Tahra Luther

Activation of an IL6 Inflammatory Loop Mediates Trastuzumab Resistance in HER2+ Breast Cancer by Expanding the Cancer Stem Cell Population

Although inactivation of the PTEN gene has been implicated in the development of resistance to the HER2 targeting antibody trastuzumab, the mechanisms mediating this resistance remain elusive. We generated trastuzumab resistant cells by knocking down PTEN expression in HER2 overexpressing breast cancer cell lines and demonstrate that development of trastuzumab resistance in these cells is mediated by activation of an IL6 inflammatory feedback loop leading to expansion of the cancer stem cell (CSC) population. Long term trastuzumab treatment generates highly enriched CSCs which display an EMT phenotype secreting over 100-fold more IL6 than parental cells. An IL6 receptor antibody interrupted this inflammatory feedback loop reducing the cancer stem cell population resulting in decreased tumor growth and metastasis in mouse xenographs. These studies demonstrate that trastuzumab resistance may be mediated by an IL6 inflammatory loop and suggest that blocking this loop may provide alternative strategy to overcome trastuzumab resistance.

HER2 Drives Luminal Breast Cancer Stem Cells in the Absence of HER2 Amplification: Implications for Efficacy of Adjuvant Trastuzumab

Although current breast cancer treatment guidelines limit the use of HER2-blocking agents to tumors with HER2 gene amplification, recent retrospective analyses suggest that a wider group of patients may benefit from this therapy. Using breast cancer cell lines, mouse xenograft models and matched human primary and metastatic tissues, we show that HER2 is selectively expressed in and regulates self-renewal of the cancer stem cell (CSC) population in estrogen receptor-positive (ER(+)), HER2(-) luminal breast cancers. Although trastuzumab had no effects on the growth of established luminal breast cancer mouse xenografts, administration after tumor inoculation blocked subsequent tumor growth. HER2 expression is increased in luminal tumors grown in mouse bone xenografts, as well as in bone metastases from patients with breast cancer as compared with matched primary tumors. Furthermore, this increase in HER2 protein expression was not due to gene amplification but rather was mediated by receptor activator of NF-κB (RANK)-ligand in the bone microenvironment. These studies suggest that the clinical efficacy of adjuvant trastuzumab may relate to the ability of this agent to target the CSC population in a process that does not require HER2 gene amplification. Furthermore, these studies support a CSC model in which maximal clinical benefit is achieved when CSC targeting agents are administered in the adjuvant setting. Cancer Res; 73(5); 1635-46. ©2012 AACR.

Notch Reporter Activity in Breast Cancer Cell Lines Identifies a Subset of Cells with Stem Cell Activity

Developmental pathways such as Notch play a pivotal role in tissue-specific stem cell self-renewal as well as in tumor development. However, the role of Notch signaling in breast cancer stem cells (CSC) remains to be determined. We utilized a lentiviral Notch reporter system to identify a subset of cells with a higher Notch activity (Notch+) or reduced activity (Notch−) in multiple breast cancer cell lines. Using in vitro and mouse xenotransplantation assays, we investigated the role of the Notch pathway in breast CSC regulation. Breast cancer cells with increased Notch activity displayed increased sphere formation as well as expression of breast CSC markers. Interestingly Notch+ cells displayed higher Notch4 expression in both basal and luminal breast cancer cell lines. Moreover, Notch+ cells demonstrated tumor initiation capacity at serial dilutions in mouse xenografts, whereas Notch− cells failed to generate tumors. γ-Secretase inhibitor (GSI), a Notch blocker but not a chemotherapeutic agent, effectively targets these Notch+ cells in vitro and in mouse xenografts. Furthermore, elevated Notch4 and Hey1 expression in primary patient samples correlated with poor patient survival. Our study revealed a molecular mechanism for the role of Notch-mediated regulation of breast CSCs and provided a compelling rationale for CSC-targeted therapeutics. Mol Cancer Ther; 14(3); 779–87. ©2015 AACR.

MicroRNA100 inhibits self-renewal of breast cancer stem-like cells and breast tumor development.

miRNAs are essential for self-renewal and differentiation of normal and malignant stem cells by regulating the expression of key stem cell regulatory genes. Here, we report evidence implicating the miR100 in self-renewal of cancer stem-like cells (CSC). We found that miR100 expression levels relate to the cellular differentiation state, with lowest expression in cells displaying stem cell markers. Utilizing a tetracycline-inducible lentivirus to elevate expression of miR100 in human cells, we found that increasing miR100 levels decreased the production of breast CSCs. This effect was correlated with an inhibition of cancer cell proliferation in vitro and in mouse tumor xenografts due to attenuated expression of the CSC regulatory genes SMARCA5, SMARCD1, and BMPR2. Furthermore, miR100 induction in breast CSCs immediately upon their orthotopic implantation or intracardiac injection completely blocked tumor growth and metastasis formation. Clinically, we observed a significant association between miR100 expression in breast cancer specimens and patient survival. Our results suggest that miR100 is required to direct CSC self-renewal and differentiation.

RAD51 Mediates Resistance of Cancer Stem Cells to PARP Inhibition in Triple-Negative Breast Cancer.

Introduction: PARP inhibitors have shown promising results in early studies for treatment of breast cancer susceptibility gene (BRCA)–deficient breast cancers; however, resistance ultimately develops. Furthermore, the benefit of PARP inhibitors (PARPi) in triple-negative breast cancers (TNBC) remains unknown. Recent evidence indicates that in TNBCs, cells that display “cancer stem cell” properties are resistant to conventional treatments, mediate tumor metastasis, and contribute to recurrence. The sensitivity of breast cancer stem cells (CSC) to PARPi is unknown. Experimental Design: We determined the sensitivity of breast CSCs to PARP inhibition in BRCA1-mutant and -wild-type TNBC cell lines and tumor xenografts. We also investigated the role of RAD51 in mediating CSC resistance to PARPi in these in vitro and in vivo models. Results: We demonstrated that the CSCs in BRCA1-mutant TNBCs were resistant to PARP inhibition, and that these cells had both elevated RAD51 protein levels and activity. Downregulation of RAD51 by shRNA sensitized CSCs to PARP inhibition and reduced tumor growth. BRCA1–wild-type cells were relatively resistant to PARP inhibition alone, but reduction of RAD51 sensitized both CSC and bulk cells in these tumors to PARPi treatment. Conclusions: Our data suggest that in both BRCA1-mutant and BRCA1–wild-type TNBCs, CSCs are relatively resistant to PARP inhibition. This resistance is mediated by RAD51, suggesting that strategies aimed at targeting RAD51 may increase the therapeutic efficacy of PARPi. Clin Cancer Res; 23(2); 514–22. ©2016 AACR.

Abstract 2311: Targeting EMT and MET breast cancer stem cell states through simultaneous inhibition of glycolytic and antioxidant pathways

Cancer stem cells (CSCs) drive tumor growth as well as mediating metastasis and treatment resistance. In breast cancer (BC), CSCs exist in alternative mesenchymal (EMT) and epithelial (MET)-like states characterized by the expression of CD44+CD24- and aldehyde dehydrogenase (ALDH) respectively. BCSCs display phenotypic plasticity allowing them to transition between EMT and MET states in a process regulated by the tumor microenvironment. The plasticity of BCSCs suggests that it may be necessary to simultaneously target alternative BCSC states to achieve maximal eradication of these cell populations. In order to develop strategies to target these BCSC states, we measured the cellular bioenergetics of EMT and MET BCSCs compared to non-stem bulk tumor cells as well as the sensitivity of distinct BCSC states to inhibitors of glucose and hydroperoxide metabolism. Using a Seahorse XCF instrument, we found that both MET and EMT BCSCs display higher glycolytic potential than bulk tumor cells. Increased glycolysis correlated with elevated expression of hexokinase 2, a rate limiting glycolytic enzyme in cancer. Interestingly, the glycolytic inhibitor, 2-deoxyglucose (2DG), specifically inhibited EMT BCSCs in a dose dependent fashion; however, MET BCSCs were completely refractory to this treatment. Proteomic and RNA-seq analyses revealed that two important arms of hydroperoxide metabolism, the thioredoxin- and glutathione-mediated antioxidant pathways, were robustly up-regulated in MET BCSCs, suggesting that these cells are rendered resistant to glycolysis inhibition via an enhanced anti-oxidant defense. Pharmacologic inhibition of this antioxidant defense by Auranofin (an inhibitor of thioredoxin reductase) is sufficient to deplete MET BCSCs in SUM149 BC cells which were rescued by NAC, a ROS scavenger, or catalase, a specific enzyme capable of degrading intracellular H2O2. Finally, administration of 2DG together with Auranofin and BSO (an inhibitor of glutathione metabolism) synergistically suppressed tumor growth in a patient derived xenograft (PDX) model by suppressing both EMT and MET BCSCs. This study suggests that utilizing metabolic inhibition to simultaneously target EMT and MET BCSCs is a viable therapeutic strategy with important clinical implications. Citation Format: Ming Luo, April Davis, Sean McDermott, Evelyn Jiagge, Michael Brooks, Elizabeth Gheordunescu, Tahra Luther, Shawn G. Clouthier, Sarah Conley, Douglas R. Spitz, Max S. Wicha. Targeting EMT and MET breast cancer stem cell states through simultaneous inhibition of glycolytic and antioxidant pathways. [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 2311. doi:10.1158/1538-7445.AM2015-2311

Abstract 3015: Annexin A3 is selectively expressed in MET-like as compared to EMT-like breast cancer stem cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Increasing evidence has demonstrated that breast cancers are driven by a cellular subset that displays stem cell properties. These cells mediate tumor metastasis and contribute to therapeutic resistance. We recently reported that breast cancer stem cells (BCSCs) are capable of inter-converting between two cellular states in a process regulated by the tumor microenvironment. CD44+/CD24- CSCs are characterized by expression of epithelial-mesenchymal transition (EMT) genes, invasive and largely quiescent, whereas mesenchymal-epithelial-like (MET) stem cells are characterized by expression of Aldehyde dehydrogenase (ALDH), epithelial morphology and self-renewal. In order to further characterize these two alternative populations of BCSCs, we carried out gene expression analysis of sorted ALDH+ and CD44+/24- populations from a series of primary human breast cancers and established cell lines. We used the CSC markers to fractionate MCF7 cells and performed proteomic analysis utilizing liquid chromatography-tandem mass spectrometry. Differentially expressed proteins were identified using t-test and ANOVA based on spectra counts. Both microarray and proteomic analysis identified Annexin A3 (ANXA3) to be consistently upregulated in ALDH/MET stem cell populations compared to matched CD44+/24-/EMT-like CSCs. These results were validated through qPCR and western blotting in utilizing the luminal cell line MCF7 as well as the claudinlow cell line SUM 159. In the latter, ANXA3 was upregulated 2-5 fold at both the mRNA and protein levels in ALDH+ compared to ALDH- populations. Further, immunofluorescence studies using confocal microscopy demonstrated co-expression of ANXA3 and ALDH in MCF7 and SUM 159 cells. To determine the clinical significance of ANXA3 in breast cancer, we performed immunohistochemical analyses on tissue microarrays of breast cancer patients. ANXA3 expression was found to be significantly higher in breast cancers compared to normal breast tissue with over 75% of primary human tumors demonstrating ANXA3 staining. ANXA3 was expressed at significantly higher levels in luminal compared to basal and claudinlow tumors. Furthermore, 42% of HER2+ tumors also showed moderate to high expression of ANXA3. These results suggest that ANXA3 is predominantly expressed in luminal breast cancers, where it marks the ALDH+ epithelial-like CSC populations. The functional role of ANXA3 in breast CSCs is currently under investigation. Citation Format: Deol S. Yadwinder, Sean McDermott, David M. Lubman, Jenny C. Chang, Song Nie, Yang Cong, Alice Turdo, Ebrahim Azizi, Tahra K. Luther, Shawn G. Clouthier, Max Wicha. Annexin A3 is selectively expressed in MET-like as compared to EMT-like breast cancer stem cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3015. doi:10.1158/1538-7445.AM2014-3015

Stem Cells in Breast Development and Cancer

Abstract The concept of the stem cell of origin for cancers was first proposed more than 100 years ago. In this theoretical model, certain cells with self-renewal capacity would form tumors from “embryonic rests.” More evidence has accumulated since that time as a result of extensive research that strongly supports the cancer stem cell (CSC) hypothesis, suggesting the existence of self-renewing cells that generate heterogenous populations of cells within the tumor mass. The preponderance of evidence now suggests that the majority of cancers are hierarchically organized and sustained by a population of cells that display stem cell properties—CSCs. As is the case for normal tissue stem cells, CSCs are able to self-renew and differentiate, generating cells that comprise the tumor bulk. Furthermore, preclinical and clinical studies demonstrate that CSCs mediate tumor metastasis and contribute to resistance to chemotherapy and radiation therapy. The CSC hypothesis has fundamental biological and clinical implications that are discussed in detail in this chapter.

Abstract 3310: Riboflavin targets autofluorescent-positive breast cancer stem cells upon light exposure

Most tumors are hierarchically organized and driven by a population of cells that display stem cell properties. These cancer stem cells (CSCs) have been characterized by in vitro assays such as sphere formation and in vivo by tumor initiation. In addition cell surface markers have been utilized to enrich for CSC populations. More recently a subtype of CSCs have been reported to display autofluorescence (AF), a property mediated by cellular uptake and concentration of riboflavin. In order to characterize the molecular heterogeneity of CSCs, we studied AF positive sorted single cells in different subtypes of breast cancer cell lines and also patient derived xenograft (PDX) breast tumor cells. Exposure of cell lines and PDXs to riboflavin significantly increased the percentage of AF+ cells 3-5 fold. These sorted AF+ cells were enriched for tumorsphere forming capacity in vitro. Flow cytometry analysis revealed partial overlap of AF+ cells with the previously characterized EMT-CSC phenotype CD44+/CD24- cells. We also utilized the microfluidic C1 and BioMark HD instruments to determine the expression patterns of 96 target genes using TaqMan assays in a multiplex RT-qPCR setting at single cell resolution. Single cell gene expression data showed a distinct signature in AF+ cells with high expression levels of MKI67, PCNA, ABCG2, BRCA1, Jag2, EZH2, and MMP9 genes. Since AF is mediated by specific cellular uptake and concentration of riboflavin, and the report that riboflavin is capable of generating cytotoxic free radicals upon light exposure, we determined whether this property could be exploited to selectively target CSCs. The AF+ cells were cultured in the presence and absence of riboflavin, exposed to visible light and analyzed by MTT cytotoxicity assay. Data revealed significant cytotoxicity of riboflavin on AF+ cells that were exposed to visible light. In conclusion, AF+ cells show distinct cancer stem cell features and are sensitive to light-activated riboflavin. These findings will help better understanding of tumor biology to identify new target therapies for treatment of cancer patients. Citation Format: Shamileh Fouladdel, Tahra Luther, Kaitlin Harvey, Rachel Martin-Trevino, Jill Granger, Ebrahim Azizi, Max S. Wicha. Riboflavin targets autofluorescent-positive breast cancer stem cells upon light exposure. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3310.

Abstract 1518: Multiethnic triple negative breast cancer comparisons of gene expression shows enrichment of distinct pathways in ALDH1+ subpopulations compared to CD44+/CD24-/EpCAM+

Triple negative breast cancers are a heterogeneous group with diverse, usually poorer survival outcomes and are more prevalent in individuals of African descent. Given the 8-12% higher mortality in breast cancers in individuals of African descent, we set out to identify differences in pathway enrichment in patients with TNBC of Caucasian, African American, and African (Ghanaian) ethnicity. Moreover, we analyzed subpopulations of cells previously found to exhibit stem-like characteristics of self-renewal, within each tumor in order delineate the heterogeneity of gene expression patterns also in different subpopulations of cells within tumors of patients of diverse ethnicities. Based on previous work, we hypothesized that there are distinct sub populations of rare cells, which exhibit distinct signaling pathway enrichment that may confer to TNBCs their highly metastatic and drug resistant characteristics. We developed PDX models from TNBC of 5 Ghanaian, 5 African American and 5 Caucasian patients. We collected bulk cells from the PDXs and used fluorescence-activated cell sorting (FACS) to select and collect ALDH+ cells and CD24-CD44+EpCAM+ (CD44+) cells. We extracted RNA from the sorted cell populations and performed RNA-sequencing using the Illumina Next Generation Sequencing platform. Our results showed that the bulk, ALDH1+ and CD44+ populations within each tumor segregate closely together, and we noted no distinct ethnic separation when we performed the analyses either of the bulk or special subpopulations. Upon centering on the mean for each sample in each tumor, thereby abrogating differences between individual tumors, we observe a significant separation between the ALDH1+ and the CD44+/CD24- sub populations. In particular, the following pathways are most differentially enriched in these subpopulations. The numbers between parentheses indicate the numbers of genes significantly altered that contribute to each pathway. ALDH1+ : Wnt (10), MAPK (13), axon guidance (6), GnRH signaling (7), TGFbeta (9), endocytosis(5) CD44+/CD24-/EpCAM+: tRNA synthesis (5), N-glycan biosynthesis (7), RNA degradation (6) We are focusing our experiments on verifying whether the above pathways are indeed active in these subpopulations and we are exploring their biological significance with regards to tumor growth and metastases. Although we did not detect any specific clustering of tumors by ethnicity, our work suggests the presence of an ALDH1+ subpopulation of cells that exhibit characteristic enrichment of pathways distinct from those enriched in CD44+/CD24-/EpCAM+ cells. Citation Format: Evelyn M. Jiagge, Qingxuan Song, Shukmei Wong, Tahra Luther, Michele Dziubinski, Shawn Clouthier, Sean McDermott, Lisa Newman, John Carpten, Jun Li, Max WIcha, Sofia Merajver. Multiethnic triple negative breast cancer comparisons of gene expression shows enrichment of distinct pathways in ALDH1+ subpopulations compared to CD44+/CD24-/EpCAM+. [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 1518. doi:10.1158/1538-7445.AM2015-1518