Normal Mammary Cells Research Articles

Cells-of-Origin of Breast Cancer and Intertumoral Heterogeneity.

Both intrinsic and extrinsic mechanisms underpin the profound intertumoral heterogeneity in breast cancer. Increasing evidence suggests that the intrinsic characteristics of breast epithelial precursor cells may influence tumour phenotype. These "cells-of-origin" of cancer preside in normal breast tissue and are uniquely susceptible to mutagenesis upon exposure to distinct oncogenic stimuli. Notably, molecular profiling studies have revealed strong concordance between the gene expression profiles of breast cancer subtypes and discrete cell types within the normal breast epithelium. Further characterisation of cells-of-origin of breast cancer requires comprehensive delineation of the normal mammary stem cell hierarchy. To this end, mouse models have provided valuable tools for exploring stem and progenitor cell function and identifying potential targets of neoplastic transformation via in vivo lineage-tracing studies. Nonetheless, the murine mammary differentiation hierarchy does not fully recapitulate human biology, and complementary studies using patient-direct breast tissue are critical. There is also accumulating evidence that extrinsic factors such as the microenvironment of premalignant cells can influence tumour initiation, highlighting opportunities for targeting cancer cells-of-origin via deconvolution of the premalignant epithelial niche. Pertinently, the identification of premalignant clones and targetable molecular perturbations responsible for driving their oncogenic transformation has critical implications for disease management and prevention.

MiR-379-5p affects breast cancer cell behavior by targeting UBE2E3 ubiquitin conjugating enzyme

MicroRNAs (miRNAs) play an increasingly recognized role in modulating cancer development. Due to their function in regulating gene expression, miRNAs can suppress or promote tumorigenesis. miR-379-5p expression is downregulated in multiple human cancers, including breast and bladder cancers. However, the mRNAs targeted by miR-379-5p that promote cancer development have not been fully identified. Our goal was to identify a gene whose expression is regulated by miR-379-5p, and which may contribute to cancer development in cells where miR-379-5p expression is reduced. Bioinformatics analysis showed the UBE2E3 ubiquitin conjugating enzyme gene to be a potential target for miR-379-5p. To verify that UBE2E3 is a target, we transfected normal human epithelial mammary cells and breast adenocarcinoma cell lines with a miR-379-5p mimic. The mimic reduced UBE2E3 mRNA and protein levels, as would be predicted for a miR-379-5p target. To determine if UBE2E3 is a direct target of miR-379-5p, we engineered two luciferase reporter gene constructs to contain either a wild-type putative miR-379-5p binding sequence isolated from the 3’UTR of the UBE2E3 gene, or a scrambled sequence. The luciferase assay showed that the miR-379-5p mimic suppressed luciferase activity for the WT binding sequence reporter, but not for the scrambled reporter, showing that the effect of miR-379-5p on UBE2E3 expression is likely to be direct. Finally, to determine if the effect of miR-379-5p on UBE2E3 is related to cellular behaviors that play a role in cancer development, we measured cell viability by resazurin assay, cell proliferation by BrdU assay, and apoptosis by caspase 3/7 activation assay. The miR-379-5p mimic and silencing UBE2E3 expression both resulted in significantly diminished cell viability, while silencing UBE2E3 demonstrated both higher proliferation and apoptotic rates. Overall, these results suggest that while the overall effect of miR-379-5p is to inhibit breast cell viability and proliferation, the effect of silencing its target UBE2E3 is more complex because it induces both cell proliferation and apoptosis.

12347 Cholesterol Biosynthesis Inhibitor Ro 48-8071 Suppresses Growth Of Triple-negative Breast Cancer Cells By Inducing Apoptosis And Suppressing Angiogenesis

Abstract Disclosure: Y. Liang: None. S.M. Hyder: None. Triple-negative breast cancers (TNBC) lack ER, PR and Her2neu, proteins that are commonly targeted in breast cancer therapies. Women who suffer from TNBC have limited treatment options and are administered toxic chemotherapeutic agents. They have poor prognosis due to the emergence of drug-resistant cells that lead to tumor metastasis. New non-toxic therapeutic strategies to control TNBC progression, and prevent metastasis are urgently needed. Cholesterol is an essential structural and functional component of cell membranes necessary for tumor growth and progression. Therefore, inhibiting cholesterol production is an attractive therapeutic strategy since tumor cells grow rapidly, and need a greater supply of cholesterol. We targeted oxidosqualene cyclase (OSC), an enzyme that occurs downstream of HMGCoA-reductase in the cholesterol biosynthetic pathway, to disrupt tumor growth. RO 48-8071 (4′-[6-(allylmethylamino)hexyloxy]-4-bromo-2′-fluorobenzophenone fumarate) (RO), has emerged as a useful chemotherapeutic agent for treating several forms of primary tumors but its effect on TNBC is not known. Pharmacological doses of RO potently reduced the viability of both MDA-MB-231 and BT20 TNBC cells in a dose dependent manner (1-50 μM). IC50 values for loss of cell viability was approximately 10 μM for both cell lines following 48 h exposure to RO. Doses close to the IC50 for OSC (1-10 nM) also reduced cell viability following exposure of TNBC cells to RO for a week. Importantly, RO had no effect on the viability of normal human mammary cells. FACS analysis revealed that RO dose-dependently induced apoptosis of TNBC cell lines in vitro. In vivo, administration of 5-10 mg/kg RO to mice with BT-20 TNBC cell-derived xenografts (100 mm3) inhibited tumor growth with no apparent toxicity. Immunohistochemical analysis of tumor tissues at the end of treatment showed that RO increased TUNEL expression, suggesting that the in vivo effects of RO were also exerted through mechanisms affecting apoptosis. Furthermore, IHC of tumor sections showed that RO also reduced levels of the angiogenic markers VEGF and CD-31 (blood vessels) in vivo, indicating that suppression of angiogenesis is also part of the mechanism through which RO exerts its anti-tumor effects. We conclude that RO is a potent inhibitor of TNBC and that its anti-tumor effects involve mechanisms that induce apoptosis and suppress angiogenesis. Presentation: 6/2/2024

Vitamin D receptor is associated with prognostic characteristics of breast cancer after neoadjuvant chemotherapy-an observational study.

Breast cancer (BC) is the most commonly diagnosed malignant tumor in women. The disease and its subsequent treatment pose a serious burden on the quality of life of patients. Neoadjuvant chemotherapy (NAC) has become one of the crucial strategies for the management of BC. Since the identification of the vitamin D receptor (VDR) in mammary tissues, extensive mechanistic research has been conducted on its function. The expression of VDR in BC cells and the tumor microenvironment could be a new prognostic factor for BC after NAC. This observational, single-center study compared data from clinical and histopathological records of 111 female subjects with the expression of VDR in different cellular and tissue components of breast specimens obtained from surgery after NAC. VDR expression was evaluated using an immunoreactive score assigned after immunohistochemistry. Intergroup comparisons and logistic regression were used to identify associations between VDR expression and clinicopathological features of BC. We found that the expression of VDR is associated with various clinical features (i.e., age, menopausal status, and NAC cycle number) and characteristics of prognostic significance, such as residual cancer burden class. Logistic regression analysis revealed that the expression of VDR in the nuclei and cytoplasm of surrounding normal mammary cells predicted vascular invasion and lymph node involvement. The expression of VDR in tumor cells and their microenvironment is related to the clinicopathological characteristics of BC after NAC.

Syndecan-1 Plays a Role in the Pathogenesis of Sjögren's Disease by Inducing B-Cell Chemotaxis through CXCL13-Heparan Sulfate Interaction.

In Sjögren's disease (SjD), the salivary glandular epithelial cells can induce the chemotaxis of B cells by secreting B-cell chemokines such as C-X-C motif chemokine ligand 13 (CXCL13). Syndecan-1 (SDC-1) is a major transmembrane heparan sulfate proteoglycan (HSPG) predominantly expressed on epithelial cells that binds to and regulates heparan sulfate (HS)-binding molecules, including chemokines. We aimed to determine whether SDC-1 plays a role in the pathogenesis of SjD by acting on the binding of HS to B-cell chemokines. To assess changes in glandular inflammation and SDC-1 concentrations in the submandibular gland (SMG) and blood, female NOD/ShiLtJ and sex- and age-matched C57BL/10 mice were used. In the SMG of NOD/ShiLtJ mice, inflammatory responses were identified at 8 weeks of age, but increased SDC-1 concentrations in the SMG and blood were observed at 6 weeks of age, when inflammation had not yet started. As the inflammation of the SMG worsened, the SDC-1 concentrations in the SMG and blood increased. The expression of the CXCL13 and its receptor C-X-C chemokine receptor type 5 (CXCR5) began to increase in the SMG at 6 weeks of age and continued until 12 weeks of age. Immunofluorescence staining in SMG tissue and normal murine mammary gland cells confirmed the co-localization of SDC-1 and CXCL13, and SDC-1 formed a complex with CXCL13 in an immunoprecipitation assay. Furthermore, NOD/ShiLtJ mice were treated with 5 mg/kg HS intraperitoneally thrice per week for 6-10 weeks of age, and the therapeutic effects in the SMG were assessed at the end of 10 weeks of age. NOD/ShiLtJ mice treated with HS showed attenuated salivary gland inflammation with reduced B-cell infiltration, germinal center formation and CXCR5 expression. These findings suggest that SDC-1 plays a pivotal role in the pathogenesis of SjD by binding to CXCL13 through the HS chain.

Flow cytometric analysis for Ki67 assessment in formalin-fixed paraffin-embedded breast cancer tissue

BackgroundPathologists commonly employ the Ki67 immunohistochemistry labelling index (LI) when deciding appropriate therapeutic strategies for patients with breast cancer. However, despite several attempts at standardizing the Ki67 LI, inter-observer and inter-laboratory bias remain problematic. We developed a flow cytometric assay that employed tissue dissociation, enzymatic treatment and a gating process to analyse Ki67 in formalin-fixed paraffin-embedded (FFPE) breast cancer tissue.ResultsWe demonstrated that mechanical homogenizations combined with thrombin treatment can be used to recover efficiently intact single-cell nuclei from FFPE breast cancer tissue. Ki67 in the recovered cell nuclei retained reactivity against the MIB-1 antibody, which has been widely used in clinical settings. Additionally, since the method did not alter the nucleoskeletal structure of tissues, the nuclei of cancer cells can be enriched in data analysis based on differences in size and complexity of nuclei of lymphocytes and normal mammary cells. In a clinical study using the developed protocol, Ki67 positivity was correlated with the Ki67 LI obtained by hot spot analysis by a pathologist in Japan (rho = 0.756, P < 0.0001). The number of cancer cell nuclei subjected to the analysis in our assay was more than twice the number routinely checked by pathologists in clinical settings.ConclusionsThe findings of this study showed the application of this new flow cytometry method could potentially be used to standardize Ki67 assessments in breast cancer.

Primary Human Breast Cancer-Associated Endothelial Cells Favor Interactions with Nanomedicines.

Cancer nanomedicines predominately rely on transport processes controlled by tumor-associated endothelial cells to deliver therapeutic and diagnostic payloads into solid tumors. While the dominant role of this class of endothelial cells for nanoparticle transport and tumor delivery is established in animal models, the translational potential in human cells needs exploration. Using primary human breast cancer as a model, the differential interactions of normal and tumor-associated endothelial cells with clinically relevant nanomedicine formulations are explored and quantified. Primary human breast cancer-associated endothelial cells exhibit up to ≈2times higher nanoparticle uptake than normal human mammary microvascular endothelial cells. Super-resolution imaging studies reveal a significantly higher intracellular vesicle number for tumor-associated endothelial cells, indicating a substantial increase in cellular transport activities. RNA sequencing and gene expression analysis indicate the upregulation of transport-related genes, especially motor protein genes, in tumor-associated endothelial cells. Collectively, the results demonstrate that primary human breast cancer-associated endothelial cells exhibit enhanced interactions with nanomedicines, suggesting a potentially significant role for these cellsin nanoparticle tumor delivery in human patients. Engineering nanoparticles that leverage the translational potential of tumor-associated endothelial cell-mediated transport into human solid tumors may lead to the development of safer and more effective clinical cancer nanomedicines.

Abstract PO1-24-06: Prolactin Hormone-Induced Mammary Differentiation and Anti-tumorigenic Role in Breast Cancer

Abstract The premise of differentiation therapy in cancer is a strategy that aims at engaging-forward differentiation and cellular reprograming restricting the proliferative, tumor repopulation, stemness, EMT and metastatic capacities of tumor cells leading to the cessation of the aggressive tumor phenotype and offering the cancer patients improved survival for decades. Therefore, deciphering molecular mechanisms deriving differentiation in normal mammary and breast cancer cells may allow the discovery of innovative differentiation-based biomarkers and therapeutics in breast cancer. Lactation is an intricate process that results in the ability of the breast cells to produce a complex nutritious biological fluid to nurse the new-born. While the beneficial effects of breastfeeding to the infant is irrefutable, its effects on maternal health are less investigated especially in relation to breast cancer that still affects 1 in 8 women in high income countries and 1 in 20 globally. Importantly, epidemiological studies have linked breastfeeding to reduced risk of breast cancer by promoting terminal differentiation of the breast epithelial cells. While breastfeeding process is regulated by multiple hormones, growth factors, and transcriptional regulators, the lactation hormone prolactin (PRL) is known to be directly implicated in the hormonal control of breastfeeding. Extensive research including our own work has shown that PRL/PRL receptor (PRLR) pathway derives mammary gland development and importantly mammary epithelial cell terminal differentiation allowing successful lactation. In contrast the role of PRL in breast cancer is still to be fully defined. Importantly, we have accumulated compelling evidence using in-silico publicly available patient data sets and molecular data implicating this pathway as a clinically relevant pro-differentiation pathway driving differentiation and cellular reprograming suppressing stemness, EMT, metastasis and tumorigenesis in breast cancer. To better characterize PRL signaling mediating its pro-differentiation effects, we used large-scale unbiased proteomics analysis of PRLR/interacting proteins in breast cancer cells. Our analyses revealed several novel PRLR-interactors. We have further confirmed these interactions through co-immunoprecipitations/western blotting analyses in breast cancer cells representative the different breast cancer subtypes. We also examined their functional contributions to mammary differentiation and tumorigenesis using in vitro and in vivo assays. Moreover, we defined their potential clinical relevance using bioinformatics in silico databases of breast cancer. Together our study delineates novel PRL/PRLR-downstream signaling mediators important for mammary differentiation and tumorigenesis and may thus be useful as biomarkers and/or therapeutic targets in breast cancer. Citation Format: Dana Hamam, Suhad Ali. Prolactin Hormone-Induced Mammary Differentiation and Anti-tumorigenic Role in 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 PO1-24-06.

Abstract SY19-03: Mechanisms of sensitivity and resistance to CDK2 inhibitors

Abstract CDK2 is a core cell-cycle kinase that phosphorylates many substrates to drive progression through the cell cycle. We used several CDK2 inhibitors in clinical development to interrogate CDK2 substrate phosphorylation, cell-cycle progression, and drug adaptation in preclinical models. In normal mammary cells and estrogen receptor-positive breast cancer cells, CDK2 inhibition causes a rapid loss of substrate phosphorylation that is rapidly restored by a hard-wired cell-cycle buffering mechanism. In this mechanism, CDK4/6 backstops Rb1 hyper-phosphorylation to maintain Cyclin A2 expression, enabling re-activation of CDK2 in the presence of CDK2 inhibitor. By contrast, CCNE1-amplified ovarian cancers do not exhibit this rapid rebound. Our results augment our understanding of CDK plasticity and CDK dependencies, delineate the genetic backgrounds in which this adaptive rebound upon CDK2 inhibition can occur, and suggest contexts in which CDK2 inhibition will be most effective as a cancer therapy. Citation Format: Sabrina L. Spencer. Mechanisms of sensitivity and resistance to CDK2 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr SY19-03.

Association between Single Nucleotide Polymorphisms of PRKD1 and KCNQ3 Gene and Milk Quality Traits in Gannan Yak (Bos grunniens).

Protein kinase D1 (PRKD1) functions primarily in normal mammary cells, and the potassium voltage-gated channel subfamily Q member 3 (KCNQ3) gene plays an important role in controlling membrane potential and neuronal excitability, it has been found that this particular gene is linked to the percentage of milk fat in dairy cows. The purpose of this study was to investigate the relationship between nucleotide polymorphisms (SNPs) of PRKD1 and KCNQ3 genes and the milk quality of Gannan yak and to find molecular marker sites that may be used for milk quality breeding of Gannan yak. Three new SNPs were detected in the PRKD1 (g.283,619T>C, g.283,659C>A) and KCNQ3 gene (g.133,741T>C) of 172 Gannan lactating female yaks by Illumina yak cGPS 7K liquid-phase microarray technology. Milk composition was analyzed using a MilkoScanTM milk composition analyzer. We found that the mutations of these three loci significantly improved the lactose, milk fat, casein, protein, non-fat milk solid (SNF) content and acidity of Gannan yaks. The lactose content of the TC heterozygous genotype population at g.283,619T>C locus was significantly higher than that of the TT wild-type population (p < 0.05); the milk fat content of the CA heterozygous genotype population at g.283,659C>A locus was significantly higher than that of the CC wild-type and AA mutant populations (p < 0.05); the casein, protein and acidity of the CC mutant and TC heterozygous groups at the g.133,741T>C locus were significantly higher than those of the wild type (p < 0.05), and the SNF of the TC heterozygous group was significantly higher than that of the mutant group (p < 0.05). The results showed that PRKD1 and KCNQ3 genes could be used as candidate genes affecting the milk traits of Gannan yak.

Differential Effect of Novel Plant Cystatins on the Adhesive Behaviour of Normal and Cancer Breast Cells

In the present work, we have investigated a novel recombinant cystatin dgECP1 and its mutant form, dgECP1m1, focused on their impact on the adhesive behaviour of two breast cell lines: the cancerous, MDA-MB-231, and the normal, MCF-10A. DgECP1 cystatin is intriguing with its RGD motif, responsible for cell adhesion and typical for mammalian extracellular matrix proteins but uncommon for plant cystatins. The presence of the RGD sequence suggests the potential of the dgECP1 to influence the adhesion of cancer cells and, respectively, cancer metastasis. A mutant form of the dgECP1cystatin, dgECP1m1, where RGD is replaced with HGD tripeptide, was also investigated. We found that both phytocystatins exerted differential effects on the adhesion behaviour of normal and cancer cells. In the case of dgECP1 cystatins, the effect on cancer cell adhesion also depends on the mode of administration of the cystatin to cells. When dgECP1 is pre-adsorbed on a substrate, it improves the attachment of breast cancer cells and induces cell aggregation, which is more typical for normal breast cells, and oppositely suppressed adhesion of cancer cells when added to the medium. The mutant form, dgECP1m1, inhibited cancer cell adhesion independently on the way of administration. On the other hand, both plant cystatins only slightly reduced the adhesion of normal mammary cells pointing to the higher sensitivity of cancer cells to both cystatins. These preliminary results open the possibility of considering the plant cystatin dgECP1 for anti-cancer strategies.

Selection of internalizing RNA aptamers into human breast cancer cells derived from primary sites.

Breast cancer is the most common cancer in women. Although chemotherapy is still broadly used in its treatment, adverse effects remain a challenge. In this scenario, aptamers emerge as a promising alternative for theranostic applications. Studies using breast cancer cell lines provide useful information in laboratory and preclinical investigations, most of which use cell lines established from metastatic sites. However, these cell lines correspond to cell populations of the late stage of tumor progression. On the other hand, studies using breast cancer cells established from primary sites make it possible to search for new theranostic approaches in the early stages of the disease. Therefore, this work aimed to select RNA aptamers internalized by MGSO-3 cells, a human breast cancer cell line, derived from a primary site previously established in our laboratory. Using the Cell-Internalization SELEX method, we have selected two candidate aptamers (ApBC1 and ApBC2). We evaluated their internalization efficiencies, specificities, cellular localization by Reverse Transcription-qPCR (RT-qPCR) and confocal microscopy assays. The results suggest that both aptamers were efficiently internalized by human breast cancer cells, MACL-1, MDA-MB-231, and especially by MGSO-3 cells. Furthermore, both aptamers could effectively distinguish human breast cancer cells derived from normal human mammary cell (MCF 10A) and prostate cancer cell (PC3) lines. Therefore, ApBC1 and ApBC2 could be promising candidate molecules for theranostic applications, even in the early stages of tumor progression.

Inhibitory Effects of Alpha-Connexin Carboxyl-Terminal Peptide on Canine Mammary Epithelial Cells: A Study on Benign and Malignant Phenotypes.

Mammary cancer is highly prevalent in non-castrated female dogs. Cell-to-cell communication is an important mechanism to maintain homeostasis, and connexins are proteins that assemble to form the communicating gap junctions. In many cancers, communication capacity is reduced; several approaches are being tested in order to increase the communication capacity in cancer cells and, therefore, alter their viability. This study analyzed the effects of the alpha-connexin carboxyl-terminal peptide (αCT1) on canine mammary non-neoplastic and neoplastic epithelial cells. Seven canine epithelial mammary cell lines were used. Among these, one was a normal canine epithelial mammary cell line (LOEC-NMG), two canine mammary adenomas (LOEC-MAd1 and LOEC-MAd2), and four canine mammary adenocarcinomas (LOEC-MCA1, LOEC-MCA2, LOEC-MCA3 and CF41). The αCT1 corresponds to a short Cx43 C-terminal sequence linked to an internalization sequence called the antennapedia. After 24 h of incubation, the medium containing different αCT1 peptide concentrations was added to the cells, and only the culture medium was used for control. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test was used to quantify cell viability before treatment and 48, 72, and 96 h after the treatment. Results showed that the normal mammary epithelial cell line (LOEC-NMG) was resistant to treatment with αCT1, which is consistent with a previous study on human mammary cell lines. One of the adenoma cell lines (LOEC-MAd2) was also resistant to treatment with αCT1, although the other (LOEC-MAd1) was susceptible to treatment, mostly at 72 h after treatment. Regarding the four canine adenocarcinoma cell lines, they differ regarding the susceptibility to the treatment with αCT1. Three cell lines, canine mixed adenocarcinoma (LOEC-MCA1), canine complex adenocarcinoma (LOEC-MCA2), and commercial canine mammary adenocarcinoma cell line CF41, were susceptible to treatment with αCT1, while one canine mammary adenocarcinoma cell line (LOEC-MCA3) was resistant to treatment. In most αCT1 treated cell lines, Cx43 was strongly detected in cell membranes by immunofluorescence. We propose that αCT1 restored the cell-to-cell communication capacity of neoplastic cells and induced inhibitory effects on cell viability.

Abstract IA16: Creation and preliminary analyses of a full spectrum of breast cancers initiated de novo from normal human mammary cells

Abstract Human breast cancers (BCs) are well recognized for the diagnostic and treatment challenges posed by their extensive and continuously evolving genetic and biologic heterogeneity. Although much progress has been made in identifying features that may serve as new targets, most of these have not been found to be usefully applied to metastatic BCs characterized by a lack of cellular expression of ER, PR, HER2 and a high proliferative activity. Accordingly, these generally incurable BCs, thus identified as “triple-negative” (TNBCs) are in particular need of strategies to model their development in ways that could facilitate the design and testing of effective treatments. Given the drawbacks historically encountered in using cell lines, mouse models, and patient-derived xenografts for this purpose, we launched experiments to assess the potential of creating a useful human model of TNBC de novo. We first discovered that forced expression of KRASG12D (K) alone is sufficient to produce low grade, but persistent human tumors in transplanted immunodeficient NRG mice from 2 of the 3 cell types that constitute and maintain the normal adult human female mammary gland. Histologically, these YB-1-dependent K–induced tumors were classified as variably human ER-, PR- and HER2-positive, invasive ductal carcinomas, also able to produce derivative small tumors in secondary hosts. Subsequent experiments to test whether the indolent properties of these tumors can be modified (i.e., by additional forced expression of human hepatocyte growth factor, or maintenance of transplanted mice on an obesity-inducing diet) showed that neither of these treatments enhance the growth of the K-induced tumors. However, in sharp contrast, transplantation of female NRG mice with the same types of freshly isolated normal adult female mammary cells transduced with lentivirally-encoded BMI1, MYC and TP53R273C and K (KBMT) produced growths containing an increased frequency of Ki67+ cells within a week, and subsequently, produced still YB-1-elevated, but continuously expanding TNBCs. Interestingly, K was required in addition to all 3 of these oncogenes to obtain aggressively expanding TNBCs and, when any one of the BMI1, MYC, or TP53R273C oncogenes was omitted, variably diminished outputs were obtained. FACS analyses showed the surface marker phenotypes of the cells present in any of the more rapidly growing tumors to be remarkably homogeneous with features of normal basal cells. In addition, despite any gross evidence of metastatic capability, cells isolated from all KBMT-induced tumors tested could be expanded in vitro and generated fast growing tumors in transplanted secondary mice. Taken together, these findings demonstrate the feasibility of rapidly and reproducibly creating a model of human TNBC de novo from initially normal human mammary cells, thus offering a new and powerful platform for potentially developing novel prevention, diagnostic, as well as treatment approaches applicable to this disease. Citation Format: Susanna Tan, Davide Pellacani, Sylvain Lefort, Amal El-Naggar, Shengsen Ding, Poul H Sorensen, Martin Hirst, Connie Eaves. Creation and preliminary analyses of a full spectrum of breast cancers initiated de novo from normal human mammary 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 IA16.

Abstract A078: Characterization of normal human male mammary cells and their de novo transformed derivatives

Abstract Men normally develop and maintain bi-layered mammary glands that appear structurally and histologically similar to those that develop in women, but with reported deficient lobule formation. Men also develop breast cancer, mostly of the ER+ subtype and an age-associated increasing onset, but at a 100-fold lower incidence and overall worse outcome. However, the classification and treatment of male breast cancers are largely based on strategies developed for female patients, despite their known hormonal and other differences. This is likely due, at least in part, to the fact that adult male mice, unlike humans do not develop full mammary glands. Thus, experimental access to mouse models have been lacking and studies of “normal” male mammary tissue have thus been largely restricted to analysis of human male gynecomastia samples. We now report the utility of multiple systems for analyzing normal adult human female mammary cell properties to this source of freshly obtained and viably cryopreserved normal human male mammary tissue. Histological examination of male mammary tissues (3 donors) confirmed their reported bi-layered structure surrounded by fibroblasts and lack of gross evidence of lobules. Use of procedures created for analyzing female breast tissue indicates that the human male mammary glands contain the same subsets but in different relative proportions; i.e., the proportion of EpCAM+CD49f- mammary cells (Luminal Cells, LCs) is relatively increased and of the EpCAM+CD49f+ cells (Luminal Progenitors, LPs) and EpCAM-CD49f+ cells (Basal Cells, BCs) is correspondingly decreased, consistent with a lack of alveolae. However, the freshly isolated male LPs and BCs contain a similar frequency of EGF-responsive colony-forming cells (CFCs) as female LPs and BCs. Subcutaneous transplantation of the male mammary cells in both male and female immunodeficient mice resulted in the generation of hollow mammary structures 4 weeks later as previously shown for female cells, and these were also found to contain LC, LP and BC populations including some with detectable levels of CFCs suggesting their derivation from a male mammary stem cell population (Eirew et al Nat Med 2008). We have also found evidence from qPCR and RNA-seq analyses of reduced progesterone signaling and alveologenesis in male LPs and LCs. These findings suggest hormonal differences may play a major role in regulating the altered ratios of cell types present in the normal adult human male mammary gland in comparison to premenopausal female. Interestingly, preliminary experiments also indicate that normal human male mammary cells transduced with KRASG12D without, or with BMI1+MYC+TP53R273C , generate analogous slowly-growing ER+, and rapidly-growing, highly aggressive tumours, respectively, in male immunodeficient mice. Taken together, our findings demonstrate the potential of established cell culture, xenotransplantation and molecular analytical tools to reveal the altered mechanisms that account for the different structure and biology of human male mammary tissue. Citation Format: Shengsen Ding, Susanna Tan, Ebrahim Eskandari-Nasab, Shrinka Sen, Martin Hirst, Connie Eaves. Characterization of normal human male mammary cells and their de novo transformed derivatives [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 A078.

Abstract A023: High glucose increases DNA damage and elevates the expression of multiple DDR genes

Abstract The DNA Damage Response (DDR) pathways sense DNA damage and coordinate robust DNA repair and bypass mechanisms. A series of repair proteins are recruited depending on the type of breaks and lesions to ensure overall survival. An increase in glucose levels was shown to induce genome instability, yet the links between DDR and glucose are still not well investigated. In this study, we aimed to identify dysregulation in the transcriptome of normal and cancerous breast cell lines upon changing glucose levels. We first performed bioinformatics analysis using a microarray dataset containing the triple-negative breast cancer (TNBC) MDA-MB-231 and the normal human mammary epithelium MCF10A cell lines grown in high glucose (HG) or in the presence of the glycolysis inhibitor 2-deoxyglucose (2DG). Interestingly, multiple DDR genes were significantly upregulated in both cell lines grown in HG. In the wet lab, we remarkably found that HG results in severe DNA damage to TNBC cells as observed using the comet assay. In addition, several DDR genes were confirmed to be upregulated using qPCR analysis in the same cell line. Our results propose a strong need for DDR pathways in the presence of HG to oppose the severe DNA damage induced in cells. Citation Format: Mai A. Rahmoon, Reem A. Elghaish, Aya A. Ibrahim, Zina Alaswad, Mohamed Z. Gad, Sherif F. El-Khamisy, Menattallah Elserafy. High glucose increases DNA damage and elevates the expression of multiple DDR genes [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr A023.

Cytotoxic effects of crotoxin from Crotalus durissus terrificus snake in canine mammary tumor cell lines.

Mammary gland tumors are the most prevalent neoplasm in intact female dogs, and they are good natural models to study comparative oncology. Most canine mammary malignancies, as in women, are commonly refractory to conventional therapies and demand continuous new therapeutic approaches. Crotalus durissus terrificus, also called rattlesnake, has more than 60 different proteins in its venom with multiple pharmaceutical uses, such as antitumor, antiviral, and antimicrobial action. Crotoxin, a potent β-neurotoxin formed by the junction of two subunits, a basic subunit (CB-PLA2) and an acidic subunit (crotapotin), has already been reported to have anticancer properties in different types of cancers. In this work, we describe the cytotoxic potential of crotoxin and its subunits compared to doxorubicin (drug of choice) in two canine mammary carcinoma cell lines. Crotoxin, CB-PLA2, crotalic venom, and doxorubicin decreased cell viability and the ability to migrate in a dose-dependent manner, and crotapotin did not present an antitumoral effect. For all compounds, the predominant cell death mechanism was apoptosis. In addition, crotoxin did not show toxicity in normal canine mammary gland cells. Therefore, this work showed that crotoxin and CB-PLA2 had cytotoxic activity, migration inhibition, and pro-apoptotic potential in canine mammary gland carcinoma cell lines, making their possible use in cancer research.

Sinensetin suppresses breast cancer cell progression via Wnt/β-catenin pathway inhibition.

Although there are many treatments for breast cancer, such as surgery, radiotherapy, chemotherapy, estrogen receptor antagonists, immune checkpoint inhibitors and so on. However, safer and more effective therapeutic drugs for breast cancer are needed. Sinensetin, a safer therapeutic drugs, come from citrus species and medicinal plants used in traditional medicine, while its role and underlying mechanism in breast cancer remain unclear. Our study aimed to investigate the role and mechanism of sinensetin in breast cancer. Cell Counting Kit-8 (CCK-8) was used to determine the safe concentration of sinensetin in MCF-10A, MCF7 and MDA-MB-231 cells; 120 μM sinensetin was used in subsequent experiments. Real time polymerase chain reaction (RT-PCR), Western blotting, Terminal Deoxynucleotidyl Transferase mediated dUTP Nick-End Labeling (TUNEL) apoptosis assay, Transwell invasion assay and Clone formation assay were used in this study to determine cell viability, mRNA expression, protein levels, apoptosis, proliferation, invasion and so on. Herein, our results showed that 120 μM sinensetin suppressed the cell viability and promoted apoptosis of MCF7 and MDA-MB-231 cells. Treatment with 120 µM sinensetin for 24 h showed no significant toxicity to normal mammary cells; 120 μM sinensetin decreased cell proliferation, invasion, and epithelial-mesenchymal transition (EMT), and downregulated β-catenin, lymphatic enhancing factor 1 (LEF1), T-cell factor (TCF) 1/TCF7, and TCF3/TCF7L1 expression in MCF7 and MDA-MB-231 cells. The Wnt agonist SKL2001 reversed the inhibitory effect of sinensetin on cell survival, metastasis, and EMT. Sinensetin-induced downregulation of β-catenin, LEF1, and TCF1/TCF7 expression were upregulated by SKL2001 in MCF7 and MDA-MB-231 cells. In summary, sinensetin suppressed the metastasis of breast cancer cell via inhibition of Wnt/β-catenin pathway and there were no adverse effects on normal breast cells. Our study confirmed the role of sinensetin in breast cancer cells and provided a better understanding of the underlying mechanism.

Evaluation of a Synthetic Polyethyleneimine Based Polymeric Vector for ING4 Gene Delivery to MCF-7 Breast Cancer Cells.

Breast cancer is the most common type of cancer among women and the second most common cause of death after lung cancer. The inhibitor of growth (ING) transcript levels are often suppressed in cancer cells, making it a promising candidate for cancer therapy. In this study, we aimed to formulate a polyplex that effectively carries and delivers pING4 to breast cancer cells. Polyethyleneimine (PEI)-based non-viral vectors were synthesized and characterized for plasmid DNA delivery. Complexation was achieved via electrostatic interactions between the synthesized polymeric vectors and plasmid DNA. Characterization studies were conducted by testing Sodium dodecyl sulfate-induced complexation, Deoxyribonuclease I protection, and serum stability of the polyplexes. Subsequently, polyplexes were tested on MCF-7 cells for anticancer activity using the XTT cell viability assay. Western blot analysis was performed for the ING4 protein. Polyplexes carrying the ING4 gene exhibited significantly lower cell viability than control polyplexes (p=0.0067). During the 5-day viability assay, the lowest cell viability was observed on day 4. Approximately 69.11±2.18% cell viability was observed with ING4 treatment and the control group showed no cell death on day 4 (101.53±5.06%). The prepared delivery systems did not show a toxic effect on MCF-7 cells treated alone. In addition, the MCF10A normal mammary cell line was used as a positive control. Western blotting was performed to confirm the overexpression of ING4 protein in the treatment groups. Unlike in the control groups, the overexpression of ING4 was clear in the wells of the treatment group. Our findings suggest that ING4 gene delivery using prepared PEI-based nonviral delivery systems is a promising approach for breast cancer treatment.

SAT635 Role Of The Human Prolactin Receptor I-tail In The Pathogenesis Of Breast Cancer

Abstract Disclosure: C.V. Clevenger: None. S. Shen: None. Evidence from epidemiological, cellular, and genetic analyses indicate that the hormone prolactin (PRL) and its receptor (hPRLr) in humans are significantly involved in breast cancer pathogenesis. The intermediate form of hPRLr (hPRLrI) induces significant increases in proliferation and anchorage-independent growth of normal mammary epithelia in vitro when co-expressed with the long form hPRLr (hPRLrL). However, the exact mechanism of how the hPRLrI transforms mammary epithelium is not well understood. The hPRLrI is produced by alternative splicing that induces a frameshift, resulting in a premature stop codon and a novel 13 amino acid tail ("I-Tail") gain. The ubiquitin-like protein neural precursor cell expressed developmentally down-regulated protein 8 (NEDD8) was found to be associated with the I-tail by yeast two-hybrid analysis. Treatment with a selective NEDD8 inhibitor resulted in the altered stability of the hPRLrL and the death of breast cancer cells. Given this, we hypothesized that the hPRLrI I-tail contributes to mammary transformation. hPRLrL, hPRLrI, hPRLrIΔ13 (I-tail removed mutant due to loss of 13 C-terminal amino acid resides) were delivered to MCF10AT cells by lentivirus transduction and subsequently analyzed. Anchorage-independent cell growth was determined by soft agar assay, cell proliferation was determined with a Incucyte analysis, and cell motility was examined by wound healing assay. In addition, signaling pathway and differential gene expression alterations mediated by the I-tail were determined by RNA-seq and signaling analysis. Successful expression of the hPRLrL+hPRLrIΔ13 in the MCF10AT transfectant resulted in significant reductions or alterations of anchorage-independent growth, cell proliferation, ERK1/2 activation, and PRL-induced different gene expression. These findings demonstrate that the hPRLrI I-tail contributes to the transformation of normal mammary epithelia cells. Presentation: Saturday, June 17, 2023