Helen Wong

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.

Cancer stem cell markers are enriched in normal tissue adjacent to triple negative breast cancer and inversely correlated with DNA repair deficiency

IntroductionWe hypothesized that cells present in normal tissue that bear cancer stem cell markers may represent a cancer cell of origin or a microenvironment primed for tumor development, and that their presence may correlate with the clinically defined subtypes of breast cancer that show increased tumorigenicity and stem cell features.MethodsNormal tissues sampled at least 5 cm from primary tumors (normal adjacent tissue) were obtained from 61 chemotherapy-naive patients with breast cancer treated with mastectomy. Samples were stained simultaneously with immunofluorescence for CD44/CD49f/CD133/2 stem cell markers. We assessed the association between CD44+CD49f+CD133/2+ staining in normal adjacent tissue and breast cancer receptor subtype (defined by the expression of the estrogen (ER), progesterone (PR), or human epidermal growth factor-2 (Her2) receptors). We also examined the correlation between CD44+CD49f+CD133/2+ immunofluorescence and each of two previously published gene signatures, one derived from stem-cell enriched tissue and one from BRCA mutated tissue expected to have defective DNA repair.ResultsPatients with triple negative breast cancer (ER–/PR–/HER2–) expressed CD44+CD49f+CD133/2+ in 9 of 9 normal adjacent tissue samples compared with 7 of 52 ER+ and/or Her2+ tumors (P < 0.001). Further, expression of CD44+CD49f+CD133/2+ by normal adjacent tissue correlated positively with a stem cell-derived tumorigenic signature (P <0.001) and inversely with a defective DNA-repair signature (P <0.001).ConclusionNormal cells bearing cancer stem cell markers are associated with the triple negative receptor subtype of breast cancer. This study suggests stem cell staining and gene expression signatures from normal breast tissues represent novel tissue-based risk biomarkers for triple negative breast cancer. Validation of these results in additional studies of normal tissue from cancer-free women could lay the foundation for future targeted triple negative breast cancer prevention strategies.

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

Role of RPL39 in Metaplastic Breast Cancer.

Abstract Background: Metaplastic breast cancer is one of the most therapeutically challenging forms of breast cancer because of its highly heterogeneous and chemoresistant nature. We have previously demonstrated that ribosomal protein L39 (RPL39) and its gain-of-function mutation A14V have oncogenic activity in triple-negative breast cancer and this activity may be mediated through inducible nitric oxide synthase (iNOS). The function of RPL39 and A14V in other breast cancer subtypes is currently unknown. The objective of this study was to determine the role and mechanism of action of RPL39 in metaplastic breast cancer. Methods: Both competitive allele-specific and droplet digital polymerase chain reaction were used to determine the RPL39 A14V mutation rate in metaplastic breast cancer patient samples. The impact of RPL39 and iNOS expression on patient overall survival was estimated using the Kaplan-Meier method. Co-immunoprecipitation and immunoblot analyses were used for mechanistic evaluation of RPL39. Results: The RPL39 A14V mutation rate was 97.5% (39/40 tumor samples). High RPL39 (hazard ratio = 0.71, 95% confidence interval = 0.55 to 0.91, P = .006) and iNOS expression (P = .003) were associated with reduced patient overall survival. iNOS inhibition with the pan-NOS inhibitor NG-methyl-L-arginine acetate decreased in vitro proliferation and migration, in vivo tumor growth in both BCM-4664 and BCM-3807 patient-derived xenograft models (P = .04 and P = .02, respectively), and in vitro and in vivo chemoresistance. Mechanistically, RPL39 mediated its cancer-promoting actions through iNOS signaling, which was driven by the RNA editing enzyme adenosine deaminase acting on RNA 1. Conclusion: NOS inhibitors and RNA editing modulators may offer novel treatment options for metaplastic breast cancer.

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.

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 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

Abstract 237: Chloroquine inhibits cancer stem cells in triple negative breast cancer via regulation of DNA methylation.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Post-treatment enrichment of the tumor initiating CD44+/CD24- breast cancer stem cell (CSC) population is believed to be responsible for breast tumor recurrence and metastasis. Among breast cancer subtypes, triple negative breast cancer (TNBC) particularly is known to have an abundant CSC population, and is characterized by frequent metastatic recurrence. Chloroquine (CQ), an anti-malarial drug, is a lysotropic reagent that inhibits autophagy. Recently, CQ has been shown to reduce the CSC population in various cancers and has been tested in clinical studies. Concurrently, CQ was identified as a potential CSC inhibitor discovered from gene expression signatures of the CD44+/CD24- CSC population. However, little, aside from inhibition of autophagy, is known about the working mechanism of chloroquine in reducing CSCs, particularly in TNBCs. Based on recent recommendations that low doses of CQ be used to limit toxicities in the heart and retina, we investigated how low doses of CQ enhance the antitumor effects of paclitaxel (PTX) and reduce the CSC population. CSC population changes and apoptosis were analyzed using flow cytometry analysis (CSC: CD44+/CD24-; apoptosis: annexin V) and western blot analysis for cleaved caspase 3, and CSC function was measured by mammosphere formation efficiency in SUM159PT, MDA-MB-231, MDA-MB-468, and Hs578T TNBC cell lines. We observed enhanced cytotoxic effects and significant reduction of the CSC population by combined treatment of PTX and CQ compared to single treatment of either PTX (5nM) or CQ (1 or 5 uM) in SUM159, Hs578T, and MDA-MB-231 cells. The enhanced cytotoxicity by co-treatment of CQ and PTX correlated well with inhibition of autophagy, as indicated by cleavage of LC3B, increased expression of p62, and accumulation of autophagosomes. Moreover, the combined treatment inhibited PTX induced STAT3 activation in CSCs and epigenetically regulated gene expression critical in maintenance of CSCs via repression of DNMT1 expression. Finally, we observed enhanced therapeutic efficacy in vivo with the combination of CQ (10-20 mg/kg, daily) and PTX (30 mg/kg, two times per week) when compared to either CQ or PTX alone (p< 0.05). Herein, we demonstrate effective reduction of the CD44+/CD24- CSC population by combined CQ and PTX treatment through autophagy inhibition. Moreover, we found that the low-dose combined treatment of CQ with PTX was able to regulate gene expression by altering DNA methylation, subsequently reducing CSCs in the TNBC cancer cells. Thus, a low dose treatment of CQ along with chemotherapy may be effective in treating TNBC patients, lend further support for in-depth studies on the mechanism of CQ among the subgroups of TNBC. Citation Format: Dong Soon Choi, Elvin Blanco, Sergio M. Granados-Principal, Bhuvanesh Dave, Melissa Landis, Helen Wong, Jenny Chang. Chloroquine inhibits cancer stem cells in triple negative breast cancer via regulation of DNA methylation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 237. doi:10.1158/1538-7445.AM2013-237

Abstract LB-106: Expression of stem cell biomarkers in benign breast tissue from patients with triple negative breast cancer

Numerous studies suggest that breast cancers contain a subpopulation of cancer stem cells that are thought to maintain the heterogeneity and self-renewing potential of the tumor. In our previously published data, we had described a breast cancer stem cell signature comprised of 493 statistically significant differentially expressed genes, which were present in a tumorigenic subpopulation of cells. Cells from patient samples with the cell surface markers CD44+CD49f+CD133/2+ have been shown to have self-renewal and tumorigenic properties – characteristics of breast cancer stem cells. In this study, we investigated the benign tissue from 42 patients with breast cancer treated with mastectomy to examine the incidence CD44+CD49f+CD133/2+ positive cells. Benign tissue was obtained at a distant site, at least 5 cm from the primary tumor, and examined with hematoxylin-eosin by a breast pathologist (AS), only samples containing normal breast tissue with at least 60% epithelial content were selected. A significant increase in expression of CD44+CD49f+CD133/2+ by immunofluorescence was identified in the benign tissue associated with patients with triple negative (ER negative/PR negative/Her2 negative) disease (8 out of 8), compared with ER positive and Her2 positive tumors combined (5 out of 36), P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-106. doi:10.1158/1538-7445.AM2011-LB-106