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XLSX file - 16K, List of mouse C3(1)-T-antigen gene expression microarrays used to derive the murine gene lists.

PDF file - 42K, Summary of drugs used in this study.

PDF file - 68K

PDF file - 1539K, Kaplan-Meier analyses for the prediction of Distant Relapse Free Survival.

Purpose: To use genetically engineered mouse models (GEMM) and orthotopic syngeneic murine transplants (OST) to develop gene expression-based predictors of response to anticancer drugs in human tumors. These mouse models offer advantages including precise genetics and an intact microenvironment/immune system.Experimental Design: We examined the efficacy of 4 chemotherapeutic or targeted anticancer drugs, alone and in combination, using mouse models representing 3 distinct breast cancer subtypes: Basal-like (C3(1)-T-antigen GEMM), Luminal B (MMTV-Neu GEMM), and Claudin-low (T11/TP53−/− OST). We expression-profiled tumors to develop signatures that corresponded to treatment and response, and then tested their predictive potential using human patient data.Results: Although a single agent exhibited exceptional efficacy (i.e., lapatinib in the Neu-driven model), generally single-agent activity was modest, whereas some combination therapies were more active and life prolonging. Through analysis of RNA expression in this large set of chemotherapy-treated murine tumors, we identified a pair of gene expression signatures that predicted pathologic complete response to neoadjuvant anthracycline/taxane therapy in human patients with breast cancer.Conclusions: These results show that murine-derived gene signatures can predict response even after accounting for common clinical variables and other predictive genomic signatures, suggesting that mice can be used to identify new biomarkers for human patients with cancer. Clin Cancer Res; 19(17); 4889–99. ©2013 AACR.

XLSX file - 60K, Gene lists obtained from a study of carboplatin/paclitaxel treated tumors.

PDF file - 46K, Univariate and Multivariate Analysis for pCR.

XLSX file - 32K, Modules/expression signatures analysis of carboplatin/paclitaxel treated C3(1)-T-antigen mouse tumors.

PDF file - 3255K, Hierarchical clustering analysis of the untreated and responding murine chemotherapy signature using 337 human breast tumors.

Supplemental Table 2 from Fibroblast Growth Factor Receptor Signaling Dramatically Accelerates Tumorigenesis and Enhances Oncoprotein Translation in the Mouse Mammary Tumor Virus–Wnt-1 Mouse Model of Breast Cancer

Supplemental Fig 5 from Fibroblast Growth Factor Receptor Signaling Dramatically Accelerates Tumorigenesis and Enhances Oncoprotein Translation in the Mouse Mammary Tumor Virus–Wnt-1 Mouse Model of Breast Cancer

Supplementary Figure Legends 1-6, Table 1 Legend from Fibroblast Growth Factor Receptor Signaling Dramatically Accelerates Tumorigenesis and Enhances Oncoprotein Translation in the Mouse Mammary Tumor Virus–Wnt-1 Mouse Model of Breast Cancer

Supplemental Fig 2 from Fibroblast Growth Factor Receptor Signaling Dramatically Accelerates Tumorigenesis and Enhances Oncoprotein Translation in the Mouse Mammary Tumor Virus–Wnt-1 Mouse Model of Breast Cancer

Supplemental Fig 3 from Fibroblast Growth Factor Receptor Signaling Dramatically Accelerates Tumorigenesis and Enhances Oncoprotein Translation in the Mouse Mammary Tumor Virus–Wnt-1 Mouse Model of Breast Cancer

Data Supplement from Fibroblast Growth Factor Receptor Signaling Dramatically Accelerates Tumorigenesis and Enhances Oncoprotein Translation in the Mouse Mammary Tumor Virus–Wnt-1 Mouse Model of Breast Cancer

Supplementary Materials and Methods PDF File - 113K, Supplemental materials and methods section

Fibroblast growth factor (FGF) cooperates with the Wnt/β-catenin pathway to promote mammary tumorigenesis. To investigate the mechanisms involved in FGF/Wnt cooperation, we genetically engineered a model of inducible FGF receptor (iFGFR) signaling in the context of the well-established mouse mammary tumor virus–Wnt-1 transgenic mouse. In the bigenic mice, iFGFR1 activation dramatically enhanced mammary tumorigenesis. Expression microarray analysis did not show transcriptional enhancement of Wnt/β-catenin target genes but instead showed a translational gene signature that also correlated with elevated FGFR1 and FGFR2 in human breast cancer data sets. Additionally, iFGFR1 activation enhanced recruitment of RNA to polysomes, resulting in a marked increase in protein expression of several different Wnt/β-catenin target genes. FGF pathway activation stimulated extracellular signal-regulated kinase and the phosphorylation of key translation regulators both in vivo in the mouse model and in vitro in a human breast cancer cell line. Our results suggest that cooperation of the FGF and Wnt pathways in mammary tumorigenesis is based on the activation of protein translational pathways that result in, but are not limited to, increased expression of Wnt/β-catenin target genes (at the level of protein translation). Further, they reveal protein translation initiation factors as potential therapeutic targets for human breast cancers with alterations in FGF signaling. Cancer Res; 70(12); 4868–79. ©2010 AACR.

Supplementary Figure 2 PDF File - 600K, Ectopic expression of FOXC2 induces EMT in transformed human mammary epithelial cells (HMLER). (a) Quantification of EMT marker mRNA expression by quantitative real-time PCR analysis. Data is represented as the expression of EMT markers in HMLER-FOXC2 (FOXC2) cells relative to HMLER-Vector control cells (Vector). GAPDH was used as an internal normalization control. Columns indicate the mean (n = 3); error bars indicate SEM. (b) Western blot analysis of EMT marker protein expression upon FOXC2 overexpression in HMLER cells. Actin was used as a loading control. (c) Phase contrast and immunofluroescence images of HMLER-Vector and HMLER-FOXC2 cells. Overlayed images are shown for respective EMT markers (red) and DAPI nuclear stain (blue). Scale bar indicates 50 �m

Supplemental Fig 8 from Fibroblast Growth Factor Receptor Signaling Dramatically Accelerates Tumorigenesis and Enhances Oncoprotein Translation in the Mouse Mammary Tumor Virus–Wnt-1 Mouse Model of Breast Cancer

Supplemental Fig 4 from Fibroblast Growth Factor Receptor Signaling Dramatically Accelerates Tumorigenesis and Enhances Oncoprotein Translation in the Mouse Mammary Tumor Virus–Wnt-1 Mouse Model of Breast Cancer

Supplementary Figures 1-9 - Germline mutation of Brca1 transforms p18-deficient luminal tumors into basal-like tumors with induction of EMT (Suppl. Fig. 1); Germline mutation of Brca1 activates EMT-TFs in mammary and tumor development (Suppl. Fig. 2); Specific deletion of Brca1 in mammary epithelia impairs luminal differentiation (Suppl. Fig. 3); Haploid loss of Brca1 in mammary epithelium inhibits the expression of luminal genes but stimulates the expression of basal genes and EMT-TFs in p18 deficient LSCs (Suppl. Fig. 4); Ck5 and Ck8 double positive epithelial cells are frequently detected in p18-/-;Brca1f/-;MC mammary glands (Suppl. Fig. 5); Characterization of human breast cancer cell lines (Suppl. Fig. 6); BRCA1 inhibits the expression of TWIST and FOXC2 in 293T cells (Suppl. Fig. 7); Knockdown of BRCA1 in T47D cells activates EMT (Suppl. Fig. 8); BRCA1 and TWIST mRNA levels are inversely correlated in human claudin-low type breast cancers (Suppl. Fig. 9).

Supplementary Figure 3 PDF File - 326K, Attenuation of FOXC2 expression leads to reduced levels of PDGFR- �. (a) Immunofluorescene staining for PDGFR-� (red) detected with Alexa Fluor 548 anti-mouse secondary antibody in HMLE, HMLE-Snail-shCntrl and HMLE-Snail-shFOXC2 cells. Nuclei were counterstained with DAPI and overlayed images are shown.Scale bar indicates 100 �m. (b) FACS analysis of PDGFR-� cell surface expression using anti- PDGFR-�-PE antibody in cells described in (a). The histogram represents the intensity of PE signal in the FL2 detector on the x-axis

Supplemental Fig 7 from Fibroblast Growth Factor Receptor Signaling Dramatically Accelerates Tumorigenesis and Enhances Oncoprotein Translation in the Mouse Mammary Tumor Virus–Wnt-1 Mouse Model of Breast Cancer

Supplementary Figure 1 PDF File - 22K, FOXC2 does not regulate the expression of other EMT factors. Quantification of EMT marker mRNA expression by quantitative real-time PCR analysis. Data is represented as the expression of EMT markers in HMLER-FOXC2 (FOXC2) cells relative to HMLER control cells GAPDH was used as an internal normalization control. Columns indicate the mean (n = 3); error bars indicate SEM.

Supplemental Table 3 from Fibroblast Growth Factor Receptor Signaling Dramatically Accelerates Tumorigenesis and Enhances Oncoprotein Translation in the Mouse Mammary Tumor Virus–Wnt-1 Mouse Model of Breast Cancer

Supplemental Table 1 from Fibroblast Growth Factor Receptor Signaling Dramatically Accelerates Tumorigenesis and Enhances Oncoprotein Translation in the Mouse Mammary Tumor Virus–Wnt-1 Mouse Model of Breast Cancer

Supplementary Figure 4 PDF File - 107K, Cells expressing endogenous FOXC2 exhibit increased sensitivity to sunitinib (a) Quantification of HMLE-Snail and (b) SUM159 cell viability expressing either shControl or shFOXC2, following culture for 96 hours in increasing concentrations of sunitinib. Cell viability was assessed using an MTS assay. (c) In vitro quantification of mammospheres formed by cells described above grown in the presence of sunitinib. Data represents the number of mammospheres formed per 1000 cells seeded after 7 days of culture. Error bars indicate SEM. * P

Legends for Supplementary Figures 1-9.

Supplemental Fig 6 from Fibroblast Growth Factor Receptor Signaling Dramatically Accelerates Tumorigenesis and Enhances Oncoprotein Translation in the Mouse Mammary Tumor Virus–Wnt-1 Mouse Model of Breast Cancer

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 ...

The purpose of this review is to highlight several areas of lncRNA biology and cancer that we hope will provide some new insights for future research. These include the relationship of lncRNAs and the epithelial to mesenchymal transition (EMT) with a focus on transcriptional and alternative splicing mechanisms and mRNA stability through miRNAs. In addition, we highlight the potential role of enhancer e-lncRNAs, the importance of transposable elements in lncRNA biology, and finally the emerging area of using antisense oligonucleotides (ASOs) and small molecules to target lncRNAs and their therapeutic implications.

Ductal carcinoma in situ (DCIS) is a nonobligate precursor to invasive breast cancer. Only a small percentage of DCIS cases are predicted to progress; however, there is no method to determine which DCIS lesions will remain innocuous from those that will become invasive disease. Therefore, DCIS is treated aggressively creating a current state of overdiagnosis and overtreatment. There is a critical need to identify functional determinants of progression of DCIS to invasive ductal carcinoma (IDC). Interrogating biopsies from five patients with contiguous DCIS and IDC lesions, we have shown that expression of the long noncoding RNA BHLHE40-AS1 increases with disease progression. BHLHE40-AS1 expression supports DCIS cell proliferation, motility, and invasive potential. Mechanistically, BHLHE40-AS1 modulates interleukin (IL)-6/signal transducer and activator of transcription 3 (STAT3) activity and a proinflammatory cytokine signature, in part through interaction with interleukin enhancer-binding factor 3. These data suggest that BHLHE40-AS1 supports early breast cancer progression by engaging STAT3 signaling, creating an immune-permissive microenvironment.

Background Increased breast cancer screening over the past four decades has led to a substantial rise in the diagnosis of ductal carcinoma in situ (DCIS). Although DCIS lesions precede invasive ductal carcinoma (IDC), they do not always transform into cancer. The current standard-of-care for DCIS is an aggressive course of therapy to prevent invasive and metastatic disease resulting in over-diagnosis and over-treatment. Thus, there is a critical need to identify functional determinants of progression of DCIS to IDC to allow discrimination between indolent and aggressive disease. Recent studies show that super-enhancers, in addition to promoting other gene transcription, are themselves transcribed producing super-enhancer associated long noncoding RNAs (SE-lncRNAs). These SE-lncRNAs can interact with their associated enhancer regions in cis and influence activities and expression of neighboring genes. Furthermore, they represent a novel, untapped group of therapeutic targets. Methods With an integrative analysis of enhancer loci with global expression of SE-lncRNAs in the MCF10A progression series, we have identified differentially expressed SE-lncRNAs which can identify mechanisms for DCIS to IDC progression. Furthermore, cross-referencing these SE-lncRNAs with patient samples in the The Cancer Genome Atlas (TCGA) database, we have unveiled 27 clinically relevant SE-lncRNAs that potentially interact with their enhancer to regulate nearby gene expression. To complement SE-lncRNA expression studies, we conducted an unbiased global analysis of super-enhancers that are acquired or lost in progression. Results Here we designate SE-lncRNAs RP11-379F4.4 and RP11-465B22.8 as potential markers of progression of DCIS to IDC through regulation of the expression of their neighboring genes (RARRES1 and miR-200b, respectively). Moreover, we classified 403 super-enhancer regions in MCF10A normal cells, 627 in AT1, 1053 in DCIS, and 320 in CA1 cells. Comparison analysis of acquired/lost super-enhancer regions with super-enhancer regions classified in 47 ER positive patients, 10 triple negative breast cancer (TNBC) patients, and 11 TNBC cell lines reveal critically acquired pathways including STAT signaling and NF-kB signaling. In contrast, protein folding, and local estrogen production are identified as major pathways lost in progression. Conclusion Collectively, these analyses identify differentially expressed SE-lncRNAs and acquired/lost super-enhancers in progression of breast cancer important for promoting DCIS lesions to IDC.

Ductal carcinoma

The interactions of chemotherapeutic drugs with nanocage protein apoferritin (APO) are the key features in the effective encapsulation and release of highly toxic drugs in APO-based controlled drug delivery systems. The encapsulation enables mitigating the drugs side effects, collateral damage to healthy cells, and adverse immune reactions. Herein, the interactions of anthracycline drugs with APO were studied to assess the effect of drug lipophilicity on their encapsulation excess n and in vitro activity. Anthracycline drugs, including doxorubicin (DOX), epirubicin (EPI), daunorubicin (DAU), and idarubicin (IDA), with lipophilicity P from 0.8 to 15, were investigated. We have found that in addition to hydrogen-bonded supramolecular ensemble formation with n = 24, there are two other competing contributions that enable increasing n under strong polar interactions (APO(DOX)) or under strong hydrophobic interactions (APO(IDA) of the highest efficacy). The encapsulation/release processes were investigated using UV-Vis, fluorescence, circular dichroism, and FTIR spectroscopies. In vitro cytotoxicity/growth inhibition tests and flow cytometry corroborate high apoptotic activity of APO(drugs) against targeted MDA-MB-231 adenocarcinoma and HeLa cancer cells, and low activity against non-tumorigenic MCF10A cells, demonstrating targeting ability of nanodrugs. A model for molecular interactions between anthracyclines and APO nanocarriers was developed, and the relationships derived compared with experimental results.

Ductal carcinoma in situ (DCIS) is a pre-cancerous 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. Here, we developed an organotypic mammary duct model by molding a channel within a collagen matrix and lining it with a basement membrane. By adjusting the concentration of collagen, we effectively modulated the stiffness and morphological properties of the matrix and examined how an assortment of breast cells responded to changing density and stiffness of the matrix. We first validated the model using two established, phenotypically divergent breast cancer cell lines by demonstrating the ability of the cells to either invade (MDA-MB-231) or cluster (MCF7). We then examined how cells of the isogenic MCF10 series—spanning the range from healthy to aggressive—behaved within our model and observed distinct characteristics of breast cancer progression such as hyperplasia and invasion, in response to collagen concentration. 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 STATEMENTThe 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. Here, 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.

Increased emphasis on breast cancer screening has led to a dramatic increase in diagnosis of ductal carcinoma in situ (DCIS). DCIS lesions are nonobligate precursors of invasive ductal carcinoma (IDC) and thus, the current standard-of-care is aggressive therapy to prevent invasive and metastatic disease. However, only ˜40% of DCIS cases are predicted to progress leading to a current state of overtreatment and overdiagnosis. Thus, there is a critical need to identify functional determinants of progression of DCIS to IDC to allow discrimination between indolent and aggressive breast cancers and refine patient treatment strategies. We propose that long noncoding RNAs (lncRNAs) functionally drive breast cancer progression and their expression can discriminate between innocuous and potentially invasive DCIS. Using biopsies from women with tandem DCIS and IDC lesions, we identified the lncRNA BHLHE40-AS1 as enriched in patient IDC. Furthermore, BHLHE40-AS1 is enriched in multiple breast cancer progression models, in HER2+ cell lines, and can be induced by expression of HER2. BHLHE40-AS1 is transcribed from a known super-enhancer that we find becomes rewired in breast cancer progression. In addition, the lncRNA is found antisense to BHLHE40, a transcription factor critical to many core processes (apoptosis, EMT, circadian rhythm). Depletion of the lncRNA attenuates expression of BHLHE40. Future studies are focused on mechanistically elaborating its function, its impact on the associated enhancer and chromatin looping, and determining the utility of BHLHE40-AS1 as a clinically relevant biomarker and therapeutic target in invasive breast cancer. Citation Format: DeVaux RKS, Schimjawicz H, Coarfa C, Behbod F, Herschkowitz JI. BHLHE40-AS1 is an enhancer associated noncoding RNA critical to breast cancer progression [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-07-11.

Targeted therapies against basal-like breast tumors, which are typically ‘triple-negative breast cancers (TNBCs)’, remain an important unmet clinical need. Somatic TP53 mutations are the most common genetic event in basal-like breast tumors and TNBC. To identify additional drivers and possible drug targets of this subtype, a comparative study between human and murine tumors was performed by utilizing a murine Trp53-null mammary transplant tumor model. We show that two subsets of murine Trp53-null mammary transplant tumors resemble aspects of the human basal-like subtype. DNA-microarray, whole-genome and exome-based sequencing approaches were used to interrogate the secondary genetic aberrations of these tumors, which were then compared to human basal-like tumors to identify conserved somatic genetic features. DNA copy-number variation produced the largest number of conserved candidate personalized drug targets. These candidates were filtered using a DNA-RNA Pearson correlation cut-off and a requirement that the gene was deemed essential in at least 5% of human breast cancer cell lines from an RNA-mediated interference screen database. Five potential personalized drug target genes, which were spontaneously amplified loci in both murine and human basal-like tumors, were identified: Cul4a, Lamp1, Met, Pnpla6 and Tubgcp3. As a proof of concept, inhibition of Met using crizotinib caused Met-amplified murine tumors to initially undergo complete regression. This study identifies Met as a promising drug target in a subset of murine Trp53-null tumors, thus identifying a potential shared driver with a subset of human basal-like breast cancers. Our results also highlight the importance of comparative genomic studies for discovering personalized drug targets and for providing a preclinical model for further investigations of key tumor signaling pathways., Editors' choice: Personalized therapeutic targets for triple-negative breast cancers remain an important unmet clinical need. Genomic profiling of murine Trp53-null tumors identified a number of promising drug targets conserved in humans.

Increased emphasis on breast cancer screening has led to a dramatic surge in diagnosis of pre-cancerous ductal carcinoma in situ (DCIS) over the past 30 years. Unfortunately, diagnosis of late stage invasive and metastatic disease has not proportionally declined, suggesting significant over diagnosis and over treatment of innocuous DCIS. Due to a lack of biomarkers, the heterogeneity of DCIS lesions, and an insufficient understanding of mechanisms of invasive progression, there is currently no clinically relevant method of predicting which DCIS lesions will advance to invasive disease. As a result, all DCIS patients undergo an aggressive treatment regimen to prevent disease progression. Therefore, there is a critical need to determine the underlying mechanisms driving breast cancer progression to better inform patient treatment options and nominate novel therapeutic entry points for treatment of invasive disease. Recently, long noncoding RNAs (lncRNAs) have gained attention as critical regulators of epigenetic states and gene expression. Although the study of lncRNAs is in its infancy, they are being uncovered as pivotal regulators of development and tumorigenesis, thus they are a rich source of potential drivers of breast cancer progression. We propose that lncRNAs functionally drive breast cancer invasion and their expression can discriminate between innocuous and potentially invasive DCIS. Using biopsies from women that exhibit tandem DCIS and invasive breast cancer lesions, we have identified a cohort of long noncoding RNAs (lncRNAs) that are enriched in the invasive biopsy. From this cohort we have identified the lncRNA BHLHE40-AS1 as increasing in a step-wise manner in a breast cancer progression series that escalates from normal, non-transformed cells to highly invasive disease. Preliminary evidence suggests that BHLHE40-AS1 expression regulates invasive potential in vitro. Using a GeneChip® Human Transcriptome 2.0 Array (HTA) we have identified several targets previously associated with driving breast cancer invasion as being potentially regulated by BHLHE40-AS1. Future directions will focus on determining the effect of BHLHE40-AS1 expression on tumor cell invasion in an orthotopic xenograft model, mechanistically elaborating its function, and determining the utility of BHLHE40-AS1 as a clinically relevant biomarker of invasive breast cancer. Citation Format: DeVaux RKS, Davis S, Sheftel J, Coarfa C, Behbod F, Herschkowitz JI. The long noncoding RNA BHLHE40-AS1 as a functional biomarker of invasive breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-05-14.

Ductal Carcinoma in Situ (DCIS) is an early breast cancer lesion that is considered a nonobligate precursor to development of invasive ductal carcinoma (IDC). Although only a small subset of DCIS lesions are predicted to progress into a breast cancer, distinguishing innocuous from minacious DCIS lesions remains a clinical challenge. Thus, patients diagnosed with DCIS will undergo surgery with the potential for radiation and hormone therapy. This has led to a current state of overdiagnosis and overtreatment. Interrogating the transcriptome alone has yet to define clear functional determinants of progression from DCIS to IDC. Epigenetic changes, critical for imprinting and tissue specific development, in the incorrect context can lead to global signaling rewiring driving pathological phenotypes. Epigenetic signaling pathways, and the molecular players that interpret and sustain their signals, are critical to understanding the underlying pathology of breast cancer progression. The types of epigenetic changes, as well as the molecular players, are expanding. In addition to DNA methylation, histone modifications, and chromatin remodeling, we must also consider enhancers as well as the growing field of noncoding RNAs. Herein we will review the epigenetic interactions that have been uncovered in early stage lesions that impact breast cancer progression, and how these players may be utilized as biomarkers to mitigate overdiagnosis and overtreatment.

Ty Santiago,1 Rebecca Sinnott DeVaux,2 Katarzyna Kurzatkowska,1 Ricardo Espinal,1 Jason I Herschkowitz,2 Maria Hepel1 1Department of Chemistry, State University of New York at Potsdam, Potsdam, 2Department of Biomedical Sciences, Cancer Research Center, University at Albany, State University of New York, Rensselaer, NY, USA Abstract: Advanced and metastatic cancer forms are extremely difficult to treat and require high doses of chemotherapeutics, inadvertently affecting also healthy cells. As a result, the observed survival rates are very low. For instance, gemcitabine (GEM), one of the most effective chemotherapeutic drugs used for the treatment of breast and pancreatic cancers, sees only a 20% efficacy in penetrating cancer tissue, resulting in

Mechanical biomarkers associated with cytoskeletal structures have been reported as powerful label-free cell state identifiers. In order to measure cell mechanical properties, traditional biophysical (e.g., atomic force microscopy, micropipette aspiration, optical stretchers) and microfluidic approaches were mainly employed; however, they critically suffer from low-throughput, low-sensitivity, and/or time-consuming and labor-intensive processes, not allowing techniques to be practically used for cell biology research applications. Here, a novel inertial microfluidic cell stretcher (iMCS) capable of characterizing large populations of single-cell deformability near real-time is presented. The platform inertially controls cell positions in microchannels and deforms cells upon collision at a T-junction with large strain. The cell elongation motions are recorded, and thousands of cell deformability information is visualized near real-time similar to traditional flow cytometry. With a full automation, the entire cell mechanotyping process runs without any human intervention, realizing a user friendly and robust operation. Through iMCS, distinct cell stiffness changes in breast cancer progression and epithelial mesenchymal transition are reported, and the use of the platform for rapid cancer drug discovery is shown as well. The platform returns large populations of single-cell quantitative mechanical properties (e.g., shear modulus) on-the-fly with high statistical significances, enabling actual usages in clinical and biophysical studies.

The HER2 (ERBB2) and MYC genes are commonly amplified in breast cancer, yet little is known about their molecular and clinical interaction. Using a novel chimeric mammary transgenic approach and in vitro models, we demonstrate markedly increased self-renewal and tumour-propagating capability of cells transformed with Her2 and c-Myc. Coexpression of both oncoproteins in cultured cells led to the activation of a c-Myc transcriptional signature and acquisition of a self-renewing phenotype independent of an epithelial-mesenchymal transition programme or regulation of conventional cancer stem cell markers. Instead, Her2 and c-Myc cooperated to induce the expression of lipoprotein lipase, which was required for proliferation and self-renewal in vitro. HER2 and MYC were frequently coamplified in breast cancer, associated with aggressive clinical behaviour and poor outcome. Lastly, we show that in HER2(+) breast cancer patients receiving adjuvant chemotherapy (but not targeted anti-Her2 therapy), MYC amplification is associated with a poor outcome. These findings demonstrate the importance of molecular and cellular context in oncogenic transformation and acquisition of a malignant stem-like phenotype and have diagnostic and therapeutic consequences for the clinical management of HER2(+) breast cancer.