ALDH1A3 promotes invasion and metastasis in triple-negative breast cancer by regulating the plasminogen activation pathway.

Aldehyde dehydrogenase 1A3 (ALDH1A3) is a cancer stem cell marker that promotes metastasis. Triple-negative breast cancer (TNBC) progression has been linked to ALDH1A3-induced gene expression changes. To investigate the mechanism of ALDH1A3-mediated breast cancer metastasis, we assessed the effect of ALDH1A3 on the expression of proteases and the regulators of proteases that degrade the extracellular matrix, a process that is essential for invasion and metastasis. This revealed that ALDH1A3 regulates the plasminogen activation pathway; it increased the levels and activity of tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA). This resulted in a corresponding increase in the activity of serine protease plasmin, the enzymatic product of tPA and uPA. The ALDH1A3 product all-trans-retinoic acid similarly increased tPA and plasmin activity. The increased invasion of TNBC cells by ALDH1A3 was plasminogen-dependent. In patient tumours, ALDH1A3 and tPA are co-expressed and their combined expression correlated with the TNBC subtype, high tumour grade and recurrent metastatic disease. Knockdown of tPA in TNBC cells inhibited plasmin generation and lymph node metastasis. These results identify the ALDH1A3-tPA-plasmin axis as a key contributor to breast cancer progression.

LncRNA PART1 Promotes Proliferation and Migration, Is Associated with Cancer Stem Cells, and Alters the miRNA Landscape in Triple-Negative Breast Cancer.

Triple-negative breast cancers (TNBCs) are aggressive, lack targeted therapies and are enriched in cancer stem cells (CSCs). Novel therapies which target CSCs within these tumors would likely lead to improved outcomes for TNBC patients. Long non-coding RNAs (lncRNAs) are potential therapeutic targets for TNBC and CSCs. We demonstrate that lncRNA prostate androgen regulated transcript 1 (PART1) is enriched in TNBCs and in Aldefluorhigh CSCs, and is associated with worse outcomes among basal-like breast cancer patients. Although PART1 is androgen inducible in breast cancer cells, analysis of patient tumors indicates its androgen regulation has minimal clinical impact. Knockdown of PART1 in TNBC cell lines and a patient-derived xenograft decreased cell proliferation, migration, tumor growth, and mammosphere formation potential. Transcriptome analyses revealed that the lncRNA affects expression of hundreds of genes (e.g., myosin-Va, MYO5A; zinc fingers and homeoboxes protein 2, ZHX2). MiRNA 4.0 GeneChip and TaqMan assays identified multiple miRNAs that are regulated by cytoplasmic PART1, including miR-190a-3p, miR-937-5p, miR-22-5p, miR-30b-3p, and miR-6870-5p. We confirmed the novel interaction between PART1 and miR-937-5p. In general, miRNAs altered by PART1 were less abundant than PART1, potentially leading to cell line-specific effects in terms miRNA-PART1 interactions and gene regulation. Together, the altered miRNA landscape induced by PART1 explains most of the protein-coding gene regulation changes (e.g., MYO5A) induced by PART1 in TNBC.

An in vivo genome-wide shRNA screen identifies BCL6 as a targetable biomarker of paclitaxel resistance in breast cancer.

Paclitaxel is a common breast cancer drug; however, some tumors are resistant. The identification of biomarkers for paclitaxel resistance or sensitivity would enable the development of strategies to improve treatment efficacy. A genome-wide in vivo shRNA screen was performed on paclitaxel-treated mice with MDA-MB-231 tumors to identify genes associated with paclitaxel sensitivity or resistance. Gene expression of the top screen hits was associated with tumor response (resistance or sensitivity) among patients who received neoadjuvant chemotherapy containing paclitaxel. We focused our validation on screen hit B-cell lymphoma 6 (BCL6), which is a therapeutic target in cancer but for which no effects on drug response have been reported. Knockdown of BCL6 resulted in increased tumor regression in mice treated with paclitaxel. Similarly, inhibiting BCL6 using a small molecule inhibitor enhanced paclitaxel treatment efficacy both in vitro and in vivo in breast cancer models. Mechanism studies revealed that reduced BCL6 enhances the efficacy of paclitaxel by inducing sustained G1/S arrest, concurrent with increased apoptosis and expression of target gene cyclin-dependent kinase inhibitor 1A. In summary, the genome-wide shRNA knockdown screen has identified BCL6 as a potential targetable resistance biomarker of paclitaxel response in breast cancer.

Decitabine Response in Breast Cancer Requires Efficient Drug Processing and Is Not Limited by Multidrug Resistance.

Dysregulation of DNA methylation is an established feature of breast cancers. DNA demethylating therapies like decitabine are proposed for the treatment of triple-negative breast cancers (TNBC) and indicators of response need to be identified. For this purpose, we characterized the effects of decitabine in a panel of 10 breast cancer cell lines and observed a range of sensitivity to decitabine that was not subtype specific. Knockdown of potential key effectors demonstrated the requirement of deoxycytidine kinase (DCK) for decitabine response in breast cancer cells. In treatment-naïve breast tumors, DCK was higher in TNBCs, and DCK levels were sustained or increased post chemotherapy treatment. This suggests that limited DCK levels will not be a barrier to response in patients with TNBC treated with decitabine as a second-line treatment or in a clinical trial. Methylome analysis revealed that genome-wide, region-specific, tumor suppressor gene-specific methylation, and decitabine-induced demethylation did not predict response to decitabine. Gene set enrichment analysis of transcriptome data demonstrated that decitabine induced genes within apoptosis, cell cycle, stress, and immune pathways. Induced genes included those characterized by the viral mimicry response; however, knockdown of key effectors of the pathway did not affect decitabine sensitivity suggesting that breast cancer growth suppression by decitabine is independent of viral mimicry. Finally, taxol-resistant breast cancer cells expressing high levels of multidrug resistance transporter ABCB1 remained sensitive to decitabine, suggesting that the drug could be used as second-line treatment for chemoresistant patients.

ALDH1A3-regulated long non-coding RNA NRAD1 is a potential novel target for triple-negative breast tumors and cancer stem cells.

To discover novel therapeutic targets for triple-negative breast cancer (TNBC) and cancer stem cells (CSCs), we screened long non-coding RNAs (lncRNAs) most enriched in TNBCs for high expression in CSCs defined by high Aldefluor activity and associated with worse patient outcomes. This led to the identification of non-coding RNA in the aldehyde dehydrogenase 1 A pathway (NRAD1), also known as LINC00284. Targeting NRAD1 in TNBC tumors using antisense oligonucleotides reduced cell survival, tumor growth, and the number of cells with CSC characteristics. Expression of NRAD1 is regulated by an enzyme that causes Aldefluor activity in CSCs, aldehyde dehydrogenase 1A3 (ALDH1A3) and its product retinoic acid. Cellular fractionation revealed that NRAD1 is primarily nuclear localized, which suggested a potential function in gene regulation. This was confirmed by transcriptome profiling and chromatin isolation by RNA purification, followed by sequencing (ChIRP-seq), which demonstrated that NRAD1 has enriched chromatin interactions among the genes it regulates. Gene Ontology enrichment analysis revealed that NRAD1 regulates expression of genes involved in differentiation and catabolic processes. NRAD1 also contributes to gene expression changes induced by ALDH1A3; thereby, the induction of NRAD1 is a novel mechanism through which ALDH1A3 regulates gene expression. Together, these data identify lncRNA NRAD1 as a downstream effector of ALDH1A3, and a target for TNBCs and CSCs, with functions in cell survival and regulation of gene expression.

The Flick of a Switch: Conferring Survival Advantage to Breast Cancer Stem Cells Through Metabolic Plasticity.

Within heterogeneous tumors, cancer stem cell (CSC) populations exhibit the greatest tumor initiation potential, promote metastasis, and contribute to therapy resistance. For breast cancer specifically, CSCs are identified by CD44highCD24low cell surface marker expression and increased aldehyde dehydrogenase activity. In general, bulk breast tumor cells possess altered energetics characterized by aerobic glycolysis. In contrast, breast CSCs appear to have adaptive metabolic plasticity that allows these tumor-initiating cells to switch between glycolysis and oxidative phosphorylation, depending on factors present in the tumor microenvironment (e.g., hypoxia, reactive oxygen species, availability of glucose). In this article, we review the regulatory molecules that may facilitate the metabolic plasticity of breast CSCs. These regulatory factors include epigenetic chromatin modifiers, non-coding RNAs, transcriptional repressors, transcription factors, energy and stress sensors, and metabolic enzymes. Furthermore, breast cancer cells acquire CSC-like characteristics and altered energetics by undergoing epithelial-mesenchymal transition (EMT). This energy costly process is paired with reprogrammed glucose metabolism by epigenetic modifiers that regulate expression of both EMT and other metabolism-regulating genes. The survival advantage imparted to breast CSCs by metabolic plasticity suggests that targeting the factors that mediate the energetic switch should hinder tumorigenesis and lead to improved patient outcomes.

Retinoic acid and arsenic trioxide induce lasting differentiation and demethylation of target genes in APL cells.

Acute promyelocytic leukemia (APL) is characterized by arrested differentiation of promyelocytes. Patients treated with all-trans retinoic acid (ATRA) alone experience relapse, while patients treated with ATRA and arsenic trioxide (ATO) are often relapse-free. This suggests sustained changes have been elicited by the combination therapy. To understand the lasting effects of the combination therapy, we compared the effects of ATRA and ATO on NB4 and ATRA-resistant NB4-MR2 APL cells during treatment versus post treatment termination. After treatment termination, NB4 cells treated with ATRA or ATO reverted to non-differentiated cells, while combination-treated cells remained terminally differentiated. This effect was diminished in NB4-MR2 cells. This suggests combination treatment induced more permanent changes. Combination treatment induced higher expression of target genes (e.g., transglutaminase 2 and retinoic acid receptor beta), which in NB4 cells was sustained post treatment termination. To determine whether sustained epigenetic changes were responsible, we quantified the enrichment of histone modifications by chromatin immunoprecipitation, and CpG methylation by bisulfite-pyrosequencing. While ATRA and combination treatment induced similar histone acetylation enrichment, combination treatment induced greater demethylation of target genes, which was sustained. Therefore, sustained demethylation of target genes by ATRA and ATO combination treatment is associated with lasting differentiation and gene expression changes.

Epigenetic Silencing of TAP1 in Aldefluor+ Breast Cancer Stem Cells Contributes to Their Enhanced Immune Evasion.

Avoiding detection and destruction by immune cells is key for tumor initiation and progression. The important role of cancer stem cells (CSCs) in tumor initiation has been well established, yet their ability to evade immune detection and targeting is only partly understood. To investigate the ability of breast CSCs to evade immune detection, we identified a highly tumorigenic population in a spontaneous murine mammary tumor based on increased aldehyde dehydrogenase activity. We performed tumor growth studies in immunocompetent and immunocompromised mice. In immunocompetent mice, growth of the spontaneous mammary tumor was restricted; however, the Aldefluor+ population was expanded, suggesting inherent resistance mechanisms. Gene expression analysis of the sorted tumor cells revealed that the Aldefluor+ tumor cells has decreased expression of transporter associated with antigen processing (TAP) genes and co-stimulatory molecule CD80, which would decrease susceptibility to T cells. Similarly, the Aldefluor+ population of patient tumors and 4T1 murine mammary cells had decreased expression of TAP and co-stimulatory molecule genes. In contrast, breast CSCs identified by CD44+ CD24- do not have decreased expression of these genes, but do have increased expression of C-X-C chemokine receptor type 4. Decitabine treatment and bisulfite pyrosequencing suggests that DNA hypermethylation contributes to decreased TAP gene expression in Aldefluor+ CSCs. TAP1 knockdown resulted in increased tumor growth of 4T1 cells in immunocompetent mice. Together, this suggests immune evasion mechanisms in breast CSCs are marker specific and epigenetic silencing of TAP1 in Aldefluor+ breast CSCs contributes to their enhanced survival under immune pressure. Stem Cells 2018;36:641-654.