Case report: Primary vulvar adenocarcinoma of mammary gland type-its genetic characteristics by focused next-generation sequencing.

Mammary-like vulvar adenocarcinoma (MLVA) is an exceedingly rare subtype of vulvar adenocarcinoma that shares features with mammary gland tissue. Due to its rarity and lack of consensus, MLVA presents diagnostic challenges to pathologists. We present the case of a 59-year-old female with an ulcerated mass on the right side of the external genitalia, diagnosed as MLVA. Comprehensive immunohistochemistry (IHC) and gene sequencing studies were performed to characterize the tumor. IHC analysis revealed triple expression of hormonal receptors (estrogen receptor, progesterone receptor, and HER2), supporting the mammary gland origin of the tumor. Gene sequencing identified unique genetic mutations associated with the expression of hormonal markers. One fusion gene (ERBB2-NAGLU) has not been reported in any tumors, and other mutations with unique mutation types have not been previously reported in MLVA. Our findings shed light on the molecular characteristics of MLV and may help improve the diagnosis and treatment of this rare type of vulvar adenocarcinoma.

Synergistic Anti-Cancer Effects of ERB-041 and Genistein through Estrogen Receptor Suppression-Mediated PI3K/AKT Pathway Downregulation in Canine Mammary Gland Tumor Cells.

Canine-mammary-gland tumors (CMTs) are prevalent in female dogs, with approximately 50% of them being malignant and often presenting as inoperable owing to their size or metastasis. Owing to poor outcomes, effective alternatives to conventional chemotherapy for humans are necessary. Two estrogen receptors, estrogen receptor alpha (ERα) and estrogen receptor beta (ERß), which act in opposition to each other, are involved, and CMT growth involves ERα through the phosphoinositide 3-kinases (PI3K)/AKT pathway. In this study, we aimed to identify the synergistic anti-cancer effects of ERB-041, an ERß agonist, and genistein, an isoflavonoid from soybeans known to have ERß-specific pseudo-estrogenic actions, on CMT-U27 and CF41.Mg CMT cell lines. ERB-041 and genistein synergistically inhibited cell proliferation and increased the number of annexin V-positive cells in both cell lines. Furthermore, we observed a synergistic increase in the Bax/Bcl-2 ratio and cleaved caspase-3 expression. Additionally, cell-cycle arrest occurred through the synergistic regulation of cyclin D1 and cyclin-dependent kinase 4 (CDK4). We also found a synergistic decrease in the expression of ERα, and the expression of proteins involved in the PI3K/AKT pathway, including p-PI3K, phosphatase and tensin homolog (PTEN), AKT, and mechanistic target of rapamycin (mTOR). In conclusion, ERB-041 and genistein exhibited a synergistic anticancer effect on CMTs, suggesting that cotreatment with ERB-041 and genistein is a promising treatment for CMTs.

Kv 11.1 Expression Is Associated With Malignancy of Canine Mammary Gland Tumors.

BACKGROUND/AIM: The expression level of the voltage-dependent potassium channel Kv 11.1 was shown to be associated with the clinicopathological features, aggressiveness, and prognosis of human breast cancer. Canine mammary gland tumor (cMGT) is the most common tumor type in intact female dogs; however, the significance of Kv 11.1 in cMGT is unknown. The aim of this study was to identify Kv 11.1 expression in 57 benign and malignant cMGT tissues from dogs and to investigate the correlation of Kv 11.1 expression with the clinicopathological parameters and prognosis of cMGT. MATERIALS AND METHODS: A total of 57 samples were collected from cMGTs surgically resected at the Veterinary Medical Teaching Hospital, Seoul National University and subjected to immunohistochemistry assay using rabbit anti-Kv 11.1 polyclonal antibody. Immunohistochemical staining results were evaluated as the sum of intensity and percentage scores. The correlation between immunohistochemistry scores and clinicopathological parameters was investigated. RESULTS: Immunohistochemical analysis revealed that Kv 11.1 immunoreactivity was higher in benign cMGTs than in malignant cMGTs. Kv 11.1 expression was significantly associated with tumor malignancy (p<0.001), tumor size (p<0.001), histological grade (p<0.05), and age at the time of mastectomy (p<0.05). CONCLUSION: This study presents the first evidence of Kv 11.1 expression in cMGTs and indicates an inverse correlation between Kv 11.1 expression and tumor malignancy. Kv 11.1 expression can be used as a prognostic biomarker and a tool for the management of cMGTs.

In vitro evaluation of the antitumor activity of axitinib in canine mammary gland tumor cell lines.

BACKGROUND: Axitinib, a potent and selective inhibitor of vascular endothelial growth factor (VEGF) receptor (VEGFR) tyrosine kinase 1,2 and 3, is used in chemotherapy because it inhibits tumor angiogenesis by blocking the VEGF/VEGFR pathway. In veterinary medicine, attempts have been made to apply tyrosine kinase inhibitors with anti-angiogenic effects to tumor patients, but there are no studies on axitinib in canine mammary gland tumors (MGTs). OBJECTIVES: This study aimed to confirm the antitumor activity of axitinib in canine mammary gland cell lines. METHODS: We treated canine MGT cell lines (CIPp and CIPm) with axitinib and conducted CCK, wound healing, apoptosis, and cell cycle assays. Additionally, we evaluated the expression levels of angiogenesis-associated factors, including VEGFs, PDGF-A, FGF-2, and TGF-ß1, using quantitative real-time polymerase chain reaction. Furthermore, we collected canine peripheral blood mononuclear cells (PBMCs), activated them with concanavalin A (ConA) and lipopolysaccharide (LPS), and then treated them with axitinib to investigate changes in viability. RESULTS: When axitinib was administered to CIPp and CIPm, cell viability significantly decreased at 24, 48, and 72 h (p < 0.001), and migration was markedly reduced (6 h, p < 0.05; 12 h, p < 0.005). The apoptosis rate significantly increased (p < 0.01), and the G2/M phase ratio showed a significant increase (p < 0.001). Additionally, there was no significant change in the viability of canine PBMCs treated with LPS and ConA. CONCLUSION: In this study, we confirmed the antitumor activity of axitinib against canine MGT cell lines. Accordingly, we suggest that axitinib can be applied as a new treatment for patients with canine MGTs.

Calcitriol promotes M2 polarization of tumor-associated macrophages in 4T1 mouse mammary gland cancer via the induction of proinflammatory cytokines.

Our research found that vitamin D3 (VD3) treatment increased lung metastasis in mice with 4T1 murine breast cancer (BC). This study aims to investigate the impact of VD3 on the activation of tumor-associated macrophages (TAMs) in BC. Mice bearing 4T1, E0771, 67NR BC cells, and healthy mice, were fed diets with varying VD3 contents (100-deficient, 1000-normal, and 5000 IU/kg-elevated). Some mice in the 1000 and 100 IU/kg groups received calcitriol. We studied bone metastasis and characterized TAMs and bone marrow-derived macrophages (BMDMs). 4T1 cells had higher bone metastasis potential in the 5000 IU/kg and calcitriol groups. In the same mice, an elevated tumor osteopontin level and M2 polarization of TAMs (MHCIIlow CD44high phenotype) were observed. Gene expression analysis confirmed M2 polarization of 4T1 (but not 67NR) TAMs and BMDMs, particularly in the 100 IU + cal group (increased Mrc1, Il23, and Il6). This polarization was likely due to COX-2/PGE2 induction in 4T1 calcitriol-treated cells, leading to increased proinflammatory cytokines like IL-6 and IL-23. Future studies will explore COX-2/PGE2 as a primary mediator of calcitriol-stimulated inflammation in the BC microenvironment, especially relevant for BC patients with VD3 deficiency and supplementation.

Anticancer effect of superoxide dismutase on canine mammary gland tumour in vitro.

BACKGROUND: Reactive oxygen species (ROS) have been shown to promote tumour growth and metastasis in human cell lines. The superoxide anion (•O2 - ) is produced during ROS formation and is involved in tumour cell signalling. OBJECTIVES: Superoxide dismutase (SOD) has been applied to canine mammary gland tumours to investigate its antitumour effects in vitro. METHODS: Cell proliferation, cell cycle cell migration assays, reverse transcription-quantitative polymerase chain reaction, and western blot analysis were performed to determine the effects of SOD on canine mammary tumour cell line. RESULTS: SOD treatment resulted in anti-proliferative effects and mediated cell cycle arrest in the canine mammary gland tumour cell lines (CIPp and CIPm). It also downregulated the expression of N-cadherin and Vimentin. CONCLUSIONS: The results confirmed that SOD inhibits tumour cell proliferation and migration, thus supporting the potential applications of SOD as a chemotherapeutic agent for canine mammary gland tumours.

Anti-cancer effect of palmatine through inhibition of the PI3K/AKT pathway in canine mammary gland tumor CMT-U27 cells.

Canine mammary gland tumors (CMTs) are the most common and lethal cancers in female dogs. Dysregulated phosphoinositide 3-kinases (PI3K)/AKT pathway reportedly was involved in the growth and metastasis of CMTs. However, there are few studies on therapeutic strategies for targeting the PI3K pathway in CMTs. In this study, we aimed to determine whether palmatine, a natural isoquinoline alkaloid with anti-cancer properties, could inhibit the growth of CMTs and whether the inhibitory effect was mediated through the PI3K/AKT pathway. Our in vitro experiments on CMT-U27, a CMT cell line, showed that palmatine reduced cell proliferation and induced cell death. Western blotting results revealed that palmatine decreased the protein expression of PI3K, PTEN, AKT, and mechanistic target of rapamycin in the PI3K/AKT pathway, which was supported by the results of immunocytochemistry. Additionally, palmatine suppressed the migration and tube formation of canine aortic endothelial cells as well as the migration of CMT U27 cells. Our in vivo results showed that palmatine inhibited tumor growth in a CMT-U27 mouse xenograft model. We observed a decreased expression of proteins in the PI3K/AKT pathway in tumor tissues, similar to the in vitro results. Furthermore, palmatine significantly disrupted the tumor vasculature and inhibited metastasis to adjacent lymph nodes. In conclusion, our findings demonstrate that palmatine exerts anti-cancer effects against CMTs by inhibiting PI3K/AKT signaling pathway, suggesting that palmatine has potential as a canine-specific PI3K inhibitor for the treatment of CMTs.

Therapeutic trial of fluvastatin in a cell line xenograft model of canine mammary gland cancer.

The Hippo signalling pathway is involved in breast cancer and canine mammary tumour (CMT). This study sought to evaluate the efficacy of fluvastatin on the Hippo pathway and its main effectors, YAP and TAZ, in vivo in a murine CMT cell line xenograft model. On treatment day 1, mice were divided into four groups: vehicle, fluvastatin, doxorubicin or a combination therapy. Tumour volumes were monitored with callipers and tissues harvested on day 28th of treatment. Histopathological examination of tumour tissues and major organs was performed as well as tumour evaluation of necrosis, apoptosis, cellular proliferation, expression of YAP, TAZ and the mRNA levels of four of their target genes (CTGF, CYR61, ANKRD1 and RHAMM2). Results showed a statistically significant variation in tumour volumes only for the combination therapy and final tumour weight only for the doxorubicin group compared to control. There was no significant difference in tumour necrosis, expression of CC3, ki-67, YAP and TAZ measured by immunohistochemistry and in the mRNA levels of the target genes. Unexpectedly, lung metastases were found in the control group (9) and not in the fluvastatin treated group (7). In addition, mass spectrometry-based quantification of fluvastatin reveals concentrations comparable to levels reported to exert therapeutic effects. This study shows that fluvastatin tumours concentration reached therapeutic levels without having an effect on the hippo pathway or various tumour parameters. Interestingly, only the control group had lung metastases. This study is the first to explore the repurposing of statins for cancer treatment in veterinary medicine.

Does Diffusely Infiltrating Lobular Carcinoma of the Breast Arise from Epithelial-Mesenchymal Hybrid Cells?

Classic diffusely infiltrating lobular carcinoma has imaging features divergent from the breast cancers originating from the terminal ductal lobular units and from the major lactiferous ducts. Although the term "invasive lobular carcinoma" implies a site of origin within the breast lobular epithelium, we were unable to find evidence supporting this assumption. Exceptional excess of fibrous connective tissue and the unique cell architecture combined with the aberrant features at breast imaging suggest that this breast malignancy has not originated from cells lining the breast ducts and lobules. The only remaining relevant component of the fibroglandular tissue is the mesenchyme. The cells freshly isolated and cultured from diffusely infiltrating lobular carcinoma cases contained epithelial-mesenchymal hybrid cells with both epithelial and mesenchymal properties. The radiologic and histopathologic features of the tumours and expression of the mesenchymal stem cell positive markers CD73, CD90, and CD105 all suggest development in the direction of mesenchymal transition. These hybrid cells have tumour-initiating potential and have been shown to have poor prognosis and resistance to therapy targeted for malignancies of breast epithelial origin. Our work emphasizes the need for new approaches to the diagnosis and therapy of this highly fatal breast cancer subtype.

Glycosylation in breast cancer progression and mammary development: Molecular connections and malignant transformations.

INTRODUCTION: The cellular behavior in normal mammary gland development and the progression of breast cancer is like the relationship between an object and its mirror image: they may appear similar, but their essence is completely different. Breast cancer can be considered as temporal and spatial aberrations of normal development in mammary gland. Glycans have been shown to regulate key pathophysiological steps during mammary development and breast cancer progression, and the glycoproteins that play a key role in both processes can affect the normal differentiation and development of mammary cells, and even cause malignant transformation or accelerate tumorigenesis due to differences in their type and level of glycosylation. KEY FINDINGS: In this review, we summarize the roles of glycan alterations in essential cellular behaviors during breast cancer progression and mammary development, and also highlight the importance of key glycan-binding proteins such as epidermal growth factor receptor, transforming growth factor ß receptors and other proteins, which are pivotal in the modulation of cellular signaling in mammary gland. Our review takes an overall view of the molecular interplay, signal transduction and cellular behaviors in mammary gland development and breast cancer progression from a glycobiological perspective. SIGNIFICANCE: This review will give a better understanding of the similarities and differences in glycosylation between mammary gland development and breast cancer progression, laying the foundation for elucidating the key molecular mechanisms of glycobiology underlying the malignant transformation of mammary cells.

Pharmacodynamic structure of deer antler base protein and its mammary gland hyperplasia inhibition mechanism by mediating Raf-1/MEK/ERK signaling pathway activation.

Mammary gland hyperplasia (MGH) is a common mammary disease whose main pathogenesis is the disruption of estradiol (E2) and progesterone (P) secretion, thereby causing overproliferation of mammary epithelial cells and mammary gland tissue hyperplasia. Deer antler base is a traditional Chinese medicine that has been used for many years to treat MGH. However, its pharmacological mechanism and pharmacodynamic material basis are unclear. In this study, we for the first time used the graded salting method to classify deer antler base protein (CNCP) as CNCP-A, CNCP-B, and CNCP-C and explored the pharmacological mechanism of the anti-MGH properties of CNCP. We found that CNCP could regulate the hormonal levels of E2, P, and follicle stimulating hormone (FSH) and improve the histopathological condition. The potential mechanism might be related to the recombinant C-Raf proto oncogene serine/threonine protein kinase/mitogen-activated protein/extracellular regulated protein kinase (Raf-1/MEK/ERK) signaling pathway. By upregulating the protein expression of the follicle stimulating hormone receptor (FSHR), cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) inhibited the activation of the downstream Raf-1/MEK/ERK signaling pathway, which in turn inhibited the proliferation of mammary epithelial cells. We analyzed the physicochemical properties of CNCP-A, CNCP-B, and CNCP-C and obtained CNCP-C-I by column chromatographic purification of the best pharmacophore protein CNCP. Using high-performance liquid gel filtration chromatography (HPGFC), we determined the molecular weight of CNCP-C-I and identified it by high-performance liquid tandem mass spectrometry (LC-MS/MS) to obtain the first match for a high confidence protein KRT1. This study provides a theoretical basis for the development of effective traditional Chinese medicines with low toxicity levels for the prevention and treatment of mammary gland diseases.

Preneoplastic stromal cells promote BRCA1-mediated breast tumorigenesis.

Women with germline BRCA1 mutations (BRCA1+/mut) have increased risk for hereditary breast cancer. Cancer initiation in BRCA1+/mut is associated with premalignant changes in breast epithelium; however, the role of the epithelium-associated stromal niche during BRCA1-driven tumor initiation remains unclear. Here we show that the premalignant stromal niche promotes epithelial proliferation and mutant BRCA1-driven tumorigenesis in trans. Using single-cell RNA sequencing analysis of human preneoplastic BRCA1+/mut and noncarrier breast tissues, we show distinct changes in epithelial homeostasis including increased proliferation and expansion of basal-luminal intermediate progenitor cells. Additionally, BRCA1+/mut stromal cells show increased expression of pro-proliferative paracrine signals. In particular, we identify pre-cancer-associated fibroblasts (pre-CAFs) that produce protumorigenic factors including matrix metalloproteinase 3 (MMP3), which promotes BRCA1-driven tumorigenesis in vivo. Together, our findings demonstrate that precancerous stroma in BRCA1+/mut may elevate breast cancer risk through the promotion of epithelial proliferation and an accumulation of luminal progenitor cells with altered differentiation.

Characterization of Hyaluronan Localization in the Developing Mammary Gland and Mammary Tumors.

The extracellular matrix (ECM) is biochemically and biomechanically important for the structure and function of the mammary gland, which undergoes vast structural changes throughout pubertal and reproductive development. Although hyaluronan (HA) is a ubiquitous glycosaminoglycan (GAG) of the mammary gland ECM, extensive characterization of HA deposition in the mammary gland is lacking. Understanding physiologic HA metabolism is critical as this tightly controlled system is often hijacked in cancer. In the current studies, we characterize HA regulation throughout mammary gland development to better understand subsequent dysregulation of HA in mammary tumors. Using immunofluorescence (IF) imaging, we demonstrate that organized HA-rich septa exist in the mammary gland stroma throughout puberty, pregnancy, and involution. Furthermore, we find heterogeneous HA deposition within two murine models of breast cancer. Using cell specific isolation techniques, we characterize expression of genes associated with HA binding, synthesis, and degradation within EpCAM + epithelial cells, CD90.2 + fibroblasts, and F4/80 + macrophages isolated from mammary glands and tumors. Most notably, we identify elevated levels of the hyaluronidases Hyal1 and Hyal2 in tumor-association macrophages (TAMs), suggesting a role for TAM-mediated turnover of HA in the tumor microenvironment (TME). Gene expression is supported functionally by in vitro experiments in which macrophages treated with tumor-cell conditioned media exhibit increased hyaluronidase activity. These findings link TAMs to the direct degradation of HA within the TME of mammary tumors, which has negative implications for patient survival.

HOXB7 induces STAT3-mediated transformation and lung metastasis in immortalized mammary gland NMuMG cells.

The homeobox family genes are often dysregulated in various cancer types. Particularly HOXB7 amplification and overexpression correlate with poor prognosis in various cancer such as gastric, pancreatic, and lung cancers. Moreover, HOXB7 is known to contribute to cancer progression by promoting epithelial to mesenchymal transition, anticancer drug resistance, and angiogenesis. In this study, we show that HOXB7 is coamplified with ERBB2 in a subset of breast cancer patients and HOXB7 expression correlates with poor prognosis in HER2-positive breast cancer patients. This clinical observation is supported by the following results-HOXB7 overexpression in an immortalized murine mammary gland epithelial cell line NMuMG induces cellular transformation in vitro, tumorigenesis, and lung metastasis through the activation of JAK-STAT signaling.

Expression of receptor-type tumour endothelial marker 8 in carcinoma cells showing luminal progenitor-like phenotypes in canine mammary gland carcinomas.

This study aimed to investigate the expression of receptor-type tumour endothelial marker 8 (TEM8RT) in canine mammary gland carcinomas (CMGCs) using immunohistochemistry and to evaluate the association between carcinoma cell TEM8RT expression and tumour histological features, histological grades and the differentiation status of neoplastic epithelial cells. TEM8RT expression was more frequently detected in simple carcinomas (tubular and tubulopapillary) than in solid carcinomas, and it was significantly correlated with histological grade Ⅰ tumours and a low mitotic index. Additionally, TEM8RT+ carcinoma cells were more frequently found in CMGCs showing luminal progenitor-like phenotypes, such as Notch1+, CK19+/CK5+/CD49f+ and CK19+/CK5-/CD49f+. Double-labelling immunofluorescence detection techniques confirmed that most TEM8RT+ carcinoma cells expressed CD49f, Notch1 and CK19. However, TEM8RT immunoreactivity was not found in carcinoma cells expressing GATA3, which upregulates mature luminal cell differentiation. Furthermore, TEM8RT+ carcinoma cells were detected in a few CMGCs showing basal/stem cell-like phenotypes such as CK19-/CK5+/CD49f+ and CK19-/CK5+/CD49f-. These findings indicate that TEM8RT is expressed in luminal progenitor-like carcinoma cells in CMGCs. Since TEM8 enhances self-renewal in human mammary stem/progenitor cells, it also may be involved in maintenance of luminal progenitor-like carcinoma cells, resulting in prevention of their transition to basal/stem cell-like carcinoma cells and development of less malignant CMGCs. Therefore, TEM8RT may be useful for indicating prognostic outcomes and identifying the possible ontogeny of carcinoma cells in mammary gland tumours.

Hormonal regulation of mammary gland development and lactation.

Lactation is critical to infant short-term and long-term health and protects mothers from breast cancer, ovarian cancer and type 2 diabetes mellitus. The mammary gland is a dynamic organ, regulated by the coordinated actions of reproductive and metabolic hormones. These hormones promote gland development from puberty onwards and induce the formation of a branched, epithelial, milk-secreting organ by the end of pregnancy. Progesterone withdrawal following placental delivery initiates lactation, which is maintained by increased pituitary secretion of prolactin and oxytocin, and stimulated by infant suckling. After weaning, local cytokine production and decreased prolactin secretion trigger large-scale mammary cell loss, leading to gland involution. Here, we review advances in the molecular endocrinology of mammary gland development and milk synthesis. We discuss the hormonal functions of the mammary gland, including parathyroid hormone-related peptide secretion that stimulates maternal calcium mobilization for milk synthesis. We also consider the hormonal composition of human milk and its associated effects on infant health and development. Finally, we highlight endocrine and metabolic diseases that cause lactation insufficiency, for example, monogenic disorders of prolactin and prolactin receptor mutations, maternal obesity and diabetes mellitus, interventions during labour and delivery, and exposure to endocrine-disrupting chemicals such as polyfluoroalkyl substances in consumer products and other oestrogenic compounds.

MicroRNAs: A Link between Mammary Gland Development and Breast Cancer.

Breast cancer is among the most common cancers in women, second to skin cancer. Mammary gland development can influence breast cancer development in later life. Processes such as proliferation, invasion, and migration during mammary gland development can often mirror processes found in breast cancer. MicroRNAs (miRNAs), small, non-coding RNAs, can repress post-transcriptional RNA expression and can regulate up to 80% of all genes. Expression of miRNAs play a key role in mammary gland development, and aberrant expression can initiate or promote breast cancer. Here, we review the role of miRNAs in mammary development and breast cancer, and potential parallel roles. A total of 32 miRNAs were found to be expressed in both mammary gland development and breast cancer. These miRNAs are involved in proliferation, metastasis, invasion, and apoptosis in both processes. Some miRNAs were found to have contradictory roles, possibly due to their ability to target many genes at once. Investigation of miRNAs and their role in mammary gland development may inform about their role in breast cancer. In particular, by studying miRNA in development, mechanisms and potential targets for breast cancer treatment may be elucidated.

Loss of Brca1 and Trp53 in adult mouse mammary ductal epithelium results in development of hormone receptor-positive or hormone receptor-negative tumors, depending on inactivation of Rb family proteins.

BACKGROUND: Breast cancer is a heterogenous disease with several histological and molecular subtypes. Models that represent these subtypes are essential for translational research aimed at improving clinical strategy for targeted therapeutics. METHODS: Different combinations of genetic aberrations (Brca1 and Trp53 loss, and inhibition of proteins of the Rb family) were induced in the mammary gland by injection of adenovirus expressing Cre recombinase into the mammary ducts of adult genetically engineered mice. Mammary tumors with different genetic aberrations were classified into molecular subtypes based on expression of molecular markers and RNAseq analysis. In vitro potency assays and Western blots were used to examine their drug sensitivities. RESULTS: Induction of Brca1 and Trp53 loss in mammary ductal epithelium resulted in development of basal-like hormone receptor (HR)-negative mammary tumors. Inhibition of Rb and Trp53 loss or the combination of Rb, Trp53 and Brca1 aberrations resulted in development of luminal ductal carcinoma positive for ER, PR, and Her2 expression. HR positivity in tumors with Rb, Trp53 and Brca1 aberrations indicated that functionality of the Rb pathway rather than Brca1 status affected HR status in these models. Mammary tumor gene expression profiles recapitulated human basal-like or luminal B breast cancer signatures, but HR-positive luminal cancer models were endocrine resistant and exhibited upregulation of PI3K signaling and sensitivity to this pathway inhibition. Furthermore, both tumor subtypes were resistant to CDK4/6 inhibition. CONCLUSIONS: Examination of molecular expression profiles and drug sensitivities of tumors indicate that these breast cancer models can be utilized as a translational platform for evaluation of targeted combinations to improve chemotherapeutic response in patients that no longer respond to hormone therapy or that are resistant to CDK4/6 inhibition.

Interplay of the transcription factor MRTF-A and matrix stiffness controls mammary acinar structure and protrusion formation.

BACKGROUND: Ongoing differentiation processes characterize the mammary gland during sexual development and reproduction. In contrast, defective remodelling is assumed to be causal for breast tumorigenesis. We have shown recently that the myocardin-related transcription factor A (MRTF-A) is essential for forming regular hollow acinar structures. Moreover, MRTF-A activity is known to depend on the biochemical and physical properties of the surrounding extracellular matrix. In this study we analysed the mutual interaction of different matrix stiffnesses and MRTF-A activities on formation and maintenance of mammary acini. METHODS: Human MCF10A acini and primary mature organoids isolated from murine mammary glands were cultivated in 3D on soft and stiff matrices (200-4000 Pa) in conjunction with the Rho/MRTF/SRF pathway inhibitor CCG-203971 and genetic activation of MRTF-A. RESULTS: Three-dimensional growth on stiff collagen matrices (> 3000 Pa) was accompanied by increased MRTF-A activity and formation of invasive protrusions in acini cultures of human mammary MCF10A cells. Differential coating and synthetic hydrogels indicated that protrusion formation was attributable to stiffness but not the biochemical constitution of the matrix. Stiffness-induced protrusion formation was also observed in preformed acini isolated from murine mammary glands. Acinar outgrowth in both the MCF10A acini and the primary organoids was partially reverted by treatment with the Rho/MRTF/SRF pathway inhibitor CCG-203971. However, genetic activation of MRTF-A in the mature primary acini also reduced protrusion formation on stiff matrices, whilst it strongly promoted luminal filling matrix-independently. CONCLUSION: Our results suggest an intricate crosstalk between matrix stiffness and MRTF-A, whose activity is required for protrusion formation and sufficient for luminal filling of mammary acini. Video Abstract.

Thirteenth Annual ENBDC Workshop: Methods in Mammary Gland Biology and Breast Cancer.

The thirteenth annual workshop of the European Network for Breast Development and Cancer (ENBDC) Laboratories Annual Workshop took place on the 28-30 April 2022 in Weggis, Switzerland and focused on methods in mammary gland biology and breast cancer. Sixty scientists participated in the ENBDC annual workshop which had not been held in person since 2019 due to the global COVID-19 pandemic. Topics spanned the mammary gland biology field, ranging from lactation biology and embryonic development, single cell sequencing of the human breast, and stunning cutting-edge imaging of the mouse mammary gland and human breast as well as breast cancer research topics including invasive progression of the pre-invasive DCIS stage, metabolic determinants of endocrine therapy resistance, models for lobular breast cancer, and how mutational landscapes of normal breast during age and pregnancy determine cancer risk. The latest findings from participating researchers were presented through oral presentations and poster sessions and included plenty of unpublished work.