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Background: Breast cancer's complex transcriptional landscape requires an improved understanding of cellular diversity to identify effective treatments. The study of genetic variations among breast cancer subtypes at single-cell resolution has potential to deepen our insights into cancer progression., Methods: In this study, we amalgamate single-cell RNA sequencing data from patient tumours and matched lymph metastasis, reduction mammoplasties, breast cancer patient-derived xenografts (PDXs), PDX-derived organoids (PDXOs), and cell lines resulting in a diverse dataset of 117 samples with 506 719 total cells. These samples encompass hormone receptor positive (HR+), human epidermal growth factor receptor 2 positive (HER2+), and triple-negative breast cancer (TNBC) subtypes, including isogenic model pairs. Herein, we delineated similarities and distinctions across models and patient samples and explore therapeutic drug efficacy based on subtype proportions., Results: PDX models more closely resemble patient samples in terms of tumour heterogeneity and cell cycle characteristics when compared with TNBC cell lines. Acquired drug resistance was associated with an increase in basal-like cell proportions within TNBC PDX tumours as defined with SCSubtype and TNBCtype cell typing predictors. All patient samples contained a mixture of subtypes; compared to primary tumours HR+ lymph node metastases had lower proportions of HER2-Enriched cells. PDXOs exhibited differences in metabolic-related transcripts compared to PDX tumours. Correlative analyses of cytotoxic drugs on PDX cells identified therapeutic efficacy was based on subtype proportion., Conclusions: We present a substantial multimodel dataset, a dynamic approach to cell-wise sample annotation, and a comprehensive interrogation of models within systems of human breast cancer. This analysis and reference will facilitate informed decision-making in preclinical research and therapeutic development through its elucidation of model limitations, subtype-specific insights and novel targetable pathways., Key Points: Patient-derived xenografts models more closely resemble patient samples in tumour heterogeneity and cell cycle characteristics when compared with cell lines. 3D organoid models exhibit differences in metabolic profiles compared to their in vivo counterparts. A valuable multimodel reference dataset that can be useful in elucidating model differences and novel targetable pathways., (© 2024 The Author(s). Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

Background: Breast cancer metastasis is driven by a profound remodeling of the cytoskeleton that enables efficient cell migration and invasion. Anillin is a unique scaffolding protein regulating major cytoskeletal structures, such as actin filaments, microtubules, and septin polymers. It is markedly overexpressed in breast cancer, and high anillin expression is associated with poor prognosis. The aim of this study was to investigate the role of anillin in breast cancer cell migration, growth, and metastasis., Methods: CRISPR/Cas9 technology was used to deplete anillin in highly metastatic MDA-MB-231 and BT549 cells and to overexpress it in poorly invasive MCF10AneoT cells. The effects of anillin depletion and overexpression on breast cancer cell motility in vitro were examined by wound healing and Matrigel invasion assays. Assembly of the actin cytoskeleton and matrix adhesion were evaluated by immunofluorescence labeling and confocal microscopy. In vitro tumor development was monitored by soft agar growth assays, whereas cancer stem cells were examined using a mammosphere formation assay and flow cytometry. The effects of anillin knockout on tumor growth and metastasis in vivo were determined by injecting control and anillin-depleted breast cancer cells into NSG mice., Results: Loss-of-function and gain-of-function studies demonstrated that anillin is necessary and sufficient to accelerate migration, invasion, and anchorage-independent growth of breast cancer cells in vitro. Furthermore, loss of anillin markedly attenuated primary tumor growth and metastasis of breast cancer in vivo. In breast cancer cells, anillin was localized in the nucleus; however, knockout of this protein affected the cytoplasmic/cortical events, e.g., the organization of actin cytoskeleton and cell-matrix adhesions. Furthermore, we observed a global transcriptional reprogramming of anillin-depleted breast cancer cells that resulted in suppression of their stemness and induction of the mesenchymal to epithelial trans-differentiation. Such trans-differentiation was manifested by the upregulation of basal keratins along with the increased expression of E-cadherin and P-cadherin. Knockdown of E-cadherin restored the impaired migration and invasion of anillin-deficient breast cancer cells., Conclusion: Our study demonstrates that anillin plays essential roles in promoting breast cancer growth and metastatic dissemination in vitro and in vivo and unravels novel functions of anillin in regulating breast cancer stemness and differentiation.

Background: Epidemiological studies indicate high serum 25(OH)D 3 is associated with increased survival in breast cancer patients. Pre-clinical studies attributed this to anti-tumorigenic properties of its metabolite 1α,25(OH) 2 D 3 . However, 1α,25(OH) 2 D 3 is highly calcemic and thus has a narrow therapeutic window. Here we propose another metabolite, 24R,25(OH) 2 D 3 , as an alternative non-calcemic vitamin D 3 supplement., Methods: NOD-SCID-IL2γR null female mice with MCF7 breast cancer xenografts in the mammary fat pad were treated with 24R,25(OH) 2 D 3 and changes in tumor burden and metastases were assessed. ERα66+ MCF7 and T47D cells, and ERα66- HCC38 cells were treated with 24R,25(OH) 2 D 3 in vitro to assess effects on proliferation and apoptosis. Effects on migration and metastatic markers were assessed in MCF7., Results: 24R,25(OH) 2 D 3 reduced MCF7 tumor growth and metastasis in vivo. In vitro results indicate that this was not due to an anti-proliferative effect; 24R,25(OH) 2 D 3 stimulated DNA synthesis in MCF7 and T47D. In contrast, markers of invasion and metastasis were decreased. 24R,25(OH) 2 D 3 caused dose-dependent increases in apoptosis in MCF7 and T47D, but not HCC38 cells. Inhibitors to palmitoylation, caveolae integrity, phospholipase-D, and estrogen receptors (ER) demonstrate that 24R,25(OH) 2 D 3 acts on MCF7 cells through caveolae-associated, phospholipase D-dependent mechanisms via cross-talk with ERs., Conclusion: These results indicate that 24R,25(OH) 2 D 3 shows promise in treatment of breast cancer by stimulating tumor apoptosis and reducing metastasis., General Significance: 24R,25(OH) 2 D 3 regulates breast cancer cell survival through ER-associated mechanisms similar to 24R,25(OH) 2 D 3 effects on chondrocytes. Thus, 24R,25(OH) 2 D 3 may modulate cell survival in other estrogen-responsive cell types, and its therapeutic potential should be investigated in ER-associated pathologies., (Published by Elsevier B.V.)

Breast cancer is one of the most common forms of cancer affecting women in the United States, second only to skin cancers. Although treatments have been developed to combat primary breast cancer, metastasis remains a leading cause of death. An early step of metastasis is cancer cell invasion through the basement membrane. However, this process is not yet well understood. AG73, a synthetic laminin- α 1 chain peptide, plays an important role in cell adhesion and has previously been linked to migration, invasion, and metastasis. Thus, we aimed to identify the binding partner of AG73 on breast cancer cells that could mediate cancer progression. We performed adhesion assays using MCF10A, T47D, SUM1315, and MDA-231 breast cell lines and found that AG73 binds to syndecans (Sdcs) 1, 2, and 4. This interaction was inhibited when we silenced Sdcs 1 and/or 4 in MDA-231 cells, indicating the importance of these receptors in this relationship. Through actin staining, we found that silencing of Sdc 1, 2, and 4 expression in MDA-231 cells exhibits a decrease in the length and number of filopodia bound to AG73. Expression of mouse Sdcs 1, 2, and 4 in MDA-231 cells provides rescue in filopodia, and overexpression of Sdcs 1 and 2 leads to increased filopodium length and number. Our findings demonstrate an intrinsic interaction between AG73 in the tumor environment and the Sdcs on breast cancer cells in supporting tumor cell adhesion and invasion through filopodia, an important step in cancer metastasis.

HER2 ( ERBB2 ) amplification is a driving oncogenic event in breast cancer. Clinical trials have consistently shown the benefit of HER2 inhibitors (HER2i) in treating patients with both local and advanced HER2+ breast cancer. Despite this benefit, their efficacy as single agents is limited, unlike the robust responses to other receptor tyrosine kinase inhibitors like EGFR inhibitors in EGFR -mutant lung cancer. Interestingly, the lack of HER2i efficacy occurs despite sufficient intracellular signaling shutdown following HER2i treatment. Exploring possible intrinsic causes for this lack of response, we uncovered remarkably depressed levels of NOXA, an endogenous inhibitor of the antiapoptotic MCL-1, in HER2 -amplified breast cancer. Upon investigation of the mechanism leading to low NOXA, we identified a micro-RNA encoded in an intron of HER2 , termed miR-4728 , that targets the mRNA of the Estrogen Receptor α ( ESR1 ). Reduced ESR1 expression in turn prevents ERα-mediated transcription of NOXA , mitigating apoptosis following treatment with the HER2i lapatinib. Importantly, resistance can be overcome with pharmacological inhibition of MCL-1. More generally, while many cancers like EGFR -mutant lung cancer are driven by activated kinases that when drugged lead to robust monotherapeutic responses, we demonstrate that the efficacy of targeted therapies directed against oncogenes active through focal amplification may be mitigated by coamplified genes., Competing Interests: Conflict of interest statement: J.D.L. and A.J.S. are employees and shareholders of AbbVie Inc., (Copyright © 2018 the Author(s). Published by PNAS.)

The activation of the Janus-activated kinase 2 (Jak2) tyrosine kinase following ligand binding has remained incompletely characterized at the mechanistic level. We report that the peptidyl-prolyl isomerase (PPI) cyclophilin A (CypA), which is implicated in the regulation of protein conformation, is necessary for the prolactin (PRL)-induced activation of Jak2 and the progression of human breast cancer. A direct correlation was observed between the levels or activity of CypA and the extent of PRL-induced signaling and gene expression. Loss of PRLr-CypA binding, following treatment with the PPI inhibitor cyclosporine A (CsA), or overexpression of a dominant-negative PRLr mutant (P334A) resulted in a loss of PRLr/Jak2-mediated signaling. In vitro, CsA treatment of breast cancer cells inhibited their growth, motility, invasion, and soft agar colony formation. In vivo, CsA treatment of nude mice xenografted with breast cancer cells induced tumor necrosis and completely inhibited metastasis. These studies reveal that a CypA-mediated conformational change within the PRLr/Jak2 complex is required for PRL-induced transduction and function and indicate that the inhibition of prolyl isomerases may be a novel therapeutic strategy in the treatment of human breast cancer.

Laminin-111 promotes the malignant phenotype, and a 12-mer synthetic peptide (AG73, RKRLQVQLSIRT) from the carboxyl terminus of the alpha1 chain increases B16F10 melanoma metastasis to the lung and liver. Using an antibody array, fibronectin was identified as an up-regulated protein in B16F10 cells after incubation with this peptide. The increased fibronectin is cell-associated with no increase in soluble fibronectin. The AG73 peptide increased the number and size of bone metastases with both B16F10 melanoma and MDA-231 breast carcinoma cells in an intracardiac injection model. Using siRNA transfection, we found that a reduction in fibronectin expression did not reduce bone metastasis in the presence of the metastasis-promoting peptide AG73. We conclude that the laminin peptide AG73 increases metastasis independently of fibronectin expression.

The focus of this study was to gain insight into the role(s) of osteonectin in the preferential metastasis of breast cancer cells to bone. Osteonectin was isolated from conditioned media of several cell lines including breast cancer (MDA-MB-435, MDA-MB-468), osteoblasts (hFOB1.19), non-neoplastic breast epithelial (hTERT-HME1), and vascular endothelial cells isolated from a bone biopsy (HBME-1). Chemical/physical properties of osteonectin from these five sources was analyzed to determine if unique configurations of osteonectin exist and therefore identify a chemotactic isoform. Osteonectin from all sources had a molecular weight of approximately 46 kDa, N-linked glycosylation, and undetectable phosphorylated serines, sialic acids and O-linked oligosaccharides. The cDNA for osteonectin from the breast cancer, osteoblast, and breast epithelial cell lines was identical, while the vascular endothelial cell cDNA contained point mutations that resulted in eight amino acid substitutions. Bone-derived osteonectin was then analyzed to assess its influence on breast cancer cell motility and migration. Although osteonectin increased undirected MDA-MB-231 cell motility, it did not chemoattract the same breast cancer cell line. However, the breast cancer cells did migrate toward the known chemoattractant vitronectin and to bone extracts derived from wild-type and osteonectin-null mice. Migration to vitronectin was enhanced when osteonectin was also present. We concluded that osteonectin was not a chemotactic factor. However, through its anti-adhesive properties, osteonectin induced undirected breast cancer cell motility, and may have enhanced chemoattraction to vitronectin., (Copyright 2005 Wiley-Liss, Inc.)

Skeletal metastases occur with high incidence in patients with breast cancer and cause long-term skeletal morbidity. Osteonectin (SPARC, BM-40) is a bone matrix factor that is an in vitro chemoattractant for breast and prostate cancer cells. Increased expression of osteonectin is found in malignant breast tumors. We infected MDA-231 breast cancer cells with an adenovirus expressing osteonectin to examine the role of osteonectin expression in breast cancer cells and its effect on metastasis, in particular to bone. Expression of osteonectin did not affect MDA-231 cell proliferation, apoptosis, migration, cell aggregation, or protease cleavage of collagen IV. However, in vitro invasion of these osteonectin-infected cells through Matrigel and colony formation on Matrigel was decreased. Interestingly, high osteonectin expression in MDA-231 cells inhibited metastasis in a dose-dependent manner to many different organs including bone. The reduction in metastasis may be due to decreased platelet-tumor cell aggregation, because exogenous osteonectin inhibited platelet aggregation in vitro and the high osteonectin expression in MDA-231 cells reduced tumor cell-induced thrombocytopenia in vivo compared with control-infected cells. These studies suggest that high endogenous expression of osteonectin in breast cancer cells may reduce metastasis via reduced invasive activity and reduced tumor cell-platelet aggregation.

Interactions of stromal and tumor cells with the extracellular matrix may regulate expression of proteases including the lysosomal proteases cathepsins B and D. In the present study, we determined whether the expression of these two proteases in human breast fibroblasts was modulated by interactions with the extracellular matrix component, collagen I. Breast fibroblasts were isolated from non-malignant breast tissue as well as from tissue surrounding malignant human breast tumors. Growth of these fibroblasts on collagen I gels affected cell morphology, but not the intracellular localization of vesicles staining for cathepsin B or D. Cathepsins B and D levels (mRNA or intracellular protein) were not affected in fibroblasts growing on collagen I gels or plastic, nor was cathepsin D secreted from these cells. In contrast, protein expression and secretion of cathepsin B, primarily procathepsin B, was induced by growth on collagen I gels. The induced secretion appeared to be mediated by integrins binding to collagen I, as inhibitory antibodies against alpha(1), alpha(2), and beta(1) integrin subunits prevented procathepsin B secretion from fibroblasts grown on collagen. In addition, procathepsin B secretion was induced when cells were plated on beta(1) integrin antibodies. To our knowledge, this is the first examination of cathepsin B and D expression and localization in human breast fibroblasts and their regulation by a matrix protein. Secretion of the cysteine protease procathepsin B from breast fibroblasts may have physiological and pathological consequences, as proteases are required for normal development and for lactation of the mammary gland, yet can also initiate and accelerate the progression of breast cancer.