, BC cell stemness, growth, proliferation, invasion, migration, and distant metastasis. We also emphasize that, in addition to beneficial compounds, our food is more and more contaminated by chemicals with harmful effects, which interact with each other and with endogenous proteins and lipids, resulting in synergistic or antagonistic effects. Thus, a healthy and diverse diet, combined with appropriate nutritional behaviors, can exert anti-carcinogenic effects and improve treatment efficacy, BC patient outcomes, and the overall quality of life of BC patients.

Effects of BYL-719 (alpelisib) on human breast cancer stem cells to overcome drug resistance in human breast cancer.

Breast cancer continues to be a major health concern and is currently the most commonly diagnosed cancer worldwide. Relapse, metastasis, and therapy resistance are major clinical issues that doctors need to address. We believe BYL-719, which is PI3 kinase p110а inhibitor, could also inhibit the breast cancer stem cell phenotype and epithelial-to-mesenchymal transition (EMT). In addition to the PI3K/AKT signaling pathway, BYL-719 can also inhibit essential cancer-related signaling pathways, all of which would ultimately act on the microenvironment of cancer stem cells, which is quite complicated and regulates the characteristics of tumors. These include the stemness and resistance of malignant tumors, plasticity of cancer stem cells, and anti-apoptotic features. A three-dimensional (3D) mammosphere culture method was used in vitro to culture and collect breast cancer stem cells (BCSCs). MTT, clonogenic, and cell apoptosis assays were used to detect cell viability, self-renewal, and differentiation abilities. A sphere formation assay under 3D conditions was used to detect the mammophore inhibition rate of BYL-719. The subpopulation of CD44+CD24- was detected using flow cytometry analysis while EMT biomarkers and essential signaling pathways were detected using western blotting. All the data were analyzed using GraphPad Prism 9 software. BCSC-like cells were obtained by using the 3D cell culture method in vitro. We confirmed that BYL-719 could inhibit BCSC-like cell proliferation in 3D cultures and that the stemness characteristics of BCSC-like cells were inhibited. The PI3K/AKT/mTOR signaling pathway could be inhibited by BYL-719, and the Notch, JAK-STAT and MAPK/ERK signaling pathways which have crosstalk in the tumor microenvironment (TME) are also inhibited. By comparing eribulin-resistant breast cancer cell lines, we confirmed that BYL-719 could effectively overcome drug resistance. The 3D cell culture is a novel and highly effective method for enriching BCSCs in vitro. Furthermore, the stemness and EMT of BCSCs were inhibited by BYL-719 by acting on various signaling pathways. Finally, we believe that drug resistance can be overcome by targeting the BCSCs. Conjugation of BYL-719 with other anti-neoplastic agents may be a promising treatment for this in clinic.

The Anti-Cancer Stem Cell Properties of Copper(II)-Terpyridine Complexes with Attached Salicylaldehyde Moieties.

We report the synthesis, characterisation, and anti-breast cancer stem cell (CSC) properties of two copper(II)-terpyridine complexes with bidentate salicylaldehyde moieties (2-hydroxybenzaldehyde for 1 and 2-hydroxy-1-naphthaldehyde for 2). The copper(II)-terpyridine complexes 1 and 2 are stable in biologically relevant aqueous solutions and display micromolar potency towards breast CSCs. The most effective complex 1 is 5-fold and 6.6-fold more potent towards breast CSCs than salinomycin and cisplatin, respectively. The copper(II)-terpyridine complexes 1 and 2 also decrease the formation and viability of three-dimensionally cultured mammospheres within the micromolar range. Notably complex 1 is up to 7-fold more potent towards mammospheres than salinomycin or cisplatin. Mechanistic studies suggest that the copper(II)-terpyridine complexes 1 and 2 are able to readily enter breast CSCs, elevate intracellular reactive oxygen species levels, induce DNA damage (presumably by oxidative DNA cleavage), and evoke apoptosis that is independent of caspases. This study shows that the copper(II)-terpyridine motif is a useful building block for the design of anti-breast CSC agents and reinforces the therapeutic potential of copper coordination complexes.

CAV1 inhibits Xc- system through IFNGR1 to promote ferroptosis to inhibit stemness and improves anti-PD-1 efficacy in breast cancer

Breast cancer is the most prevalent malignancy among women worldwide, with breast cancer stem cells (BCSCs) being the primary drivers of metastasis and recurrence. Numerous studies have elucidated the relationship between ferroptosis and cellular stemness, identifying the Xc- system as a key regulatory mechanism governing ferroptosis. However, the interplay between CAV1 and ferroptosis, along with its implications for stemness in breast cancer, remains inadequately understood. This gap in knowledge impedes advancements in targeted therapies for breast cancer. We employed immunohistochemistry and bioinformatics analyses to demonstrate the downregulation of CAV1 in breast cancer tissues. Additionally, we utilized CCK-8 assays, EDU staining, and Transwell assays to assess cell proliferation, migration, and invasion capabilities. Furthermore, we evaluated indicators associated with ferroptosis while examining markers related to stemness through sphere culture experiments and flow cytometry techniques. Our findings indicate that CAV1 expression can induce cell death via ferroptosis while simultaneously inhibiting both cell proliferation and features of stemness by upregulating IFNGR1 and promoting ferroptosis. Moreover, our in vivo experiments revealed that overexpression of CAV1 enhances the efficacy of anti-PD-1 therapy. In conclusion, our study elucidates the regulatory role of CAV1 on ferroptosis within breast cancer contexts; it suppresses BCSC characteristics while positioning CAV1 as a promising therapeutic target for combating this disease.

Correction: Regua et al. TrkA Interacts with and Phosphorylates STAT3 to Enhance Gene Transcription and Promote Breast Cancer Stem Cells in Triple-Negative and HER2-Enriched Breast Cancers. Cancers 2021, 13, 2340

In the original publication [...]

The Role of MicroRNA-124-3p in Breast Cancer Stem Cell Inhibition by Benzyl Isothiocyanate.

We have shown previously that benzyl isothiocyanate (BITC) derived from cruciferous vegetables inhibits self-renewal of breast cancer stem-like cells (bCSC). The current study provides insights into the mechanism of bCSC inhibition by BITC. Quantitative real time-polymerase chain reaction and western blot analysis were performed to detect microRNAs(miRNAs) and Forkhead box Q1 (FoxQ1) protein expression, respectively. The bCSC were characterized by aldehyde dehydrogenase 1 activity and flow cytometric analysis of CD49f high/CD133high fraction. BITC treatment resulted in induction of miR-124-3p expression in MDA-MB-231 and MCF-7 cells. miR-124-3p did not affect BITC-mediated inhibition of cell migration or cell proliferation but it significantly regulated bCSC in response to BITC. We also found that miR-124-3p directly targets the 3'untranslated regions (UTR) of FoxQ1 and negatively regulates its expression. The BITC-mediated inhibition of bCSC was partially attenuated by miR-124-3p inhibitor. These findings indicate that miR-124-3p plays an important role in BITC-mediated inhibition of bCSC.

8038 Targeting Breast Cancer Stem Cells With Oncolytic Mammalian Orthoreovirus (MRV)

Abstract Disclosure: N.I. Roman Ortiz: None. J. Davydova: None. P. Danthi: None. J.H. Ostrander: None. Breast cancer is the most frequently diagnosed malignant cancer in women and is the second leading cause of cancer deaths among women in the United States. Patients with ER+ breast cancer can experience recurrence for more than 10 years after initial diagnosis. Breast cancer stem-like cells (BCSC) are slowly proliferative and exist as a minority sub-population (0-5%) within the tumor. Existing treatments target rapidly dividing cells, and BCSC are unresponsive to these therapies. Due to their self-renewal capabilities, BCSCs can fuel recurrence, metastasis, and resistance to therapies. Notably, no FDA-approved therapeutics selectively target BCSCs. Mammalian orthoreovirus (MRV) is an oncolytic virus tested in clinical trials for many cancer types including metastatic breast cancer. MRV was deemed safe but lacked efficacy as standalone treatment. We tested various laboratory MRV strains (T1L, R2) with T3D, a similar strain to the one evaluated in clinical trials. Our studies suggest that T3D is less effective in killing ER+ breast cancer cells and BCSCs compared to T1L and R2. To understand how T1L and R2 are more effective at killing ER+ breast cancer we aim to test the stages of MRV infection and how MRV induces cell death in ER+ therapy sensitive and resistant cells. MRV can enter cells by receptor mediated endocytosis through attachment with Junctional Adhesion Molecule A (JAM-A) and sialic acids as co-receptors. We have found that T1L attaches to cells in a JAM-A-dependent manner in ER+ sensitive cancer cells, while ER+ therapy resistant cells have increased resistance to JAM-A mediated endocytosis. Previous work has shown that apoptosis is involved in MRV-induced cell death. We found that apoptosis inhibitors in MRV-treated cells increase cell viability, suggesting that MRV-induces apoptotic cell death in ER+ breast cancer cells. Our current studies involve using RNA sequencing as a tool to assess gene expression of ER+ therapy sensitive and resistant cells infected with MRV at early and late stages of infection to determine key genes and pathways associated with cell death.We also aim to generate novel MRV strains with enhanced BCSC killing. We used a forward genetic approach and serially passaged MRV strains in BCSC enriched tumorsphere cultures of MCF-7 paclitaxel resistant (TaxR) cells to generate a selective oncolytic virus that can infect and kill BCSCs. Preliminary data has shown that serially passaged (SP) MRV strains (T1L SP, R2 SP) are more effective in decreasing cell viability and inhibiting tumorsphere formation compared to the parental strains (T1L and R2). Current studies are focused on sequencing SP clones to identify mutations that enhance BCSC death. We expect these studies to enhance MRV virotherapeutics for combined use with clinically relevant inhibitors (i.e. CDK4/6 inhibitors), anti-estrogens, or immunotherapy to prevent and treat metastatic ER+ breast cancer. Presentation: 6/2/2024

8038 Targeting Breast Cancer Stem Cells with Oncolytic Mammalian Orthoreovirus (MRV)

Abstract Disclosure: N.I. Roman Ortiz: None. J. Davydova: None. P. Danthi: None. J.H. Ostrander: None. Breast cancer is the most frequently diagnosed malignant cancer in women and is the second leading cause of cancer deaths among women in the United States. Patients with ER+ breast cancer can experience recurrence for more than 10 years after initial diagnosis. Breast cancer stem-like cells (BCSC) are slowly proliferative and exist as a minority sub-population (0-5%) within the tumor. Existing treatments target rapidly dividing cells, and BCSC are unresponsive to these therapies. Due to their self-renewal capabilities, BCSCs can fuel recurrence, metastasis, and resistance to therapies. Notably, no FDA-approved therapeutics selectively target BCSCs. Mammalian orthoreovirus (MRV) is an oncolytic virus tested in clinical trials for many cancer types including metastatic breast cancer. MRV was deemed safe but lacked efficacy as standalone treatment. We tested various laboratory MRV strains (T1L, R2) with T3D, a similar strain to the one evaluated in clinical trials. Our studies suggest that T3D is less effective in killing ER+ breast cancer cells and BCSCs compared to T1L and R2. To understand how T1L and R2 are more effective at killing ER+ breast cancer we aim to test the stages of MRV infection and how MRV induces cell death in ER+ therapy sensitive and resistant cells. MRV can enter cells by receptor mediated endocytosis through attachment with Junctional Adhesion Molecule A (JAM-A) and sialic acids as co-receptors. We have found that T1L attaches to cells in a JAM-A-dependent manner in ER+ sensitive cancer cells, while ER+ therapy resistant cells have increased resistance to JAM-A mediated endocytosis. Previous work has shown that apoptosis is involved in MRV-induced cell death. We found that apoptosis inhibitors in MRV-treated cells increase cell viability, suggesting that MRV-induces apoptotic cell death in ER+ breast cancer cells. Our current studies involve using RNA sequencing as a tool to assess gene expression of ER+ therapy sensitive and resistant cells infected with MRV at early and late stages of infection to determine key genes and pathways associated with cell death.We also aim to generate novel MRV strains with enhanced BCSC killing. We used a forward genetic approach and serially passaged MRV strains in BCSC enriched tumorsphere cultures of MCF-7 paclitaxel resistant (TaxR) cells to generate a selective oncolytic virus that can infect and kill BCSCs. Preliminary data has shown that serially passaged (SP) MRV strains (T1L SP, R2 SP) are more effective in decreasing cell viability and inhibiting tumorsphere formation compared to the parental strains (T1L and R2). Current studies are focused on sequencing SP clones to identify mutations that enhance BCSC death. We expect these studies to enhance MRV virotherapeutics for combined use with clinically relevant inhibitors (i.e. CDK4/6 inhibitors), anti-estrogens, or immunotherapy to prevent and treat metastatic ER+ breast cancer. Presentation: 6/2/2024

7324 Regulation of Super Enhancers by Phosphorylated Progesterone Receptors Drives Breast Cancer Stem Cell Expansion

Abstract Disclosure: N. Gillis: None. C.A. Lange: None. Current treatments for estrogen receptor (ER) positive breast cancer largely target rapidly dividing tumor cells. However, aggressive breast cancers often contain long-lived and weakly proliferative cancer stem cells (CSCs) that readily escape treatments aimed at cycling cells. We previously reported that progesterone receptors (PR) and their target genes aid the growth of these CSCs and help them evade anti-estrogen therapy. As proliferation and stemness are directed by separate signaling pathways that lead to the expression of distinct gene programs, it's crucial to understand how PR facilitates this cellular transformation at the genomic level. Establishing a gene expression program that may limit proliferation but encourages stemness requires coordination between transcription factors and their coregulators to alter the nuclear microenvironment and chromatin landscape.To reveal these mechanisms, we profiled PR and ER genomic binding using CUT&RUN in the T47D luminal breast cancer cell line grown in ultra-low attachment (3D) mammosphere conditions. Our analysis of genomic binding profiles displayed a pattern of PR/ER co-occupancy at distant sites more than 10 kilobases away from the nearest transcriptional start site. We used Rank Ordering of Super Enhancer (ROSE) analysis on these PR/ER binding sites to predict which are likely super enhancer elements capable of coordinating the regulation of multi-gene subsets. We employed a genetic approach to directly test the function of novel enhancer sites linked to PR-target genes by disrupting the PR/ER binding with a dCas9-KRAB construct directed to the predicted enhancer elements. We propose that abnormal growth factor signaling in breast cancer cells permits PR/ER complex formation which promotes expression of genes that induce endocrine resistance and CSC expansion through regulation of super enhancer elements. Further studies to elucidate these mechanisms are ongoing. Understanding the intricacies of PR/ER crosstalk is the crucial next step in developing new therapies that target PR-driven processes in ER+ breast cancers. Presentation: 6/3/2024

Value of miR-31 and miR-150-3p as diagnostic and prognostic biomarkers for breast cancer.

The most prevalent malignancy among women is breast cancer (BC). MicroRNAs (miRNAs) play a role in the initiation and progression of BC by influencing breast cancer stem cells (BCSCs) but the diagnostic and prognostic roles of those miRNAs on BC patients are still unknown. It was aimed to investigate expression profiles, diagnostic and prognostic potentials of BCSC-related miRNAs in different subtypes (Luminal A and B, HER2 + and TNBC) of BC patients. Expression analysis of 15 BCSC-related miRNAs was performed in 50 breast tumor tissues and 20 adjacent non-tumor tissues obtained from BC patients using the qRT-PCR method. The expression levels of miR-31 and miR-150-3p were significantly upregulated in the tumor tissues compared to the adjacent non-tumor tissues (p < 0.05). miR-31 expression upregulated in the Luminal A and Luminal B group compared to non-tumor tissue (p < 0.05). miR-31 expression was determined to be significantly higher in the Luminal group (Luminal A and B) compared to the aggressive group (HER2 + and TNBC) (p < 0.05). According to the ROC analysis, the area under the curve (AUC) of miR-31 and miR-150-3p were 0.66 with a sensitivity of 68% and a specificity of 70%. A significant inverse correlation was observed between miR-31 expression with metastatic carcinoma status, in situ component, and Ki67 value in tumors, and high miR-150-3p expression was correlated with p63 expression (p < 0.05). miR-31 and miR-150-3p have the potential to serve as biomarkers for guiding diagnosis, evaluating prognosis, and metastatic process in patients with BC.

Epigenetic and Immune Mechanisms Linking Breastfeeding to Lower Breast Cancer Rates.

This review shows how mammary stem cells (MaSCs) influence breast development, breastfeeding, and breast cancer risk. MaSCs, which can differentiate into various cell types, are vital for breast tissue health, but also disease development in breast tissue. Research shows that breastfeeding affects MaSCs, offering protection against breast cancer through various mechanisms. Hormonal changes such as increased prolactin concentration, oxytocin secretion, lower progesterone levels, and reduced exposure to estrogen during lactation promote apoptosis in potential cancer cells, boost immune surveillance, and modulate inflammation. Key findings reveal that pregnancy at an earlier age and extended breastfeeding reduce MaSC numbers, lowering cancer risk. Additionally, breastfeeding induces various epigenetic changes, such as DNA methylation and histone modification, which provide long-term protection against the development of cancer. Components of breast milk, like alpha-lactalbumin and lactoferrin, contribute by promoting cancer cell apoptosis and inhibiting tumor growth. The dual benefits of breastfeeding are reduced breast cancer risk for mothers and immunological advantages for infants. Multicenter epidemiology research has focused particular attention on longer breastfeeding duration associated with a reduced risk of triple-negative breast cancer. This review offers comprehensive evidence that breastfeeding protects against breast cancer through various biological, hormonal, and molecular mechanisms, showing the importance of promoting breastfeeding as a natural cancer prevention method. This article reviews the role of mammary stem cells in breast development, lactation, and breast cancer.

Standard chemotherapy impacts on in vitro cellular heterogeneity in spheroids enriched with cancer stem cells (CSCs) derived from triple-negative breast cancer cell line

Triple-negative breast cancer is a heterogeneous disease with high recurrence and mortality, linked to cancer stem cells (CSCs). Our study characterized distinct cell subpopulations and signaling pathways to explore chemoresistance. We observed cellular heterogeneity among and within the cells regarding phenotyping and drug response. In untreated BT-549 cells, we noted plasticity properties in both CD44+/CD24+/CD146+ hybrid cells and CD44-/CD24+/CD146+ epithelial cells, enabling phenotypic conversion into CD44+/CD24-/CD146- epithelial-mesenchymal transition (EMT)-like like breast CSCs (BCSCs). Additionally, non-BCSCs may give rise to ALDH+ epithelial-like BCSCs. Enriched BCSCs demonstrated the potential to differentiation into CD44-/CD24-/CD146- cells and exhibited self-renewal capabilities. Similar phenotypic plasticity was not observed in untreated Hs 578T and HMT-3522 S1 cells. BT-549 cells were more resistant to paclitaxel/PTX than to doxorubicin/DOX, a phenomenon potentially linked to the presence of CD24+ cells prior to treatment. Under the CSCs-enriched spheroids model, BT-549 demonstrated extreme resistance to DOX, likely due to the enrichment of BCSCs CD44+/CD24-/CD146- and the tumor cells CD44-/CD24-/CD146-. Additionally, DOX treatment induced the enrichment of plastic and chemoresistant cells, further exacerbating resistance mechanisms. BT-549 exhibited high heterogeneity, leading to significant alterations in cell subpopulations under BCSCs enrichment, demonstrating increased phenotypic plasticity during EMT. This phenomenon appears to play a major role in DOX resistance, as indicated by the presence of the refractory cells CD44+/CD24-/CD146- BCSCs EMT-like, CD44-/CD24-/CD146- tumor cells, and elevated STAT3 expression. Gene expression data from BT-549 CSCs-enriched spheroids suggests that ferroptosis may be occurring via autophagic regulation triggered by RAB7A, highlighting this gene as a potential therapeutic target.

Amcasertib Increases Apoptosis While Decreasing Invasive and Migrating Abilities in Breast Cancer Stem Cells

Objective: A relationship exists between breast cancer stem cells (BCSCs) and the chemo-resistance and recurrence of aggressive breast tumors. Amcasertib is a small chemical compound and multiple kinase inhibitor that inhibits downstream Nanog and other cancer stem signaling pathways in cancer stem cells by targeting several serine-threonine kinases. In this study, we aimed to investigate the cytotoxicity and anticancer effects of Amcasertib on BCSCs, gaining insight into the targetability of BCSCs. Method: We used the combined xCELLigence-Real-Time Cell Analyzer (RTCA) equipment to analyze cytotoxicity and cell proliferation. We detected the IC50 dosages of Amcasertib at 24, 48, and 72 hours and examined its effects on apoptosis, cell cycle, invasion, and migration over 48 hours. We used flow cytometry for assays of apoptosis and cell cycle, and the CytoSelect 96-well Cell Migration and Invasion Assay Kit for evaluating invasion and migration. Results: Our results showed that Amcasertib has cytotoxic properties, with an IC50 dosage of 1.9 μM at the 48th hour. In addition, Amcasertib significantly induced apoptosis in BCSCs, despite not affecting the cell cycle. Moreover, Amcasertib decreased BCSCs’ invasion and migratory properties, part of epithelial-mesenchymal transition (EMT). Conclusion: In conclusion, our findings provide crucial information for understanding the potential of Amcasertib in targeting BCSCs. In addition, we suggest that Amcasertib could be a beneficial drug for breast cancer treatment by targeting BCSCs.

Novel CD44-Targeted Albumin Nanoparticles: An Innovative Approach to Improve Breast Cancer Treatment.

This study introduces novel CD44-targeted and redox-responsive nanoparticles (FNPs), proposed as doxorubicin (DOX) delivery devices for breast cancer. A cationized and redox-responsive Human Serum Albumin derivative was synthesized by conjugating Human Serum Albumin with cystamine moieties and then ionically complexing it with HA. The suitability of FNPs for cancer therapy was assessed through physicochemical measurements of size distribution (mean diameter of 240 nm), shape, and zeta potential (15.4 mV). Nanoparticles possessed high DOX loading efficiency (90%) and were able to trigger the drug release under redox conditions of the tumor environment (55% release after 2 h incubation). The use of the carrier increased the cytotoxic effect of DOX by targeting the CD44 protein. It was shown that, upon loading, the cytotoxic effect of DOX was enhanced in relation to CD44 protein expression in both 2D and 3D models. DOX@FNPs significantly decrease cellular metabolism by reducing both oxygen consumption and extracellular acidification rates. Moreover, they decrease the expression of proteins involved in the oxidative phosphorylation pathway, consequently reducing cellular viability and motility, as well as breast cancer stem cells and spheroid formation, compared to free DOX. This new formulation could become pioneering in reducing chemoresistance phenomena and increasing the specificity of DOX in breast cancer patients.