Dong Soon Choi

Microfluidics separation reveals the stem-cell-like deformability of tumor-initiating cells

Here we report a microfluidics method to enrich physically deformable cells by mechanical manipulation through artificial microbarriers. Driven by hydrodynamic forces, flexible cells or cells with high metastatic propensity change shape to pass through the microbarriers and exit the separation device, whereas stiff cells remain trapped. We demonstrate the separation of (i) a mixture of two breast cancer cell types (MDA-MB-436 and MCF-7) with distinct deformabilities and metastatic potentials, and (ii) a heterogeneous breast cancer cell line (SUM149), into enriched flexible and stiff subpopulations. We show that the flexible phenotype is associated with overexpression of multiple genes involved in cancer cell motility and metastasis, and greater mammosphere formation efficiency. Our observations support the relationship between tumor-initiating capacity and cell deformability, and demonstrate that tumor-initiating cells are less differentiated in terms of cell biomechanics.

Chloroquine eliminates cancer stem cells through deregulation of Jak2 and DNMT1.

Triple negative breast cancer (TNBC) is known to contain a high percentage of CD44+/CD24−/low cancer stem cells (CSCs), corresponding with a poor prognosis despite systemic chemotherapy. Chloroquine (CQ), an antimalarial drug, is a lysotropic reagent which inhibits autophagy. CQ was identified as a potential CSC inhibitor through in silico gene expression signature analysis of the CD44+/CD24−/low CSC population. Autophagy plays a critical role in adaptation to stress conditions in cancer cells, and is related with drug resistance and CSC maintenance. Thus, the objectives of this study were to examine the potential enhanced efficacy arising from addition of CQ to standard chemotherapy (paclitaxel) in TNBC and to identify the mechanism by which CQ eliminates CSCs in TNBCs. Herein, we report that CQ sensitizes TNBC cells to paclitaxel through inhibition of autophagy and reduces the CD44+/CD24−/low CSC population in both preclinical and clinical settings. Also, we are the first to report a mechanism by which CQ regulates the CSCs in TNBC through inhibition of the Janus‐activated kinase 2 (Jak2)—signal transducer and activator of transcription 3 signaling pathway by reducing the expression of Jak2 and DNA methyltransferase 1. Stem Cells 2014;32:2309–2323

Studying Cancer Stem Cell Dynamics on PDMS Surfaces for Microfluidics Device Design

This systematic study clarified a few interfacial aspects of cancer cell phenotypes on polydimethylsiloxane (PDMS) substrates and indicated that the cell phenotypic equilibrium greatly responds to cell-to-surface interactions. We demonstrated that coatings of fibronectin, bovine serum albumin (BSA), or collagen with or without oxygen-plasma treatments of the PDMS surfaces dramatically impacted the phenotypic equilibrium of breast cancer stem cells, while the variations of the PDMS elastic stiffness had much less such effects. Our results showed that the surface coatings of collagen and fibronectin on PDMS maintained breast cancer cell phenotypes to be nearly identical to the cultures on commercial polystyrene Petri dishes. The surface coating of BSA provided a weak cell-substrate adhesion that stimulated the increase in stem-cell-like subpopulation. Our observations may potentially guide surface modification approaches to obtain specific cell phenotypes.

Pharmacological inhibition of NOS activates ASK1/JNK pathway augmenting docetaxel-mediated apoptosis in triple negative breast cancer

Purpose: Chemoresistance in triple-negative breast cancer (TNBC) is associated with the activation of a survival mechanism orchestrated by the endoplasmic reticulum (EnR) stress response and by inducible nitric oxide synthase (iNOS). Our aim was to determine the effects of pharmacologic NOS inhibition on TNBC. Experimental Design: TNBC cell lines, SUM-159PT, MDA-MB-436, and MDA-MB-468, were treated with docetaxel and NOS inhibitor (L-NMMA) for 24, 48, and 72 hours. Apoptosis was assessed by flow cytometry using Annexin-V and propidium iodide. Western blot was used to assess ER stress and apoptosis, and rtPCR was used to evaluate s-XBP1. TNBC patient-derived xenografts (PDX) were treated either with vehicle, docetaxel, or combination therapy (NOS inhibition + docetaxel). Mouse weight and tumor volumes were recorded twice weekly. Docetaxel concentration was determined using mass spectrometry. To quantify proliferation and apoptosis, PDX tumor samples were stained using Ki67 and TUNEL assay. Results: In vitro, L-NMMA ameliorated the iNOS upregulation associated with docetaxel. Apoptosis increased when TNBC cells were treated with combination therapy. In TNBC PDXs, combination therapy significantly reduced tumor volume growth and increased survival proportions. In the BCM-5998 PDX model, intratumoral docetaxel concentration was higher in mice receiving combination therapy. Coupling docetaxel with NOS inhibition increased EnR-stress response via coactivation of ATF4 and CHOP, which triggered the pASK1/JNK proapoptotic pathway, promoting cleavage of caspases 3 and 9. Conclusions: iNOS is a critical target for docetaxel resistance in TNBC. Pharmacologic inhibition of NOS enhanced chemotherapy response in TNBC PDX models. Combination therapy may improve prognosis and prevent relapse in TNBC patients who have failed conventional chemotherapy. Clin Cancer Res; 24(5); 1152–62. ©2018 AACR.

HN1L Promotes Triple-Negative Breast Cancer Stem Cells through LEPR-STAT3 Pathway

Summary Here, we show that HEMATOLOGICAL AND NEUROLOGICAL EXPRESSED 1-LIKE (HN1L) is a targetable breast cancer stem cell (BCSC) gene that is altered in 25% of whole breast cancer and significantly correlated with shorter overall or relapse-free survival in triple-negative breast cancer (TNBC) patients. HN1L silencing reduced the population of BCSCs, inhibited tumor initiation, resensitized chemoresistant tumors to docetaxel, and hindered cancer progression in multiple TNBC cell line-derived xenografts. Additionally, gene signatures associated with HN1L correlated with shorter disease-free survival of TNBC patients. We defined HN1L as a BCSC transcription regulator for genes involved in the LEPR-STAT3 signaling axis as HN1L binds to a putative consensus upstream sequence of STAT3, LEPTIN RECEPTOR, and MIR-150. Our data reveal that BCSCs in TNBC depend on the transcription regulator HN1L for the sustained activation of the LEPR-STAT3 pathway, which makes it a potentially important target for both prognosis and BCSC therapy.

Breast Cancer Stem Cells

Cancer stem cells (CSCs) represent a heterogeneous subpopulation of cancer cells within tumors. CSCs divide asymmetrically to generate daughter cells that either have CSC characteristics including self-renewal, or differentiation potential to form neoplastic cells which constitute most of the tumor. These characteristics suggest that the cells may play an important role in tumor initiation, and development of chemo-resistance. These characteristics are evident in the ability of CSCs to seed new tumors upon transplantation in experimental animal models. In this chapter, we describe the evidence around the role of CSCs in breast cancer. A brief overview of the methods and markers used to identify these cells is also provided. More importantly, we present the data regarding the signaling pathways that are implicated in the aggressiveness associated with CSCs. Lastly, we discuss the strategies that can be used for targeting these pathways for therapeutic purposes.

Abstract B53: The role of RPL39 induced autophagy in resistance of breast cancer stem cells

Introduction: Autophagy is an evolutionarily conserved lysosomal pathway for degradation of cytoplasmic proteins, macromolecules, as well as organelles, and is a survival pathway under nutrient and environmental stress. Tumor initiating cells (TICs) survive standard radiation, and chemotherapy that ultimately cause breast tumor recurrence and metastasis. Our previous studies have identified autophagy as the mechanism of survival in treatment-resistant populations in patient samples. Recent evidence from our group has defined for the first time two previously unidentified cancer genes, ribosomal protein L39 (RPL39) and myeloid leukemia factor 2 (MLF2) that play a crucial role in tumor initiation and metastasis. Additionally, RPL39 and MLF2 genes in TIC self-renewal utilize hypoxia and reactive nitrogen species for their action. In this study, we aim to determine the mechanism by which RPL39 and MLF2 modulate TICs and their relationship to tumor microenvironment. Experimental Procedures: Analysis of clinical samples was performed using low density array analysis. This data was then confirmed using real time PCR analysis. The mechanism of action of RPL39 as related to autophagy was identified utilizing western analysis. Results and Discussion: We confirmed our in silico bioinformatics based findings by using a real time PCR low density array of 84 genes related to autophagy in 9 paired pre- vs. post-treatment clinical samples, where 60 out of 84 genes demonstrated a statistically significant change (p Citation Format: Bhuvanesh Dave, Dong Soon Choi, Helen Wong, Yi Liu, Prem Siddharth Gunamalai, Jenny C. Chang. The role of RPL39 induced autophagy in resistance of breast cancer stem cells. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B53.

Abstract P6-15-01: Triple negative breast cancer is vulnerable to Pan-HER, an antibody mixture simultaneously targeting EGFR, HER2 and HER3

Background: Triple negative breast cancer (TNBC) is a highly heterogeneous and aggressive subtype of cancer, lacking expression of estrogen and progesterone receptors as well as human epidermal growth factor receptor (HER) 2 protein. Limited standard therapeutic options, absence of effective targeted therapies, and early metastatic spread have contributed to poor prognosis and outcomes associated with this disease. Although overexpression of EGFR has been reported in nearly 80% TNBC, EGFR-targeted therapy has yielded little clinical benefit, and the outcome is still under debate. In conjunction, we also found mixed effects of EGFR-targeted therapy on TNBC xenograft tumors despite significant target engagement, suggesting that tumor heterogeneity and compensating mechanisms may contribute to the variable drug responses to the EGFR-targeted therapy. Recently, we reported superior anti-cancer effects of Pan-HER, a mixture of antibodies targeting the HER family members EGFR, HER2 and HER3 on various types of cancer by overcoming drug resistance and tumor heterogeneity. To this end, we hypothesized that Pan-HER can effectively inhibit tumor growth in TNBC by inhibiting tumor heterogeneity and drug resistance. Objective: The goal of this study is to test the effect of Pan-HER antibody mixture (Sym013) on tumor growth and recurrence of 14 patient-derived (PDX) TNBC orthotopic xenograft tumor models and to investigate molecular biomarkers which can predict drug response to Pan-HER. Methods: We evaluated in-vivo anti-tumor effects of Pan-HER (50 mg/kg, i.p. three times/week, 10 doses in total for 3 weeks) over vehicle on tumor growth and tumor recurrence on 14 PDX TNBC models with known expression levels of EGFR and HER3 (n=3/group). HER family proteins and related downstream molecules (Akt, Erk, Stat3, FAK) in the tumor tissues were evaluated by Western blot assay and immunohistochemistry analysis. Additionally, using dCHIP and ingenuity pathway analysis, we compared microarray data from the tested cohorts and other TNBC PDX models with known HER family receptor status. Results: We found that Pan-HER alone effectively inhibited tumor growth in all 14 PDX models and showed statistical significance (p=0.0103) when compared to the vehicle groups. Among these, one PDX model, BCM-3186, showed substantial tumor reduction and additional two (MC1 and BCM-4913) showed complete response with no recurrence after the last treatment of Pan-HER. The significant anti-tumor effects of Pan-HER were positively correlated with inhibition of phosphorylation and expression of EGFR, HER3, Akt, Erk, and FAK, but not Stat3, and this was consistent in all PDX models tested. Additionally, the microarray and the pathway enrichment analyses suggest that loss of PTEN expression and up-regulation of FAK and RAS pathways may be the predictive markers for the Pan-HER drug response in TNBC. Conclusion: Our in-vivo data suggest that simultaneous targeting of the three HER family receptors is a potential new approach for treatment of TNBC. Further confirmation of our in-vivo results will warrant a phase I clinical trial and lend support to single agent Pan-HER as a viable treatment strategy for TNBC patients in the clinic. Citation Format: Choi DS, Qian W, Davila-Gonzalez D, Ensor JE, Lantto J, Kragh M, Horak ID, Chang JC. Triple negative breast cancer is vulnerable to Pan-HER, an antibody mixture simultaneously targeting EGFR, HER2 and HER3. [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 P6-15-01.

Abstract LB-C09: Hematological and Neurological Expressed 1-Like (HN1L) is a Novel Therapeutic Target for Triple Negative Breast Cancer

Background and Objective: Triple negative breast cancer (TNBC: ER-,PR-,Her2-), is characterized by its aggressive clinical behavior with high incidence of visceral metastasis to the lungs and brain. These patients do not respond to hormonal therapy and show intrinsic resistance to conventional chemotherapy. Robust evidence indicates that treatment-resistance and metastases may arise from a subpopulation of cells with tumor-initiating capacity called breast cancer stem cells (BCSC). We are one of the first groups to demonstrate that residual tumors after exposure to chemotherapy are enriched for BCSC (Li et. al, J Natl Cancer Inst 100:672-9, 2008). We have previously described a tumorigenic treatment-resistant gene signature of 493 genes derived from patient biopsies (Creighton et. al, Proc Natl Acad Sci USA 106:13820-5, 2009).This finding narrowed down the possible cancer stem cell growth related genes from genome wide to only 493 genes and provided candidate genes for screening potential targets that affect BCSC self-renewal. Further screening and re-evaluation of the candidate genes determined a previously unidentified cancer gene, Hematological and Neurological Expressed 1-Like (HN1L) as the target of cancer stem cell self-renewal. HN1L is known to be involved in embryo development, but has been remained as an orphan gene. Thus, the objective of this study was to investigate the role of HN1L in in TNBC and to validate HN1L as a novel therapeutic target. Results: We found from the TCGA database that the expression of HN1L is up-regulated in more than 20% breast cancer and is negatively correlated with relapse-free survival in TNBC patients. Silencing of HN1L considerably suppressed BCSC population in vitro and in vivo, sensitized tumors to chemotherapy in vivo, and substantially reduced lung metastasis. Mechanistically, our study identified HN1L as an novel and putative transcription factor regulating expression of STAT3 and the related regulatory genes including Leptin-receptor (Lepr) and miR-150. These upstream pathways converge to result in a persistent activation of a STAT3-regulated gene network promoting downstream BCSC self-renewal pathways. Correlatively, the TNBC patients exhibiting gene signature from HN1L knockdown tend to survive longer. Conclusion: we described here for the first time that HN1L is a novel therapeutic target for triple negative breast cancer. This study highlights the importance of targeting HN1L that deactivates STAT3 and abrogates its function in BCSC self-renewal. Further investigation of HN1L in TNBC-targeted therapies will offer new strategies to treat this aggressive disease. Citation Format: Dong Soon Choi, Liu Yi, Cristian Rodriguez-Aguayo, Bhuvanesh Dave, Stephen T.C. Wong, Anil K. Sood, Jenny C. Chang. Hematological and Neurological Expressed 1-Like (HN1L) is a Novel Therapeutic Target for Triple Negative Breast Cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-C09.

Abstract 2312: Identifying novel cancer stem cell target for triple-negative breast cancer

Triple negative breast cancer is characterized by its aggressive clinical behavior with high incidence of metastasis to the lungs and brain. These patients do not respond to hormonal therapy and show intrinsic resistance to conventional chemotherapy. Robust evidence indicates that treatment-resistance and metastases may arise from a subpopulation of cells with tumor-initiating capacity called breast cancer stem cells (BCSC). We are one of the first groups to demonstrate that residual tumors after exposure to chemotherapy are enriched for BCSC. We have previously described a treatment-resistant gene signature of 493 genes derived from patient biopsies.This finding narrowed down the possible cancer stem cell growth related genes from genome wide to only 493 genes and provided candidate genes for screening potential targets that affect BCSC self-renewal. Re-evaluation of the candidate genes determined a previously unidentified cancer gene, Hematological and Neurological Expressed 1-Like (HN1L) as the target of cancer stem cell self-renewal. The objective of this study was to investigate the role of HN1L in regulating BCSC and metastasis in TNBC, and to determine the mechanism of action of HN1L in BCSC. Knocking down HN1L by shRNA in SUM159 and MDAMB231 cell lines significantly decreased mammosphere forming efficiency (MSFE) and CD44+/CD24low/- population. To assess the contribution of HN1L to BCSC and tumor growth, a patient derived human-cancer-in-mouse xenograft model and two cancer cell line xenograft models were employed. To ensure targeted delivery, siRNA was packaged into DOPC liposomes and delivered into mice via intraperitoneal injection. Results showed that silencing HN1L alone or in combination with chemotherapy in xenografts reduced tumor volume and BCSC population as measured by MSFE, CD44+/CD24low/- markers, ALDF+ cell population and limiting dilution assay. Liposomal HN1L siRNA treated mice also showed lower level of lung metastasis. A gene signature obtained from microarray analysis on HN1L siRNA treated tumor tissues highly correlated with better prognosis in TNBC patients. Overexpression of HN1L in TNBC cell lines activated STAT3 signaling, induced CD44+/CD24low/- population and cancer cell migration. Moreover, chemoresistance were also observed with HN1L overexpression. HN1L colocalized with CNTFR and STAT3 in cytoplasm and nucleus by immunofluorescence staining. Changes in HN1L expression level resulted in corresponding changes in phospho-STAT3. Furthermore, CNTFR knockdown compromised the effect of HN1L overexpression in activating STAT3.These results suggest that the HN1L gene plays an important role in BCSC through regulating CNTFR-STAT3 signaling pathway, and is associated with poor prognosis. Taken together, the results from our study demonstrate for the first time that HN1L is a novel target for TNBC. Targeting HN1L and/or CNTFR-STAT3 pathway might provide an alternative therapeutic strategy for TNBC. Citation Format: Yi Liu, Dong Soon Choi, Sergio Grandos-Principal, Wei Qian, Lacey Burey, Helen Wong, Crisitian Rodriguez-Aguayo, Anil Sood, Zheng Li, Stephen Wong, Heidi Weiss, Bhuvanesh Dave, Melissa Landis, Jenny C. Chang. Identifying novel cancer stem cell target for triple-negative breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2312. doi:10.1158/1538-7445.AM2015-2312