Stages Of Breast Cancer Progression Research Articles

M6A regulates breast cancer proliferation and migration through stage-dependent changes in Epithelial to Mesenchymal Transition gene expression.

While many factors have been implicated in breast cancer progression, effective treatments are still lacking. In recent years, it has become clear that posttranscriptional regulation plays a key role in the aberrant gene expression underlying malignancy and metastasis. For example, the mRNA modification N6-methyladenosine (m6A) is involved in numerous post-transcriptional regulation processes and has been implicated in many cancer types, including breast cancer. Despite intense study, even within a single type of cancer, there is little consensus, and often conflicting results, as to the role of m6A, suggesting other factors must influence the process. The goal of this study was to determine if the effects of m6A manipulation on proliferation and migration differed based on the stage of disease progression. Using the MCF10 model of breast cancer, we reduced m6A levels by targeting METTL3, the main cellular m6A RNA methyltransferase. Knocking down Mettl3 at different stages of breast cancer progression indeed shows unique effects at each stage. The early-stage breast cancer line showed a more proliferative phenotype with the knockdown of Mettl3 while the transformed breast cancer line showed a more migratory phenotype. Interestingly, the metastasized breast cancer cell line showed almost no effect on phenotype with the knockdown of Mettl3. Furthermore, transcriptome wide analysis revealed EMT as the probable pathway influencing the phenotypic changes. The results of this study may begin to address the controversy of m6A's role in cancer and suggest that m6A may have a dynamic role in cancer that depends on the stage of progression.

Paper-Based Coculture Platform to Evaluate the Effects of Fibroblasts on Estrogen Signaling in ER+ Breast Cancers.

Cell-based assays enable molecular-level studies of cellular responses to drug candidates or potential toxins. Transactivation assays quantify the activation or inhibition of nuclear receptors, key transcriptional regulators of gene targets in mamalian cells. One such assay couples the expression of luciferase to the transcriptional activity of estrogen receptor-alpha (ERα). While this assay is regularly used to screen for agonists and antagonists of the estrogen signaling pathway, the setup relies on monolayer cultures in which cells are plated directly onto the surface of cell-compatible plasticware. The tumor microenvironment is more than a collection of cancerous cells and is profoundly influenced by tissue architecture, the presence of extracellular matrices, and intercellular signaling molecules produced by non-cancerous neighboring cells (e.g., fibroblasts). There exists a need for three-dimensional culture platforms that can be rapidly prototyped to assess new configurations and readily produced in the large numbers needed for translational studies and screening applications. Here, we demonstrate the utility of the paper-based culture platform to probe the effects of intercellular signaling between two cell types. We used paper scaffolds to generate tumor-like environments, forming a defined volume of breast cancer cells suspended in collagen. By placing the paper scaffolds in commercial 96-well plates, we compared monocultures of only breast cancer cells with coculture configurations containing fibroblasts in different locations that mimicked the stages of breast cancer progression. We show that ERα transactivation in the T47D-KBluc cell line is affected by the presence, number, and proximity of fibroblasts, and is a consequence of intercellular signaling molecules. After screening a small library of fibroblast-secreted signaling molecules, we showed that interleukin-6 (IL-6) was the primary driver of reduced estradiol sensitivity. These effects were mitigated in the coculture configurations by the addition of an IL-6 neutralizing antibody. We also assessed estrogen receptor expression and transcriptional regulation, further demonstrating the utility of the paper-based platform for detailed mechanistic studies.

Robust Identification of Differential Gene Expression Patterns from Multiple Transcriptomics Datasets for Early Diagnosis, Prognosis, and Therapies for Breast Cancer.

Background and Objectives: Breast cancer (BC) is one of the major causes of cancer-related death in women globally. Proper identification of BC-causing hub genes (HubGs) for prognosis, diagnosis, and therapies at an earlier stage may reduce such death rates. However, most of the previous studies detected HubGs through non-robust statistical approaches that are sensitive to outlying observations. Therefore, the main objectives of this study were to explore BC-causing potential HubGs from robustness viewpoints, highlighting their early prognostic, diagnostic, and therapeutic performance. Materials and Methods: Integrated robust statistics and bioinformatics methods and databases were used to obtain the required results. Results: We robustly identified 46 common differentially expressed genes (cDEGs) between BC and control samples from three microarrays (GSE26910, GSE42568, and GSE65194) and one scRNA-seq (GSE235168) dataset. Then, we identified eight cDEGs (COL11A1, COL10A1, CD36, ACACB, CD24, PLK1, UBE2C, and PDK4) as the BC-causing HubGs by the protein-protein interaction (PPI) network analysis of cDEGs. The performance of BC and survival probability prediction models with the expressions of HubGs from two independent datasets (GSE45827 and GSE54002) and the TCGA (The Cancer Genome Atlas) database showed that our proposed HubGs might be considered as diagnostic and prognostic biomarkers, where two genes, COL11A1 and CD24, exhibit better performance. The expression analysis of HubGs by Box plots with the TCGA database in different stages of BC progression indicated their early diagnosis and prognosis ability. The HubGs set enrichment analysis with GO (Gene ontology) terms and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways disclosed some BC-causing biological processes, molecular functions, and pathways. Finally, we suggested the top-ranked six drug molecules (Suramin, Rifaximin, Telmisartan, Tukysa Tucatinib, Lynparza Olaparib, and TG.02) for the treatment of BC by molecular docking analysis with the proposed HubGs-mediated receptors. Molecular docking analysis results also showed that these drug molecules may inhibit cancer-related post-translational modification (PTM) sites (Succinylation, phosphorylation, and ubiquitination) of hub proteins. Conclusions: This study's findings might be valuable resources for diagnosis, prognosis, and therapies at an earlier stage of BC.

Neighborhood Disadvantage, African Genetic Ancestry, Cancer Subtype, and Mortality Among Breast Cancer Survivors.

Racial disparities in breast cancer (BC) survival arise from multilevel causes, which may exert influence at different stages of BC progression. Clarifying the importance of genetic and social factors could help prioritize interventions. To jointly examine associations between African genetic ancestry, social environment, and mortality from any cause and BC in Black BC survivors. This population-based cohort study enrolled self-identified Black women aged 20 to 75 years with histologically confirmed BC from June 2005 to May 2019 and followed them up until death or censoring in September 2021. Participants lived in 10 New Jersey counties. Data were analyzed between December 2022 and April 2023. A neighborhood socioeconomic status (nSES) index composed of census tract measures (education, income, wealth, employment status, and occupation) was linked to residential addresses at diagnosis. Percentage African ancestry was estimated using the ADMIXTURE program. Sequentially adjusted (age adjusted: age and interview year; fully adjusted: age adjusted with individual SES, lifestyle factors, and comorbidities) logistic regression models were fit to estimate associations with tumor subtypes (estrogen receptor-negative [ER-] vs estrogen receptor-positive [ER+]; triple-negative breast cancer [TNBC] vs luminal A), and Cox models were fit for associations with all-cause mortality (ACM) and breast cancer-specific mortality (BCSM). Models for BCSM were fit using Fine-Gray competing risks models, and robust standard errors were used to account for census tract-level clustering. Among 1575 participants, median (IQR) African ancestry was 85% (76%-90%), and median (IQR) age was 55 (46-63) years. A 10-percentage point increase in African ancestry was associated with higher odds of ER- vs ER+ (adjusted odds ratio [aOR], 1.08; 95% CI, 0.98-1.18) and TNBC vs luminal (aOR, 1.15; 95% CI, 1.02-1.31) tumors, but not with ACM or BCSM. A 1-IQR increase in nSES was associated with lower ACM (adjusted hazard ratio [aHR], 0.76; 95% CI, 0.63-0.93), and the HR for BCSM was less than 1 but not statistically significant (aHR, 0.81; 95% CI, 0.62-1.04) in age-adjusted models, but associations attenuated following further adjustment for potential mediators (individual SES, lifestyles, comorbidities). In this cohort study of Black female BC survivors, higher African ancestry was associated with aggressive tumor subtypes. Compared with genetic ancestry, mediating pathways related to social environments may be more important for survival in these patients.

A decellularized lung extracellular matrix/chondroitin sulfate/gelatin/chitosan-based 3D culture system shapes breast cancer lung metastasis

Distal metastasis of breast cancer is a primary cause of death, and the lung is a common metastatic target of breast cancer. However, the role of the lung niche in promoting breast cancer progression is not well understood. Engineered three-dimensional (3D) in vitro models capable of bridging this knowledge gap can be specifically designed to mimic crucial characteristics of the lung niche in a more physiologically relevant context than conventional two-dimensional systems. In this study, two 3D culture systems were developed to mimic the late stage of breast cancer progression at a lung metastatic site. These 3D models were created based on a novel decellularized lung extracellular matrix/chondroitin sulfate/gelatin/chitosan composite material and on a porcine decellularized lung matrix (PDLM), with the former tailored with comparable properties (stiffness, pore size, biochemical composition, and microstructure) to that of the in vivo lung matrix. The different microstructure and stiffness of the two types of scaffolds yielded diverse presentations of MCF-7 cells in terms of cell distribution, cell morphology, and migration. Cells showed better extensions with apparent pseudopods and more homogeneous and reduced migration activity on the composite scaffold compared to those on the PDLM scaffold. Furthermore, alveolar-like structures with superior porous connectivity in the composite scaffold remarkably promoted aggressive cell proliferation and viability. In conclusion, a novel lung matrix-mimetic 3D in vitro breast cancer lung metastasis model was developed to clarify the underlying correlativity between lung ECM and breast cancer cells after lung colonization. A better understanding of the effects of biochemical and biophysical environments of the lung matrix on cell behaviors can help elucidate the potential mechanisms of breast cancer progression and further improve target discovery of therapeutic strategies.

A novel computational predictive biological approach distinguishes Integrin β1 as a salient biomarker for breast cancer chemoresistance

Chemoresistance is a primary cause of breast cancer treatment failure, and protein-protein interactions significantly contribute to chemoresistance during different stages of breast cancer progression. In pursuit of novel biomarkers and relevant protein-protein interactions occurring during the emergence of breast cancer chemoresistance, we used a computational predictive biological (CPB) approach. CPB identified associations of adhesion molecules with proteins connected with different breast cancer proteins associated with chemoresistance. This approach identified an association of Integrin β1 (ITGB1) with chemoresistance and breast cancer stem cell markers. ITGB1 activated the Focal Adhesion Kinase (FAK) pathway promoting invasion, migration, and chemoresistance in breast cancer by upregulating Erk phosphorylation. FAK also activated Wnt/Sox2 signaling, which enhanced self-renewal in breast cancer. Activation of the FAK pathway by ITGB1 represents a novel mechanism linked to breast cancer chemoresistance, which may lead to novel therapies capable of blocking breast cancer progression by intervening in ITGB1-regulated signaling pathways.

Ultrasoft edge-labelled hydrogel sensors reveal internal tissue stress patterns in invasive engineered tumors

Measuring internal mechanical stresses within 3D tissues can provide important insights into drivers of morphogenesis and disease progression. Cell-sized hydrogel microspheres have recently emerged as a powerful technique to probe tissue mechanobiology, as they can be sufficiently soft as to deform within remodelling tissues, and optically imaged to measure internal stresses. However, measuring stresses at resolutions of ∼10 Pa requires ultrasoft, low-polymer content hydrogel formulations that are challenging to label with sufficiently fluorescent materials to support repeated measurements, particularly in optically dense tissues over 100 μm thick, as required in cancer tumor models. Here, we leverage thermodynamic partitioning of hydrogel components to create “edge-labelled” ultrasoft hydrogel microdroplets, in a single polymerization step. Bright and stable fluorescent nanoparticles preferentially polymerize at the hydrogel droplet interface, and can be used to repeatedly track sensor surfaces over long-term experiments, even when embedded deep in light-scattering tissues. We utilize these edge-labelled microspherical stress gauges (eMSGs) in inducible breast cancer tumor models of invasion, and demonstrate distinctive internal stress patterns that arise from cell-matrix interactions at different stages of breast cancer progression. Our studies demonstrate a long-term macroscale compaction of the tumor during matrix encapsulation, but only a short-term increase in local stress as non-invasive tumors rapidly make small internal reorganizations that reduce the mechanical stress to baseline levels. In contrast, once invasion programs are initiated, internal stress throughout the tumor is negligible. These findings suggest that internal tumor stresses may initially prime the cells to invade, but are lost once invasion occurs. Together, this work demonstrates that mapping internal mechanical stress in tumors may have utility in advancing cancer prognostic strategies, and that eMSGs can have broad utility in understanding dynamic mechanical processes of disease and development.

Adipose Cells Induce Escape from an Engineered Human Breast Microtumor Independently of their Obesity Status.

Obesity is associated with increased breast cancer incidence, recurrence, and mortality. Adipocytes and adipose-derived stem cells (ASCs), two resident cell types in adipose tissue, accelerate the early stages of breast cancer progression. It remains unclear whether obesity plays a role in the subsequent escape of malignant breast cancer cells into the local circulation. We engineered models of human breast tumors with adipose stroma that exhibited different obesity-specific alterations. We used these models to assess the invasion and escape of breast cancer cells into an empty, blind-ended cavity (as a mimic of a lymphatic vessel) for up to sixteen days. Lean and obese donor-derived adipose stroma hastened escape to similar extents. Moreover, a hypertrophic adipose stroma did not affect the rate of adipose-induced escape. When admixed directly into the model tumors, lean and obese donor-derived ASCs hastened escape similarly. This study demonstrates that the presence of adipose cells, independently of the obesity status of the adipose tissue donor, hastens the escape of human breast cancer cells in multiple models of obesity-associated breast cancer. The online version contains supplementary material available at 10.1007/s12195-022-00750-y.

Role of syndecan-4 in breast cancer pathophysiology.

Expression of the cell surface heparan sulfate proteoglycan syndecan-4 is dysregulated in breast cancer, the most frequent malignancy in women. High expression of syndecan-4 correlates with a worse survival in the subgroup of estrogen receptor negative and estrogen/progesterone-receptor negative patients. Aberrant expression of syndecan-4 in breast cancer involves both transcriptional and posttranscriptional mechanisms, including estrogen- and growth factor-dependent regulation, mutations in GAPVD1, NUP153, PDE4DIP, and RREB1, as well as targeting by microRNAs. At the functional level, syndecan-4 plays an important role in various stages of breast cancer progression by interacting with ligands as diverse as plasma proteins, extracellular matrix proteins, growth factors, and surface receptors, as well as members of the integrin family. Mechanisms including integrin recycling, ectodomain shedding, and crosstalk with other syndecans expand the repertoire of syndecan-4 function. Through these interactions, syndecan-4 regulates cellular processes such as adhesion, migration, and invasion. Additional possible functions of syndecan-4 in cells of the microenvironment contribute to the complexity of its pathophysiology. Notably, syndecan-4 expression is modulated by drugs used in breast cancer treatment, such as trastuzumab and zoledronate. Overall, these findings mark syndecan-4 as a novel pathogenesis factor and promising target for therapeutic interventions in breast cancer.

NR2F1 Is a Barrier to Dissemination of Early-Stage Breast Cancer Cells.

During early stages of breast cancer progression, HER2-mediated suppression of NR2F1 promotes dissemination by inducing EMT and a hybrid luminal/basal-like program.

An Organotypic Mammary Duct Model Capturing Matrix Mechanics-Dependent Ductal Carcinoma In Situ Progression.

Ductal carcinoma in situ (DCIS) is a precancerous stage breast cancer, where abnormal cells are contained within the duct, but have not invaded into the surrounding tissue. However, only 30-40% of DCIS cases are likely to progress into an invasive ductal carcinoma (IDC), while the remainder are innocuous. Since little is known about what contributes to the transition from DCIS to IDC, clinicians and patients tend to opt for treatment, leading to concerns of overdiagnosis and overtreatment. In vitro models are currently being used to probe how DCIS transitions into IDC, but many models do not take into consideration the macroscopic tissue architecture and the biomechanical properties of the microenvironment. In this study, we modeled an organotypic mammary duct as a channel molded in a collagen matrix and lined with basement membrane. By adjusting the concentration of collagen (4 and 8 mg/mL), we modulated the stiffness and morphological properties of the matrix and examined how an assortment of breast cells, including the isogenic MCF10 series that spans the range from healthy to aggressive, behaved within our model. We observed distinct characteristics of breast cancer progression such as hyperplasia and invasion. Normal mammary epithelial cells (MCF10A) formed a single-cell layer on the lumen surface, whereas the most aggressive (MCF10CA1) were several cell layers thick. The model captured collagen concentration-dependent protrusive behaviors by the MCF10A and MCF10CA1 cells, as well as a known invasive cell line (MDA-MB-231). The MCF10A and MCF10CA1 cells extended protrusions into the lower collagen concentration matrix, while the MDA-MB-231 cells fully invaded matrices of either collagen concentration but to a greater distance in the higher collagen concentration matrix. Our results show that the model can recapitulate different stages of breast cancer progression and that the MCF10 series is adaptable to physiologically relevant in vitro studies, demonstrating the potential of both the model and cell lines to elucidate key factors that may contribute to understanding the transition from DCIS to IDC. Impact statement The success of early preventative measures for breast cancer has left patients susceptible to overdiagnosis and overtreatment. Limited knowledge of factors driving an invasive transition has inspired the development of in vitro models that accurately capture this phenomenon. However, current models tend to neglect the macroscopic architecture and biomechanical properties of the mammary duct. In this study, we introduce an organotypic model that recapitulates the cylindrical geometry of the tissue and the altered stroma seen in tumor microenvironments. Our model was able to capture distinct features associated with breast cancer progression, demonstrating its potential to uncover novel insights into disease progression.

Is mammography screening beneficial: An individual-based stochastic model for breast cancer incidence and mortality.

The benefits of mammography screening have been controversial, with conflicting findings from various studies. We hypothesize that unmeasured heterogeneity in tumor aggressiveness underlies these conflicting results. Based on published data from the Canadian National Breast Screening Study (CNBSS), we develop and parameterize an individual-based mechanistic model for breast cancer incidence and mortality that tracks five stages of breast cancer progression and incorporates the effects of age on breast cancer incidence and all-cause mortality. The model accurately reproduces the reported outcomes of the CNBSS. By varying parameters, we predict that the benefits of mammography depend on the effectiveness of cancer treatment and tumor aggressiveness. In particular, patients with the most rapidly growing or potentially largest tumors have the highest benefit and least harm from the screening, with only a relatively small effect of age. However, the model predicts that confining mammography to populations with a high risk of acquiring breast cancer increases the screening benefit only slightly compared with the full population.

Circulating Tumor Cells in Breast Cancer.

With active screening for early detection and advancements in treatment, there has been a significant decrease in mortality from breast cancer. However, a significant proportion of patients with non-metastatic breast cancer at time of diagnosis will relapse. Therefore, it is suggested that the dissemination of bloodstream tumor cells (circulating tumor cells, CTCs) undetectable by currently available diagnostic tools occurs during the early stages of breast cancer progression, and may be the potential source of micrometastases responsible for treatment failures. Here, we review the clinical significance of CTCs, as detected by the FDA-approved CellSearch® System, in both metastatic and non-metastatic breast cancer patients. Studies so far suggest that CTCs are prognostic of poorer outcomes in breast cancer patients; however, there is currently insufficient data to support use of CTC data to guide treatment. Therefore, there are ongoing studies to evaluate the utility of assessing CTC phenotypes to develop personalized breast cancer treatment, which will be reviewed in this chapter.

A guide to hyaluronan and related enzymes in breast cancer: biological significance and diagnostic value.

Hyaluronan (HA) is a unique nonsulfated glycosaminoglycan that contributes to breast cancer cells growth and functional properties, including cell migration, invasion, adhesion, as well as tumor-associated angiogenesis in different stages of breast cancer progression and especially metastasis. Latest data show that the levels of HA and/or low molecular mass HA in blood serum and plasma of breast cancer patients may be a useful biomarker for breast cancer prognosis, differential diagnosis, and patients' treatment monitoring. Therefore, the qualitative and quantitative determination of HA in biological samples is an emerging area of research. This review gathers, categorizes, and sums up all the currently used methodologies to analyze HA and HA-related enzymes. The advantages, disadvantages, limitations in use, and the information they provide, are critically considered and discussed. Moreover, emphasis is given to the significance of HA determination in breast cancer, as well as of its related enzymes, for diagnosis and prognosis of this type of cancer.

SAT-342 Macrophage-Derived C/EBPb During Mammary Gland Development and Breast Cancer Progression

Macrophages are necessary in mammary gland development to drive ductal elongation and have been shown to promote cancer metastasis. Our laboratory has previously shown that macrophages are recruited to pre-invasive lesions to induce localized invasion during the switch from premalignant to invasive cancer. However, the mechanisms by which macrophages promote mammary gland development and early breast cancer progression are poorly understood. CCAAT/enhancer-binding protein β (C/EBPβ) is a transcription factor that regulates the growth and differentiation of myeloid and epithelial cells in various tissues. The goal of this study is to define the role of macrophage-C/EBPβ in mammopoiesis and early breast cancer progression. Conditional deletion of C/EBPβ in macrophages resulted in alterations in mammary branching and TEB formation, while ductal elongation remained normal. Interestingly, loss of C/EBPβ in macrophages using CSFR1-iCre mice induced an increase in eosinophils and overall F4/80+CD11b+, while lymphoid cells remained unchanged. Using a p53-null mouse model of premalignant progression (termed PN1a), we previously showed that macrophages are polarized by pre-invasive PN1a cells in vivo, and consequently induce a tumor-like phenotype. To determine whether C/EBPβ is required for macrophage polarization, macrophages were transduced with specific protein isoforms of C/EBPβ and various cytokines were examined. C/EBPβ-LAP2 induced an increase in TNFα and TGFβ expression, suggesting a distinct function for the individual isoforms. Polarizing Raw246.7 cells changes the expression of C/EBPβ indicating that the environment can effect C/EBPβ expression and cytokine production. Ongoing studies are aimed at delineating the consequences of C/EBPβ deletion in myeloid cells at different stages of breast cancer progression. The results of these studies will have critical implications for developing prevention strategies and for therapeutically targeting macrophages in early stage disease. These studies are supported by Susan G. Komen and NCI RO1 grants.

Early and late endothelial response in breast cancer metastasis in mice: simultaneous quantification of endothelial biomarkers using a mass spectrometry-based method.

ABSTRACTThe endothelium plays an important role in cancer metastasis, but the mechanisms involved are still not clear. In the present work, we characterised the changes in endothelial function at early and late stages of breast cancer progression in an orthotopic model of murine mammary carcinoma (4T1 cells). Endothelial function was analysed based on simultaneous microflow liquid chromatography–tandem mass spectrometry using multiple reaction monitoring (microLC/MS-MRM) quantification of 12 endothelium-related biomarkers, including those reflecting glycocalyx disruption – syndecan-1 (SDC-1), endocan (ESM-1); endothelial inflammation – vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), E-selectin (E-sel); endothelial permeability – fms-like tyrosine kinase 1 (FLT-1), angiopoietin 2 (Angpt-2); and haemostasis – von Willebrand factor (vWF), tissue plasminogen activator (t-PA), plasminogen activator inhibitor 1 (PAI-1), as well as those that are pathophysiologically linked to endothelial function – adrenomedullin (ADM) and adiponectin (ADN). The early phase of metastasis in mouse plasma was associated with glycocalyx disruption (increased SDC-1 and ESM-1), endothelial inflammation [increased soluble VCAM-1 (sVCAM-1)] and increased vascular permeability (Angpt-2). During the late phase of metastasis, additional alterations in haemostasis (increased PAI-1 and vWF), as well as a rise in ADM and substantial fall in ADN concentration, were observed. In conclusion, in a murine model of breast cancer metastasis, we identified glycocalyx disruption, endothelial inflammation and increased endothelial permeability as important events in early metastasis, while the late phase of metastasis was additionally characterised by alterations in haemostasis.

Interplay between ShcA Signaling and PGC-1α Triggers Targetable Metabolic Vulnerabilities in Breast Cancer.

The ShcA adaptor protein transduces oncogenic signals downstream of receptor tyrosine kinases. We show here that breast tumors engage the ShcA pathway to increase their metabolism. ShcA signaling enhanced glucose catabolism through glycolysis and oxidative phosphorylation, rendering breast cancer cells critically dependent on glucose. ShcA signaling simultaneously increased the metabolic rate and flexibility of breast cancer cells by inducing the PGC-1α transcriptional coactivator, a central regulator of mitochondrial metabolism. Breast tumors that engaged ShcA signaling were critically dependent on PGC-1α to support their increased metabolic rate. PGC-1α deletion drastically delayed breast tumor onset in an orthotopic mouse model, highlighting a key role for PGC-1α in tumor initiation. Conversely, reduced ShcA signaling impaired both the metabolic rate and flexibility of breast cancer cells, rendering them reliant on mitochondrial oxidative phosphorylation. This metabolic reprogramming exposed a targetable metabolic vulnerability, leading to a sensitization of breast tumors to inhibitors of mitochondrial complex I (biguanides). Genetic inhibition of ShcA signaling in the Polyoma virus middle T (MT) breast cancer mouse model sensitized mammary tumors to biguanides during the earliest stages of breast cancer progression. Tumor initiation and growth were selectively and severely impaired in MT/ShcA-deficient animals. These data demonstrate that metabolic reprogramming is a key component of ShcA signaling and serves an unappreciated yet vital role during breast cancer initiation and progression. These data further unravel a novel interplay between ShcA and PGC-1α in the coordination of metabolic reprogramming and demonstrate the sensitivity of breast tumors to drugs targeting oxidative phosphorylation.Significance: This study uncovers a previously unrecognized mechanism that links aberrant RTK signaling with metabolic perturbations in breast cancer and exposes metabolic vulnerabilities that can be targeted by inhibitors of oxidative phosphorylation. Cancer Res; 78(17); 4826-38. ©2018 AACR.

Intraoperative Resection Guidance with Photoacoustic and Fluorescence Molecular Imaging Using an Anti-B7-H3 Antibody-Indocyanine Green Dual Contrast Agent.

Purpose: Breast cancer often requires surgical treatment including breast-conserving surgical resection. However, with current postsurgical histologic margin analysis, one quarter of breast cancer patients undergo reexcision to achieve negative margins corresponding to decreased local recurrence and better outcomes. Therefore, a method with high resolution and specificity for intraoperative margin assessment is needed.Experimental Design: First, quantitative immunofluorescence staining of B7-H3 expression was assessed in four pathologic stages of breast cancer progression of the MMTV-PyMT transgenic murine model. Next, an antibody-dye contrast agent, B7-H3-ICG, was injected into mice prior to surgical resection of breast cancer. Anatomic ultrasound, spectroscopic photoacoustic (sPA), and fluorescence imaging were used to guide resection of mammary glands suspected of containing cancer. Resected tissues were processed for H&E staining and pathologic assessment and compared with sPA and fluorescence imaging signals.Results: Tissue containing DCIS (46.0 ± 4.8 a.u.) or invasive carcinoma (91.7 ± 21.4 a.u.) showed significantly higher (P < 0.05) B7-H3 expression than normal and hyperplastic tissues (1.3 ± 0.8 a.u.). During image-guided surgical resection, tissue pieces assessed as normal or hyperplastic (n = 17) showed lower average sPA (3.17 ± 0.48 a.u.) and fluorescence signal [6.83E07 ± 2.00E06 (p/s)/(μW/cm²)] than DCIS and invasive carcinoma tissue (n = 63) with an average sPA signal of 23.98 ± 4.88 a.u. and an average fluorescence signal of 7.56E07 ± 1.44E06 (p/s)/(μW/cm²) with AUCs of 0.93 [95% confidence interval (CI), 0.87-0.99] and 0.71 (95% CI, 0.57-0.85), respectively.Conclusions: It was demonstrated that sPA and fluorescence molecular imaging combined with B7-H3-ICG agent can assess the disease status of tissues with high diagnostic accuracy, intraoperatively, with high resolution, sensitivity, and specificity. Clin Cancer Res; 24(15); 3572-82. ©2018 AACR.

Cytoplasmic FOXP1 expression is correlated with ER and calpain II expression and predicts a poor outcome in breast cancer

BackgroundNuclear forkhead box protein P1 (N-FOXP1) expression in invasive breast cancer has been documented in the literature. However, the FOXP1 expression patterns at different stages of breast cancer progression are largely unknown, and the significance of cytoplasmic FOXP1 (C-FOXP1) expression in breast cancer has not been well illustrated. The aims of this study were to investigate FOXP1 expression patterns in invasive ductal carcinoma (IDC), ductal carcinoma in situ (DCIS), atypical ductal hyperplasia (ADH) and usual ductal hyperplasia (UDH), and to analyze the clinicopathological relevance of C-FOXP1 and its prognostic value in IDC.MethodsN-FOXP1 and C-FOXP1 expression in cases of IDC, DCIS, ADH and UDH was determined using immunohistochemistry. The correlation between C-FOXP1 expression and clinicopathological parameters as well as the overall survival (OS) and disease-free survival (DFS) rates of patients with IDC were analyzed.ResultsExclusive N-FOXP1 expression was found in 85.0% (17/20), 40.0% (8/20), 12.2% (5/41) and 10.8% (9/83) of UDH, ADH, DCIS, and IDC cases, respectively, and exclusive C-FOXP1 expression was observed in 0% (0/20), 0% (0/20), 4.9% (2/41), and 31.3% (26/83) of the cases, respectively. Both N- and C-FOXP1 staining were observed in 15.0% (3/20), 60.0% (12/20), 82.9% (34/41) and 48.2% (40/83) of the above cases, respectively, while complete loss of FOXP1 expression was observed in only 9.6% (8/83) of IDC cases. Estrogen receptor (ER) expression in C-FOXP1-positive IDC cases (31/66, 47.0%) was significantly lower than that in C-FOXP1-negative cases (13/17, 76.5%) (p = 0.030). Calpain II expression was observed in 83.3% (55/66) of C-FOXP1-positive IDC cases, which was significantly higher than that in C-FOXP1-negative cases (9/17, 52.9%) (p = 0.007). Calpain II was significantly associated with pAKT (p = 0.029), pmTOR (p = 0.011), p4E-BP1 (p < 0.001) and p-p70S6K (p = 0.003) expression levels. The 10-year OS and DFS rates of the C-FOXP1-positive patients were 60.5% and 48.7%, respectively, both of which were lower than those of the C-FOXP1-negative patients (93.3, 75.3%). The OS curve showed a dramatic impact of C-FOXP1 status on OS (p = 0.045).ConclusionsCytoplasmic relocalization of FOXP1 protein was a frequent event in breast IDC. Calpain II might play an important role in nucleocytoplasmic trafficking of FOXP1 and the AKT pathway might be involved in this process. C-FOXP1 expression was inversely associated with ER expression and might be a predictor of poor OS in patients with IDC.

Abstract 4782: Transforming growth factor-beta regulation of cancer stem cell dynamics at different stages of breast cancer progression

Abstract Cancer stem cells (CSCs) are a subpopulation of tumor cells that are uniquely capable of initiating and sustaining tumorigenesis, and they have also been implicated in driving disease recurrence after cancer therapy. We have previously developed a novel lentiviral-based reporter system for direct visualization, quantitation and isolation of cells with CSC properties. The construct consists of a tandemly-repeated composite SOX2-OCT4 response element (“SORE6”) driving expression of a destabilized fluorescent protein reporter (Tang et al. Stem Cell Reports, 2015). TGF-βs are pleiotropic regulatory factors with complex roles in tumorigenesis. We and others have hypothesized that TGF-β can regulate the CSC population in breast cancer with stimulatory or inhibitory effects depending the model and stage of cancer development. Using the MCF10Ca1h and MCF7 breast cancer models, which retain tumor suppressor responses to TGF-β, here we show that TGF-β treatment significantly reduces the size of the CSC population in vitro. Although TGF-β had relatively little effect on invasion and migration of the bulk population in these models, it clearly inhibited migration and invasion of the CSCs, suggesting that biological responses to TGF-β can vary depending on the position of the cell in the differentiation hierarchy. Combining our stem cell reporter with a TGF-β pathway reporter, we further show that CSCs in the MCF10Ca1h model have higher endogenous activation of the TGF-β pathway than does the bulk tumor cell population, and by time-lapse video microscopy we find that CSCs with active TGF-β signaling are relatively quiescent. Furthermore, neutralization of TGF-β in vivo leads to an increased representation of CSCs in MCF10Ca1h and MCF7 tumors. Thus our preliminary results suggest that in breast cancer models where TGF-β acts as a tumor suppressor, TGF-β signaling is preferentially activated in the CSC compartment and keeps a subpopulation of CSCs in a quiescent and stationary state. These findings have important implications for the clinical use of TGF-β pathway antagonists. In contrast, in the metastatic breast cancer MDA-MB231 model, in which TGF-β acts as a pro-progression factor, we show that TGF-β can increase the size of the CSC population and enhance invasion. In this model, the CSCs are enriched for the ability to form macroscopic lung metastases when compared with non-CSCs, and we show that this effect is due in part to a selective survival advantage of the CSCs in the first 24 hours after arrival at the metastatic site. The role of TGF-β in this process is currently under investigation. Citation Format: Binwu Tang, George Baker, Joshua Zent, Yuval Raviv, Lalage Wakefield. Transforming growth factor-beta regulation of cancer stem cell dynamics at different stages of breast cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4782. doi:10.1158/1538-7445.AM2017-4782