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Breast cancer is the most common cancer in women and a leading cause of cancer-related deaths for women worldwide. Various cell models have been developed to study breast cancer tumorigenesis, metastasis, and drug sensitivity. The MCF10A human mammary epithelial cell line is a widely used in vitro model for studying normal breast cell function and transformation. However, there is limited knowledge about whether MCF10A cells reliably represent normal human mammary cells. MCF10A cells were grown in monolayer, suspension (mammosphere culture), three-dimensional (3D) \"on-top\" Matrigel, 3D \"cell-embedded\" Matrigel, or mixed Matrigel/collagen I gel. Suspension culture was performed with the MammoCult medium and low-attachment culture plates. Cells grown in 3D culture were fixed and subjected to either immunofluorescence staining or embedding and sectioning followed by immunohistochemistry and immunofluorescence staining. Cells or slides were stained for protein markers commonly used to identify mammary progenitor and epithelial cells. MCF10A cells expressed markers representing luminal, basal, and progenitor phenotypes in two-dimensional (2D) culture. When grown in suspension culture, MCF10A cells showed low mammosphere-forming ability. Cells in mammospheres and 3D culture expressed both luminal and basal markers. Surprisingly, the acinar structure formed by MCF10A cells in 3D culture was positive for both basal markers and the milk proteins β-casein and α-lactalbumin. MCF10A cells exhibit a unique differentiated phenotype in 3D culture which may not exist or be rare in normal human breast tissue. Our results raise a question as to whether the commonly used MCF10A cell line is a suitable model for human mammary cell studies.

The revision of the eighth edition of the primary tumor, lymph node, and metastasis (TNM) classification of the American Joint Commission of Cancer (AJCC) for breast cancer was determined by a multidisciplinary team of breast cancer experts. The panel recognized the need to incorporate biologic factors, such as tumor grade, proliferation rate, estrogen and progesterone receptor expression, human epidermal growth factor 2 (HER2) expression, and gene expression prognostic panels into the staging system. AJCC levels of evidence and guidelines for all tumor types were followed as much as possible. The panel felt that, to maintain worldwide value, the tumor staging system should remain based on TNM anatomic factors. However, the recognition of the prognostic influence of grade, hormone receptor expression, and HER2 amplification mandated their inclusion into the staging system. The value of commercially available, gene-based assays was acknowledged and prognostic input added. Tumor biomarkers and low Oncotype DX recurrence scores can alter prognosis and stage. These updates are expected to provide additional precision and flexibility to the staging system and were based on the extent of published information and analysis of large, as yet unpublished databases. The eighth edition of the AJCC TNM staging system, thus, provides a flexible platform for prognostic classification based on traditional anatomic factors, which can be modified and enhanced using patient biomarkers and multifactorial prognostic panel data. The eighth edition remains the worldwide basis for breast cancer staging and will incorporate future online updates to remain timely and relevant.

MicroRNAs (miRs) are a class of small noncoding RNAs whose expression changes have been associated with cancer development and progression. Current techniques to isolate miRs for expression analysis from blood are inefficient. We developed a reverse-transcription quantitative real-time PCR (RT-qPCR) assay for direct detection of circulating miRs in serum. We hypothesized that serum concentrations of miR-21, a biomarker increased in breast tumors, would correlate with the presence and extent of breast cancer. The RT-qPCR applied directly in serum (RT-qPCR-DS) assay for circulating miR-21 was tested in sera from 102 patients with different stages of breast cancer and 20 healthy female donors. The assay was sensitive for detection of miR-21 in 0.625 μL of serum from breast cancer patients. For differentiation of samples from patients with locoregional breast cancer from those from healthy donors, the odds ratio was 1.796 and the area under the curve was 0.721. In a multivariate analysis that included standard clinicopathologic prognostic factors, high circulating miR-21 concentrations correlated significantly (P < 0.001) with visceral metastasis. A novel RT-qPCR-DS can improve the efficiency of miR assessment. Use of this assay to detect circulating miR-21 has diagnostic and prognostic potential in breast cancer.

Triple-negative breast cancer (TNBC) has high rates of local recurrence and distant metastasis, partially due to its high invasiveness. The Forkhead box C1 (FOXC1) transcription factor has been shown to be specifically overexpressed in TNBC and associated with poor clinical outcome. How TNBC’s high invasiveness is driven by FOXC1 and its downstream targets remains poorly understood. In the present study, pathway-specific PCR array assays revealed that WNT5A and matrix metalloproteinase-7 (MMP7) were upregulated by FOXC1 in TNBC cells. Interestingly, WNT5A mediates the upregulation of MMP7 by FOXC1 and the WNT5A-MMP7 axis is essential for FOXC1-induced invasiveness of TNBC cells in vitro. Xenograft models showed that the lung extravasation and metastasis of FOXC1-overexpressing TNBC cells were attenuated by knocking out WNT5A, but could be restored by MMP7 overexpression. Mechanistically, FOXC1 can bind directly to the WNT5A promoter region to activate its expression. Engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP), coupled with mass spectrometry, identified FOXC1-interacting proteins including a group of heterogeneous nuclear ribonucleoproteins involved in WNT5A transcription induction. Finally, we found that WNT5A activates NF-κB signaling to induce MMP7 expression. Collectively, these data demonstrate a FOXC1-elicited non-canonical WNT5A signaling mechanism comprising NF-κB and MMP7 that is essential for TNBC cell invasiveness, thereby providing implications toward developing an effective therapy for TNBC.

Glyceraldehyde-3-phosphate dehydrogenase （GAPDH）, initially identified as a glycolytic enzyme and considered as a housekeeping gene, is widely used as an internal control in experiments on proteins, mRNA, and DNA. However, emerging evidence indicates that GAPDH is implicated in diverse functions independent of its role in energy metabolism; the expression status of GAPDH is also deregulated in various cancer cells. One of the most common effects of GAPDH is its inconsistent role in the determination of cancer cell fate. Furthermore, studies have described GAPDH as a regulator of cell death; other studies have suggested that GAPDH participates in tumor progression and serves as a new therapeutic target. However, related regulatory mechanisms of its numerous cellular functions and deregulated expression levels remain unclear. GAPDH is tightly regulated at transcriptional and pnsttranscriptional levels, which are involved in the regulation of diverse GAPDH functions. Several cancer-related factors, such as insulin, hypoxia inducible factor-1 （HIF-1）, p53, nitric oxide （NO）, and acetylated histone, not only modulate GAPDH gene expression but also affect protein functions via common pathways. Moreover, posttranslational modifications （PTMs） occurring in GAPDH in cancer cells result in new activities unrelated to the original glycnlytic function of GAPDH. In this review, recent findings related to GAPDH transcriptional regulation and PTMs are summarized. Mechanisms and pathways involved in GAPDH regulation and its different roles in cancer cells are also described.

Background Invasive lobular breast cancer (ILC) is less common than invasive ductal breast cancer (IDC), more difficult to detect mammographically, and usually diagnosed at a later stage. Does delayed diagnosis of ILC affect survival? We used a national registry to compare outcomes of patients with stage-matched ILC and IDC. Methods Query of the National Cancer Institute’s Surveillance, Epidemiology, and End Results (SEER) tumor registry identified 263,408 women diagnosed with IDC or ILC between 1993 and 2003. Survival of patients matched by T and N stage was compared using Kaplan-Meier curves and log-rank analysis. Results When compared with IDC, ILC was more likely to be >2 cm (43.1 vs. 32.6%; P  < 0.001), lymph node positive (36.8 vs. 34.4%; P  < 0.001), and ER positive (93.1 vs. 75.6%; P  < 0.001). The 5-year disease-specific survival (DSS) was significantly better for patients with ILC than for those with IDC, before (90 vs. 88%; P  < 0.001) and after matching for stage T1N0 (98 vs. 96%; P  < 0.001), T2N0 (94 vs. 88%; P  < 0.001), and T3N0 (92 vs. 83%, P  < 0.001). The 5-year DSS for patients with nodal metastasis of ILC vs. IDC was 89% vs. 88% ( P  = NS) for stage T1N1, 81 vs. 73% ( P  < 0.001) for T2N1, and 72 vs. 56% ( P  < 0.001) for T3N1. Multivariate analysis identified a 14% survival benefit for ILC (hazard ratio 0.86, 95% confidence interval 0.80–0.92). Conclusions Stage-matched prognosis is better for patients with ILC than for those with IDC. Our findings support a different biology for ILC and are important for counseling and risk stratification.

Triple-negative breast cancer (TNBC) is associated with a high rate of early recurrence and distant metastasis, frequent development of therapeutic resistance, and a poor prognosis. There is a lack of targeted therapies for this aggressive subtype of breast cancer. Identifying novel effective treatment modalities for TNBC remains an urgent and unmet clinical need. In this study, we investigated the anti-cancer effect of triptonide, a natural compound derived from the traditional Chinese medicinal herb Tripterygium wilfordii Hook F, in TNBC. We found that triptonide inhibits human TNBC cell growth in vitro and growth of TNBC xenograft mammary tumors. It induces apoptosis and suppresses stem-like properties as indicated by reduced mammosphere formation and aldehyde dehydrogenase activity in TNBC cells. We show that triptonide downregulates multiple cancer stem cell-associated genes but upregulates SNAI1 gene expression. In support of SNAI1 induction as a negative feedback response to triptonide treatment, in vitro-derived triptonide-resistant HCC1806 cells display a markedly higher expression of SNAI1 compared with parental cells. Mechanistically, the increase of SNAI1 expression is mediated by the activation of JNK signaling, but not by ERK and AKT, two well-established SNAI1 regulators. Furthermore, knockdown of SNAI1 in the triptonide-resistant HCC1806 cells increases sensitivity to triptonide and reduces mammosphere formation. These results indicate that triptonide holds promise as a novel anti-tumor agent for TNBC treatment. Our study also reveals a SNAI1-associated feedback mechanism which may lead to acquired resistance to triptonide.

Surface ectoderm (SE) cells give rise to structures including the epidermis and ectodermal associated appendages such as hair, eye and the mammary gland. In this study, we validate a protocol that utilizes BMP4 and the γ-secretase inhibitor DAPT to induce SE differentiation from human induced pluripotent stem cells (hiPSCs). hiPSC-differentiated SE cells expressed markers suggesting their commitment to the SE lineage. Computational analyses using integrated quantitative transcriptomic and proteomic profiling reveal that TGFβ superfamily signaling pathways are preferentially activated in SE cells compared with hiPSCs. SE differentiation can be enhanced by selectively blocking TGFβ-RI signaling. We also show that SE cells and neural ectoderm cells possess distinct gene expression patterns and signaling networks as indicated by functional Ingenuity Pathway Analysis. Our findings advance current understanding of early human SE cell development and pave the way for modeling of SE-derived tissue development, studying disease pathogenesis and development of regenerative medicine approaches.

Breast cancer (BC) is the most frequently diagnosed malignancy among women globally. Although mouse models have been critical in advancing the knowledge of BC tumorigenesis and progression, human breast models comprising the breast tissue microenvironment are needed to help elucidate the underlying mechanisms of BC risk factors. As such, it is essential to identify an ex vivo human breast tissue mimetic model that can accurately pinpoint the effects of these factors in BC development. While two-dimensional models have been invaluable, they are not suitable for studying patient-specific tumor biology and drug response. Recent developments in three-dimensional (3D) models have led to the prominence of organized structures grown in a 3D environment called “organoids.” Breast organoids can accurately recapitulate the in vivo breast microenvironment and have been used to examine factors that affect signaling transduction, gene expression, and tissue remodeling. In this review, the applications, components, and protocols for development of breast organoids are discussed. We summarize studies that describe the utility of breast organoids, including in the study of normal mammary gland development and tumorigenesis. Finally, we provide an overview of protocols for development of breast organoids, and the advantages and disadvantages of different techniques in studies are described. The included studies have shown that breast organoids will continue to serve as a crucial platform for understanding of progression of BC tumors and the testing of novel therapeutics.