Normal Mammary Tissue Research Articles

Expression of tumour transcription factor GLI1 in canine mammary tumours tissue.

BackgroundMammary tumor is one of the most common diseases of canine in pet clinics.ObjectivesThis study investigates the distribution and expression of the tumor transcription factor GLI1 and the downstream proteins, Bmi1 and Sox2, in canine mammary tumors and paracancerous tissues.MethodsCancerous and paracancerous normal mammary tissues were detected using western blotting (WB), and immunohistochemistry.ResultsThe results showed that the histopathology of different types in mammary tumors by microscopic observation. GLI1/Bmi1/Sox2 expression was significantly higher in canine mammary invasive carcinoma than in ductal carcinoma and adjacent normal mammary tissues (p < 0.01). The expression of GLI1 in invasive carcinoma tissues was significantly higher than Bmi1 and Sox2, while Sox2 expression in ductal carcinoma tissues was significantly higher than GLI1 and Bmi1 (p < 0.01). GLI1/Bmi1/Sox2 all showed positive reactions in both mammary tumor and adjacent normal mammary tissues with immunohistochemistry. GLI1 and Sox2 showed strong positive staining in the cytoplasm of invasive mammary carcinoma and ductal carcinoma cells, and weak positive staining in the nuclei. The positive Bmi1 reaction was mainly concentrated in the cytoplasm of invasive carcinoma and ductal carcinoma cells, while the positive reaction on the cell membrane was weak.ConclusionsWe speculate that GLI1 and related proteins play an important role in regulating the proliferation and differentiation of tumors. Therefore, it provides important reference for the pathogenesis and pathogenicity of canine mammary tumor.

A novel patient-derived organoid model for triple-negative breast cancer patients with residual disease after neoadjuvant chemotherapy for screening personalized drugs.

e12625 Background: Triple-negative breast cancer (TNBC) patients with residual disease after neoadjuvant chemotherapy (NAC) have high risk of recurrence. In this study, we developed a novel organoid culture method for growing TNBC patient samples after NAC, with the goal of generating models that more faithfully recapitulates the key features of the primary tumor for screening personalized drugs. Methods: We established organoid lines derived from TNBC patients who had residual tumors after anthracycline and taxane±platinum based NAC as well as normal human mammary tissues. The histopathology signatures of organoid lines were characterized by H&amp;E staining and immunohistochemistry. The genetic and transcriptional features of organoids were analyzed by targeted sequencing and RNA sequencing. A set of clinical drugs were screened for their ability to suppress cancer organoids in vitro and the consistency was also analyzed between clinical response and drug efficacy of organoid models. Results: A total of 10 cancer organoid lines were successfully established, and cultured stably for more than 4 months. For comparison, 2 organoids derived from normal mammary tissues were generated as well. Our success rate for establishing cancer organoids from TNBC patient samples after NAC was 83% (10 out of 12 samples), and for organoids from normal mammary tissues, it was 100% (2 out of 2 samples). These organoid lines closely recapitulated the histopathology, genetic and transcriptional signature, and intratumor heterogeneity of the primary tissues. We found that active PI3K/AKT/mTOR pathways signaling is always required for cancer organoids from TNBC patient samples after NAC, while Wnt pathway activation is dispensable for generation of them in comparison to the organoid cultures from normal breast tissues. More importantly, we found that the anti-Trop-2 antibody-drug conjugate sacituzumab govitecan (IMMU-132) showed higher sensitivity to most cancer organoids from TNBC patient samples after NAC (8 out of 10 samples) with IC50 ranging from 0.3 nM to 86 nM in contrast to 5-FU (5 out of 10 samples) with IC50 ranging from 0.9 μM to 10.2 μM in vitro, whose prodrug capetabine is considered as a standard option in the postneoadjuvant setting. These results were also confirmed in patient-derived tumor xenograft models. The consistency of drug efficacy in the established cancer organoids and the patient responses in the clinic reached to 70% (7 out of 10 samples). Conclusions: We established a novel organoid culture system for TNBC patient samples after NAC, which was potentially a useful platform to explore molecular characteristics of TNBC patients after NAC and discover personalized drugs for intensive treatment.

Integrative Analysis of KCNK Genes and Establishment of a Specific Prognostic Signature for Breast Cancer.

Two-pore domains potassium channel subunits, encoded by KCNK genes, play vital roles in breast cancer progression. However, the characteristics of most KCNK genes in breast cancer has yet to be clarified. In this study, we comprehensively analyzed the expression, alteration, prognosis, and biological functions of various KCNKs in breast cancer. The expression of KCNK1/4/6/9/10/13 were significantly upregulated, while KCNK2/3/5/7/17 were downregulated in breast cancer tissues compared to normal mammary tissues. Increased expression of KCNK1/3/4/9 was correlated with poor overall survival, while high expression of KCNK2/7/17 predicted better overall survival in breast cancer. Eight KCNK genes were altered in breast cancer patients with a genomic mutation rate ranged from 1.9% to 21%. KCNK1 and KCNK9 were the two most common mutations in breast cancer, occurred in 21% and 18% patients, respectively. Alteration of KCNK genes was associated with the worse clinical characteristics and higher TMB, MSI, and hypoxia score. Using machine learning method, a specific prognostic signature with seven KCNK genes was established, which manifested accuracy in predicting the prognosis of breast cancer in both training and validation cohorts. A nomogram with great predictive performance was afterwards constructed through incorporating KCNK-based risk score with clinical features. Furthermore, KCNKs were correlated with the activation of several tumor microenvironment cells, including T cells, mast cells, macrophages, and platelets. Presentation of antigen, stimulation of G protein signaling and toll-like receptor cascaded were regulated by KCNKs family. Taken together, KCNKs may regulate breast cancer progression via modulating immune response which can serve as ideal prognostic biomarkers for breast cancer patients. Our study provides novel insight for future studies evaluating their usefulness as therapeutic targets.

A Spectroscopic Technique to Simultaneously Characterize Fatty Acid Uptake, Mitochondrial Activity, Vascularity, and Oxygen Saturation for Longitudinal Studies In Vivo.

Aggressive breast cancer has been shown to shift its metabolism towards increased lipid catabolism as the primary carbon source for oxidative phosphorylation. In this study, we present a technique to longitudinally monitor lipid metabolism and oxidative phosphorylation in pre-clinical tumor models to investigate the metabolic changes with mammary tissue development and characterize metabolic differences between primary murine breast cancer and normal mammary tissue. We used optical spectroscopy to measure the signal of two simultaneously injected exogenous fluorescent metabolic reporters: TMRE (oxidative phosphorylation surrogate) and Bodipy FL C16 (lipid catabolism surrogate). We leverage an inverse Monte Carlo algorithm to correct for aberrations resulting from tissue optical properties and to extract vascular endpoints relevant to oxidative metabolism, specifically oxygen saturation (SO2) and hemoglobin concentration ([Hb]). We extensively validated our optical method to demonstrate that our two fluorescent metabolic endpoints can be measured without chemical or optical crosstalk and that dual measurements of both fluorophores in vivo faithfully recapitulate the measurements of each fluorophore independently. We then applied our method to track the metabolism of growing 4T1 and 67NR breast tumors and aging mammary tissue, all highly metabolic tissue types. Our results show the changes in metabolism as a function of mammary age and tumor growth, and these changes can be best distinguished through the combination of endpoints measured with our system. Clustering analysis incorporating both Bodipy FL C16 and TMRE endpoints combined with either SO2 or [Hb] proved to be the most effective in minimizing intra-group variance and maximizing inter-group differences. Our platform can be extended to applications in which long-term metabolic flexibility is important to study, for example in tumor regression, recurrence following dormancy, and responses to cancer treatment.

Micro RNA differential expression profile in canine mammary gland tumor by next generation sequencing.

Canine mammary gland tumors are very common and represent a potential model of human breast cancer, and microRNA (miRNAs) are promising biomarkers and therapeutic targets for these tumors. Accordingly, we aimed to identify miRNAs differentially expressed in canine mammary gland tumors using next generation sequencing (NGS), with subsequent confirmatory qPCR and target gene analyses. Mammary gland tissue was collected from healthy dogs (n=7) and dogs with suspected tumors (n=80). A subset of samples was analyzed with NGS to identify differentially expressed miRNAs with CLC Genome Workbench. Normal (n=10), tumor-adjacent (n=6), and tumor-bearing (n=76) mammary gland tissue samples were analyzed for the identified miRNAs using qPCR. An in silico analysis (TargetScan) was performed to predict the miRNAs' target genes using gene ontology (GO) terms and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (DAVID). We identified four miRNAs (cfa-miR-1-3p, cfa-miR-133a-3p, cfa-miR-133b-3p, and cfa-miR-133c-3p) as down regulated in canine mammary gland tumor tissues relative to normal and tumor adjacent tissues. KEGG analysis revealed the potential target genes of cfa-miR-1-3p are related to the Rap1 signaling pathway, adherens junction, and Ras signaling pathway, and those of the miR-133 family are related to the TGF-beta signaling pathway, synaptic vesicle cycle, and sphingolipid signaling pathway. In combination, these target genes are related to the regulation of transcription and DNA binding transcription (GO analysis), and the Hippo signaling pathway, adherens junction, and endocytosis (KEGG analysis). Accordingly, we suggest these four miRNAs are promising potential biomarker candidates for canine mammary gland tumors warranting further investigation.

Interactions between the breast tissue microbiota and host gene regulation in nonpuerperal mastitis

BackgroundNonpuerperal mastitis (NPM) causes considerable psychological distress in females, since it is difficult to diagnose and treat. A spectrum of etiological factors can lead to NPM. However, the pathogenesis of NPM remains unclear. Here, we aimed to dissect the role of host gene–microbe interactions in NPM. MethodsWe compared the breast tissue microbiome between NPM patients and controls using 16S rRNA sequencing. We also compared the gut microbiome between NPM patients and healthy controls. Moreover, we investigated whether the breast tissue microbiome was associated with an altered gut microbiome in patients with NPM. We analyzed differentially expressed genes in inflammatory tissues of mammary gland from patients with NPM and normal mammary gland tissues from patients with benign and non-infectious breast disease by RNA-sequencing (RNA-seq). Lastly, we explored the association of specific bacterial taxa with differential expression of immune-related genes and differences in infiltrating immune cells. ResultsThe breast tissue microbiome from NPM and controls showed significant differences in community composition. The breast tissue shared a relatively small proportion of bacterial communities with the gut in patients with NPM. Ruminococcus (family Ruminococcaceae) of breast tissue was positively correlated with the differentially expression of immune-related genes between NPM patients and controls, including antigen processing and presentation genes (ICAM1, LGMN, THBS1, TAP1, HSPA1B and HSPA1A), cytokine receptor gene IL15RA, and chemokine gene CCN1. Rhizobium of breast tissue was negatively correlated with the differentially expression of the antigen processing and presentation gene HSPA6 between NPM patients and controls. We also found that Ruminococcus (family Ruminococcaceae), Coprococcus, and Clostridium of breast tissue positively correlated with the difference of CD8+ T cells between NPM patients and controls. ConclusionsWe preliminarily explored the potential role of host–microbe interactions in NPM. We demonstrate cross-talk between the breast tissue microbiome and the gut microbiome in patients with NPM. We suggest that NPM microbiome composition influences the immune microenvironment of the disease by affecting the transcriptome. This is an exploratory study and further investigation of host–microbe interactions and its potential mechanism in NPM development are warranted.

Abstract PD3-05: The paradoxical role of RalA and RalB in triple negative breast cancer

Abstract Background: Breast Cancer (BC) is the most common cancer and leading cause of cancer associated mortality in women worldwide. TNBC patients have the highest mortality mainly due to lack of receptors for targeted therapies. RalA and RalB are small GTPases that are known to regulate growth and metastasis in several cancers. However, roles for these GTPases in BC is poorly understood. The goal of this study was to investigate the contributions of RalA and RalB in TNBC. Methods: Control, RalA or RalB CRISPR knockout (KO) MDA-MB-231 cells were injected into the mammary gland of nod-skid-gamma (NSG) mice. Tumor growth was monitored and groups were taken as they met early removal criteria. Tumors and lungs were formalin-fixed and paraffin embedded. Tumors underwent immunohistochemical staining for Ki-67 and Cleaved caspase-3 and lungs were stained for hematoxylin and eosin and imaged on a Leica Aperio ScanScope XT to calculate lung metastasis. RalA or RalB were also depleted in MDA-MB-231 and MVT1 cells by shRNA. In addition, RalA depleted MDA-MB-231 cells were labeled with luciferase and injected into the tail vein of NSG mice and imaged on an IVIS spectrum to test seeding and lung colonization. Immunohistochemistry of patient TMAs was preformed on a Bond RX autostainer using RalA (Abcam, ab126627, 1:2000). Immunohistochemical stains were imaged on a PerkinElmer’s Vectra® Automatic Imaging System and quantified using inForm® Advanced Image Analysis software. Statistical significance of Kaplan-Meier survival curves were determined by log rank. Results: RalA knockout and depletion slowed primary orthotopic tumor growth in MDA-MB-231 and MVT1 cells. RalB KO had the opposite effect and increased growth rate compared to controls and RalA KO cells. Ki67 and cleaved caspase 3 IHC staining of tumors indicate KO of RalA decreased proliferation, whereas KO RalB increased proliferation with no change in apoptosis. RalA KO decreased the number and area of lung metastasis in both spontaneous and experimental metastasis assays. RalB KO or depletion caused an increase in the area and number of metastasis. Utilizing data from the METABRIC and TCGA BC datasets, elevated RALA, but not RALB, was prognostic of worse outcome in the overall BC populations and the TNBC populations specifically. RALA was shown to be more highly expressed in BC, particularly TNBC, relative to normal mammary tissue whereas RalB was decreased in BC and TNBC. IHC staining of a TMA comprised of all BC subtypes and a TMA of only TNBC samples confirmed RalA as a prognostic marker of patient outcome. Conclusions: RalA and RalB have important but paradoxical roles in TNBC. Citation Format: Jonathan M. Spehar, Katie A. Thies, Matthew W. Cole, Rachel E. Schafer, Dillon S. Richardson, Sarah A. Steck, Manjusri Das, Arthur W. Lian, Alo Ray, Sue E. Knoblaugh, Cynthia D. Trimmers, Gina M. Sizemore, Steven T. Sizemore. The paradoxical role of RalA and RalB in triple negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD3-05.

LncRNA RP11-214F16.8 drives breast cancer tumorigenesis via a post-translational repression on NISCH expression

Increasing research interests have been aroused in exploring the function of long non-coding RNA (lncRNA) in breast cancer and developing lncRNA-targeted diagnosis, treatment and prognosis. In GEPIA2 database, we compared the expression pattern of the lncRNA RP11-214F16.8 in normal mammary tissues and breast cancer tumors and its correlation with the overall death rate of breast cancer patients. Gain- and loss-of function assays were employed to study function of the lncRNA in breast cancer cell lines in vitro while xenograft tumor growth assay was performed to investigate its function in tumorigenesis in vivo. We also used RNA pull-down coupled with mass spectrometry to identify the lncRNA binding partner, and RIP, EMSA, ChIP and Co-IP assays as well to testify these physical interactions.We identified that up-regulation of the lncRNA RP11-214F16.8 is subtype-independently associated with a higher overall death rate in breast cancer patients. Increased RP11-214F16.8 expression endows breast cancer cells enhanced capabilities in the aspects of proliferation, invasion, migration and tumor-initiation, while loss of the lncRNA exerts the opposite effects. Mechanistically, the oncogenic property of RP11-214F16.8 lies to its post-translational repression on the tumor suppressor NISCH via recruiting SENP3-mediated de-SUMOylation and ubiquitin-proteasome-mediated protein degradation. NISCH in turn inhibits the transcription of RP11-214F16.8 through restraining the expression of the transcription factors located downstream of RAC1, PAK1 and ERK1/2 signaling transduction pathways. In all, dysregulation of RP11-214F16.8 not only stimulates activation of the proliferation- and migration-promoting signaling cascades, but also facilitates the removal of restrictions on self-transcription, which ensures the progression of tumorigenesis.

GLAD-PCR assay of DNA methylation sites in regulatory regions of some tumor-suppressor genes in breast cancer

Hypermethylation of the RcgY sites is shown for many cancer diseases. such aberrant methylation, suppressing the gene activity, occurs at early stages of carcinogenesis. Recently, using glad-pcR assay, we have detected aberrantly methylated RcgY sites, which can be considered to be epigenetic markers of colorectal, lung, and gastric cancers. in breast cancer, methylation of the regulatory regions of ALX4, BMP2, CCND2, CDH13, CDX1, FOXA1, GALR1, GATA5, GREM1, HIC1, HMX2, HS3ST2, HOXC10, ICAM5, LAMA1, RARB, RASSF1A, RUNX3, RXRG, RYR2, SFRP2, SOX17, TERT, and ZNF613 tumor-suppressor genes is reported. in the present work, we determined aberrantly methylated RcgY sites in the regulatory regions of these genes in dNa preparations from breast cancer tissues. the study of dNa samples from 30 tumor and 22 normal mammary tissue samples demonstrates a high diagnostic potential of selected R(5mc)gY sites in regulatory regions of CCND2, BMP2, GALR1, SOX17, HMX2, and HS3ST2 genes with total index of sensitivity and specificity for R(5mc)gY detection in tumor dNa 90.0 % and 100.0 %, respectively.

Assessment of Breast Cancer Stem Cell Activity Using a Spheroid Formation Assay.

Evidence is emerging that cancer cells are arranged as a hierarchy that spans from stem cells to lineage-restricted progenitor cells. The recent development of spheroid cultures with several tissue type has provided new opportunities to assess cancer stem cell (CSC) activity by allowing them to propagate under conditions that resemble the microenvironment for growth of tumors. One tissue type widely used for stem cell investigations is mammary tissue, and the sphere formation assay has been used in both normal mammary tissue and in breast cancer. Here, we describe detailed experimental methodology for generating and propagating spheres from normal mammary tissue and primary breasttumors of mice, patient derived xenografts (PDXs) and breast cancercell lines. We further describe how these sphere cultures can be employed for coculture assays to assess the effect of tumor microenvironment (TME) on self-renewal ability of CSCs in breast cancer.

Long Noncoding RNAs-LET Behave as a Noncoding Signature for Early-Onset Menarche and Late-Onset Menopause in Breast Cancer Patients

Background: Breast cancer (BC) as a major cause of cancer-related death in women shows a very complex molecular and clinical phenotype, which has reduced the effectiveness of medical interventions. Evidence suggests that long noncoding RNAs (lncRNAs) are responsible for an important part of this complexity. This study aims to assess the expression and clinical implication of lncRNA LET in the pathobiology of BC. Materials and Methods: Quantitative real-time polymerase chain reaction was used to measure the expression of lncRNA-LET in breast tumors and adjacent normal-appearing tissues from 4 BC patients, as well as normal mammary tissues. Moreover, a bioinformatics approach was applied to uncover the potential lncRNA-LET-mediated sponge regulatory network as LET/miRNA/mRNA crosstalk. Results: Our study revealed that lncRNA-LET was significantly down-expressed not only in breast tumors but also in normal appearing breast tissues samples from BC subjects compared with true normal breast tissues obtained from healthy women. The low level of lncRNA-LET was meaningfully associated with early-onset menarche (≤13 years) and late-onset menopause (≥50) in patients. Moreover, the bioinformatics analyses support that lncRNA-LET could function as a tumor suppressor miRNA sponge. Conclusion: The results indicate that normal appearing breast tissues can undergo tumor-related molecular changes. Furthermore, they reveal the potential role of the dysregulation in LET-mediated ceRNA network in the pathophysiology of BC.

Breast reconstruction using a tissue expander after enucleation of a giant fibroadenoma: A case report.

IntroductionFibroadenomas are among the most common benign tumors in women. Juvenile giant fibroadenomas account for nearly 0.5% of all fibroadenomas. Due to its size, a giant juvenile fibroadenoma leaves a large defect or deformity after its resection. The optimal surgical management strategy for giant juvenile fibroadenomas remains unclear. Here, we report a case of successful breast reconstruction without residual deformity through gradual deflation of a saline-filled tissue expander after resection of a giant juvenile fibroadenoma.Presentation of caseA 14-year-old girl with a growing tumor in her left breast presented to a private clinic. Given that the tumor was 8 cm in size, phyllodes could not be ruled out. Consequently, she was referred to our hospital for further examination and treatment. Core needle biopsy confirmed the tumor to be a fibroadenoma. We resected the tumor and inserted a tissue expander filled with 120 mL of saline, matching the area of the large defect caused by tumor resection. We removed approximately 25 mL of saline every 3 weeks to aid normal mammary tissue enlargement. After completely draining saline from the tissue expander and confirming an acceptable enlargement of the residual mammary gland, we performed an operation to remove the tissue expander. Follow-up revealed that the symmetry and contour of the breast were excellent after the second operation.ConclusionsOur observations suggest that using a tissue expander to enlarge normal mammary tissue may help reconstruct large defects caused by excision of benign tumors.

The FoxQ1 transcription factor is a novel regulator of electron transport chain complex I subunits in human breast cancer cells.

The FoxQ1 is an oncogenic transcription factor that is overexpressed in basal-like and luminal-type human breast cancers when compared to the normal mammary tissue. The FoxQ1 is implicated in mammary tumor progression. However, the mechanism by which FoxQ1 promotes mammary tumorigenesis is not fully understood. In this study, we present experimental evidence for a novel function of FoxQ1 in the regulation of complex I activity of the electron transport chain. The RNA-seq data from FoxQ1 overexpressing basal-like SUM159 cells revealed a statistically significant increase in the expression of complex I subunits NDUFS1 and NDUFS2 when compared to the empty vector (EV) transfected control cells. Consistent with these results, the basal and ATP-linked oxygen consumption rates were significantly increased by FoxQ1 overexpression in SUM159 and luminal-type MCF-7 cells. The FoxQ1 overexpression in both cell lines resulted in increased intracellular levels of pyruvate, lactate, and ATP that was associated with overexpression of pyruvate dehydrogenase and pyruvate carboxylase proteins. Activity and assembly of complex I were significantly enhanced by FoxQ1 overexpression in SUM159 and MCF-7 cells that correlated with increased mRNA and/or protein levels of complex I subunits NDUFS1, NDUFS2, NDUFV1, and NDUFV2. The chromatin immunoprecipitation assay revealed the recruitment of FoxQ1 at the promoters of both NDUFS1 and NDUFV1. The cell proliferation of SUM159 and MCF-7 cells was increased significantly by overexpression of NDUFS1 as well as NDUFV1 proteins. In conclusion, we propose that increased complex I-linked oxidative phosphorylation is partly responsible for oncogenic role of FoxQ1 at least in human breast cancer cells.

Hormone receptor expression in aging mammary tissue and carcinoma from a rodent model after xenoestrogen disruption

AimsHormone receptors are the main markers applied for prognosis of breast cancer subtypes. Among modulators, exogenous chemical agents known as endocrine disruptors interact with certain receptors, triggering molecular pathways or increasing their expression. Bisphenol A (BPA), a xenoestrogen, interacts with several hormone receptors. Thus, our aim was to characterize the hormone receptor status in the mammary gland (MG) of aged female Mongolian gerbils exposed to BPA in pregnancy and lactation. MethodsWe evaluated the expression of receptors for estrogens (ERα and ERβ), progesterone (PR), prolactin (PRL-R), HER2/ErbB2, and androgen (AR) in normal and hyperplastic mammary tissue and in carcinomas developed after BPA exposure. Key findingsBPA-exposed MG presented increased ERα, whereas ERβ, PR, and PRL-R showed lower expression. AR and HER2/ErbB2 showed similar expression in normal and hyperplastic tissue from control, vehicle, and BPA groups. Both receptors were found in cytoplasm and nucleus in BPA-induced carcinoma. We demonstrate the presence of EZH2 expression, an epigenetic and epithelial-mesenchymal transition (EMT) marker, with a high H-score in BPA-exposed MG, which was associated with poor prognosis of cancer. Co-localization of ERα and EZH2 was present in normal and carcinoma features, corroborating the installation of ERα-positive mammary cancer associated with the EMT process. Enhanced EZH2 in BPA-exposed mammary tissue could decrease ERβ expression and promote tumorigenesis progress through HER2/ErbB2. SignificanceThe present study proposes the Mongolian gerbil as an experimental model for mammary carcinogenesis studies, based on BPA disruption that triggers a phenotype of increased ERα/HER2 positivity and depletion of ERβ/PR expression.

Ribosome Proteins Represented by RPL27A Mark the Development and Metastasis of Triple-Negative Breast Cancer in Mouse and Human.

Triple-negative breast cancer (TNBC) is known to have a poor prognosis and limited treatment options. The lack of targeted therapies and poor prognosis of patients with TNBC have made it urgent to discover novel critical diagnosis and therapeutic targets in the TNBC field. Here, in the current study, we integrated the single-cell RNA-sequencing (scRNA-seq) data from four normal mouse mammary tissues and four mouse breast tumors. Comparative analysis was conducted to identify the gene profiles of normal epithelial cells and cancer cells at different models. Surprisingly, two ribosomal protein genes, Rpl27a and Rpl15, were significantly upregulated in the cancer cells in all the TNBC models. Next, we accessed the scRNA-seq data from human primary and metastatic TNBC tissues, and comparative analysis revealed gene profiles of human primary and metastatic TNBC cancer cells. Ribosomal protein genes, represented by RPL27A and RPL15, showed significantly upregulated expression in metastatic TNBC cancer cells. Pathway analysis on the upregulated genes of the metastatic TNBC cancer cells identified the key regulators and signaling pathways that were driving the metastasis of the TNBC cancer cells. Specifically, EIF2 signaling was significantly activated, and major member genes of this signaling pathway were upregulated. In vitro study revealed that targeting RPL27A or EIF2 signaling in a TNBC cell line, MDA-MB-231, significantly reduced cell migration and invasion. Altogether, these data suggested that the RPL27A gene is conducting critical functions in TNBC cancer development and metastasis and is a potential therapeutic target for TNBC.

Development of mammary cancer in γ-irradiated F1 hybrids of susceptible Sprague-Dawley and resistant Copenhagen rats, with copy-number losses that pinpoint potential tumor suppressors.

Copenhagen rats are highly resistant to mammary carcinogenesis, even after treatment with chemical carcinogens and hormones; most studies indicate that this is a dominant genetic trait. To test whether this trait is also dominant after radiation exposure, we characterized the susceptibility of irradiated Copenhagen rats to mammary carcinogenesis, as well as its inheritance, and identified tumor-suppressor genes that, when inactivated or mutated, may contribute to carcinogenesis. To this end, mammary cancer-susceptible Sprague-Dawley rats, resistant Copenhagen rats, and their F1 hybrids were irradiated with 4 Gy of γ-rays, and tumor development was monitored. Copy-number variations and allelic imbalances of genomic DNA were studied using microarrays and PCR analysis of polymorphic markers. Gene expression was assessed by quantitative PCR in normal tissues and induced mammary cancers of F1 rats. Irradiated Copenhagen rats exhibited a very low incidence of mammary cancer. Unexpectedly, this resistance trait did not show dominant inheritance in F1 rats; rather, they exhibited intermediate susceptibility levels (i.e., between those of their parent strains). The susceptibility of irradiated F1 rats to the development of benign mammary tumors (i.e., fibroadenoma and adenoma) was also intermediate. Copy-number losses were frequently observed in chromosome regions 1q52-54 (24%), 2q12-15 (33%), and 3q31-42 (24%), as were focal (38%) and whole (29%) losses of chromosome 5. Some of these chromosomal regions exhibited allelic imbalances. Many cancer-related genes within these regions were downregulated in mammary tumors as compared with normal mammary tissue. Some of the chromosomal losses identified have not been reported previously in chemically induced models, implying a novel mechanism inherent to the irradiated model. Based on these findings, Sprague-Dawley × Copenhagen F1 rats offer a useful model for exploring genes responsible for radiation-induced mammary cancer, which apparently are mainly located in specific regions of chromosomes 1, 2, 3 and 5.

Reduced hyaluronan cross-linking induces breast cancer malignancy in a CAF-dependent manner

Hyaluronan (HA) cross-linking is a conformational state of HA, a covalent complex between HA and heavy chains (HCs) from inter-α-trypsin inhibitor (I-α-I) mediated by tumor necrosis factor-induced protein 6 (TSG6). Cross-linked HA has been identified as a protective factor in physiological and inflammatory conditions. However, the state of HA cross-linking in tumor microenvironment has not been fully elucidated. As a major constituent of the extracellular matrix (ECM), HA is mainly synthesized by cancer-associated fibroblasts (CAFs). Our study aimed to clarify the role of HA cross-linking in breast cancer malignancy. Compared to normal mammary gland tissues, cross-linked HA levels were significantly decreased in breast cancer and associated with tumor malignancy. When NFbs were activated into CAFs, the levels of cross-linked HA and TSG6 were both suppressed. Through upregulating TSG6, CAFs restored the high level of cross-linked HA and significantly inhibited breast cancer malignancy, whereas NFbs promoted the malignancy when the cross-linked HA level was reduced. Furthermore, the inhibitory role of HA cross-linking in tumor malignancy was directly verified using the synthesized HA-HC complex. Collectively, our study found that the deficiency of cross-linked HA induced breast cancer malignancy in a CAF-dependent manner, suggesting that recovering HA cross-linking may be a potential therapeutic strategy.

Disposition of Dietary Polyphenols in Breast Cancer Patients' Tumors, and Their Associated Anticancer Activity: The Particular Case of Curcumin.

Some polyphenol-derived metabolites reach human breast cancer (BC) tissues at concentrations that induce cell senescence. However, this is unknown for isoflavones, curcuminoids, and lignans. Here, their metabolic profiling in normal (NT) and malignant (MT) mammary tissues of newly-diagnosed BC patients and the tissue-occurring metabolites' anticancer activity are evaluated. Patients (n = 26) consumed 3 capsules/day (turmeric, red clover, and flaxseed extracts plus resveratrol; 296.4mg phenolics/capsule) from biopsy-confirmed diagnosis to surgery (5±2 days) or did not consume capsules (n = 13). NT and MT, blood, and urine are analyzed by UPLC-QTOF-MS using targeted metabolomics. Anticancer activity was tested in MCF-7 and MDA-MB-231 BC cells. Mainly phase-II metabolites were detected (108, 84, 49, and 47 in urine, plasma, NT, and MT, respectively). Total metabolite concentrations reached 10.7±11.1 and 2.5±2.4µmolL-1 in NT and MT, respectively. Free curcumin, but not its glucuronide, was detected in the tissues (1.1±1.8 and 0.2±0.2µmolL-1 in NT and MT, respectively). Breast tissue-occurring metabolites' antiproliferation was mainly exerted in p53-wild-type MCF-7 cells by curcuminoids through cell cycle arrest, senescence, and apoptosis induction via p53/p21 induction, while isoflavone-derived metabolites exerted estrogenic-like activity. Curcuminoids could be coadjuvants that might help fight BC upon regular consumption.

Interactions Between the Breast Tissue Microbiota and Host Gene Regulation in Nonpuerperal Mastitis

Background: Nonpuerperal mastitis (NPM) causes considerable psychological distress in females, since it is difficult to diagnose and treat. A spectrum of etiological factors, ranging from infection to autoimmune disorders, can lead to NPM. However, the pathogenesis of NPM remains unclear. Here, we aimed to dissect the role of host gene-microbe interactions in NPM. Methods: We performed 16S rRNA sequencing to characterize the breast tissue microbiome and identify bacteria that were differentially abundant between NPM patients and controls. We also performed 16S rRNA sequencing to characterize the gut microbiome and identify gut microbes with different abundances between NPM patients and healthy controls. Moreover, we investigated whether the breast tissue microbiome was associated with an altered gut microbiome in patients with NPM. We analyzed differentially expressed genes in inflammatory tissues of mammary gland from patients with NPM and normal mammary gland tissues from patients with benign and non-infectious breast disease by RNA-sequencing (RNA-seq). The landscape of infiltrating immune cell subtypes in the breast tissue immune microenvironment was analyzed using CIBERSORT, based on the gene expression profiling of breast tissues. Lastly, we modeled associations of relative abundances of specific bacterial taxa with differential expression of immune-related genes and differences in infiltrating immune cells. Results: The composition and functions of the breast tissue microbiota communities differed between NPM patients and controls. The breast tissue shared a relatively small proportion of bacterial communities with the gut in patients with NPM. The relative abundance of Ruminococcus (family Ruminococcaceae) of breast tissue was positively correlated with the differentially expression of immune-related genes between NPM patients and controls, including antigen processing and presentation genes (ICAM1, LGMN, THBS1, TAP1, HSPA1B and HSPA1A), cytokine receptor gene IL15RA, and chemokine gene CCN1. The relative abundances of Rhizobium of breast tissue was negatively correlated with the differentially expression of the antigen processing and presentation gene HSPA6 between NPM patients and controls. We also found that the relative abundance of Ruminococcus (family Ruminococcaceae), Coprococcus, and Clostridium of breast tissue positively correlated with the difference of CD8+ T cells between NPM patients and controls. The relative abundance of Ruminococcus (family Ruminococcaceae) of breast tissue positively correlated with resting memory CD4+ T cells. The relative abundance of Leucobacter of breast tissue negatively correlated with CD8+ T cells. The relative abundance of Caulobacter of breast tissue positively correlated with activated memory CD4+ T cells. Conclusions: Our results explored the potential role of host-microbe interactions in the etiology of NPM. We demonstrate cross-talk between the breast tissue microbiome and the gut microbiome in patients with NPM. We suggest that NPM microbiome composition influences the immune microenvironment and natural history of the disease by affecting the transcriptome, which merits further study. Funding Information: This study was supported by the Youth Science Fund Project of Jiangxi Provincial Department of Science and Technology (20192BAB215037), the Science and Technology Project of Education Department of Jiangxi Province (GJJ180057) and the Science and Technology Project of Education Department of Jiangxi Province (GJJ180056). Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: The study was approved by the Ethics Committee of the First Affiliated Hospital of Nanchang University (Nanchang, China), and informed consent was obtained from all subjects.

Hematopoietic protein RELT stains prominently in breast cancer and binds the cytoskeletal protein Filamin A.

Abstract Receptor Expressed in Lymphoid Tissues (RELT) is a human TNFR superfamily member expressed most prominently in cells and tissues of the hematopoietic system. Mouse knockout studies indicate that RELT functions in part by negatively regulating T-cell activation. RELT has two homologous binding partners, RELL1 and RELL2, and collectively, these proteins are described as RELT family members. This study sought to identify novel protein interactions with RELT family members and to compare the expression of RELT in normal and diseased tissues. A yeast two-hybrid screen utilizing RELL1 as bait identified the cytoskeletal protein Filamin A (FlnA); physical interaction between FlnA and RELT family members was confirmed by in vitro co-immunoprecipitation. A truncated mutant of FlnA disrupts the ability of RELT family members to activate the p38 pathway. Previous reports indicate that autoantibodies against RELT are associated with breast cancer and that RELL2 prevents migration and invasion of the metastatic breast cancer cell line MDA-MB-231 (231), while multiple studies suggest a link between FlnA and breast cancer. We therefore sought to examine the association of RELT family members with breast cancer. Western blotting revealed significant expression of endogenous RELT family members in the breast cancer cell lines 231 and MCF-7. Immunohistochemistry (IHC) revealed a higher intensity of RELT staining in breast cancer tissues versus normal mammary tissue. Additionally, overexpression of RELT family members enhanced apoptosis in 231 cells. Collectively, these results further our understanding of signal transduction pathways associated with RELT family members and provide evidence that RELT is upregulated in breast cancer.