Informed by Cancer Stem Cells of Solid Tumors: Advances in Treatments Targeting Tumor-Promoting Factors and Pathways.

Cancer stem cells (CSCs) represent a subpopulation within tumors that promote cancer progression, metastasis, and recurrence due to their self-renewal capacity and resistance to conventional therapies. CSC-specific markers and signaling pathways highly active in CSCs have emerged as a promising strategy for improving patient outcomes. This review provides a comprehensive overview of the therapeutic targets associated with CSCs of solid tumors across various cancer types, including key molecular markers aldehyde dehydrogenases, CD44, epithelial cellular adhesion molecule, and CD133 and signaling pathways such as Wnt/ß-catenin, Notch, and Sonic Hedgehog. We discuss a wide array of therapeutic modalities ranging from targeted antibodies, small molecule inhibitors, and near-infrared photoimmunotherapy to advanced genetic approaches like RNA interference, CRISPR/Cas9 technology, aptamers, antisense oligonucleotides, chimeric antigen receptor (CAR) T cells, CAR natural killer cells, bispecific T cell engagers, immunotoxins, drug-antibody conjugates, therapeutic peptides, and dendritic cell vaccines. This review spans developments from preclinical investigations to ongoing clinical trials, highlighting the innovative targeting strategies that have been informed by CSC-associated pathways and molecules to overcome therapeutic resistance. We aim to provide insights into the potential of these therapies to revolutionize cancer treatment, underscoring the critical need for a multi-faceted approach in the battle against cancer. This comprehensive analysis demonstrates how advances made in the CSC field have informed significant developments in novel targeted therapeutic approaches, with the ultimate goal of achieving more effective and durable responses in cancer patients.

Metabolite profiling reveals a connection between aldehyde dehydrogenase 1A3 and GABA metabolism in breast cancer metastasis.

INTRODUCTION: Aldehyde dehydrogenase 1A3 (ALDH1A3) is a cancer stem cell (CSC) marker and in breast cancer it is associated with triple-negative/basal-like subtypes and aggressive disease. Studies on the mechanisms of ALDH1A3 in cancer have primarily focused on gene expression changes induced by the enzyme; however, its effects on metabolism have thus far been unstudied and may reveal novel mechanisms of pathogenesis. OBJECTIVE: Determine how ALDH1A3 alters the metabolite profile in breast cancer cells and assess potential impacts. METHOD: Triple-negative MDA-MB-231 tumors and cells with manipulated ALDH1A3 levels were assessed by HPLC-MS metabolomics and metabolite data was integrated with transcriptome data. Mice harboring MDA-MB-231 tumors with or without altered ALDH1A3 expression were treated with γ-aminobutyric acid (GABA) or placebo. Effects on tumor growth, and lungs and brain metastasis were quantified by staining of fixed thin sections and quantitative PCR. Breast cancer patient datasets from TCGA, METABRIC and GEO were used to assess the co-expression of GABA pathway genes with ALDH1A3. RESULTS: Integrated metabolomic and transcriptome data identified GABA metabolism as a primary dysregulated pathway in ALDH1A3 expressing breast tumors. Both ALDH1A3 and GABA treatment enhanced metastasis. Patient dataset analyses revealed expression association between ALDH1A3 and GABA pathway genes and corresponding increased risk of metastasis. CONCLUSION: This study revealed a novel pathway affected by ALDH1A3, GABA metabolism. Like ALDH1A3 expression, GABA treatment promotes metastasis. Given the clinical use of GABA mimics to relieve chemotherapy-induced peripheral nerve pain, further study of the effects of GABA in breast cancer progression is warranted.

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.

Hide-and-seek: the interplay between cancer stem cells and the immune system.

The enhanced ability of cancer stem cells (CSCs) to give rise to new tumors suggests that these cells may also have an advantage in evading immune detection and elimination. This tumor-forming ability, combined with the known plasticity of the immune system, which can play both protumorigenic and antitumorigenic roles, has motivated investigations into the interaction between CSCs and the immune system. Herein, we review the interplay between host immunity and CSCs by examining the immune-related mechanisms that favor CSCs and the CSC-mediated expansion of protumorigenic immune cells. Furthermore, we discuss immune cells, such as natural killer cells, that preferentially target CSCs and the strategies used by CSCs to evade immune detection and destruction. An increased understanding of these interactions and the pathways that regulate them may allow us to harness immune system components to create new adjuvant therapies that eradicate CSCs and improve patient survival.

Newly recruited CD11b+, GR-1+, Ly6C(high) myeloid cells augment tumor-associated immunosuppression immediately following the therapeutic administration of oncolytic reovirus.

Tumor-associated immunosuppression aids cancer cells to escape immune-mediated attack and subsequent elimination. Recently, however, many oncolytic viruses, including reovirus, have been reported to overturn such immunosuppression and promote the development of a clinically desired antitumor immunity, which is known to promote favorable patient outcomes. Contrary to this existing paradigm, in this article we demonstrate that reovirus augments tumor-associated immunosuppression immediately following its therapeutic administration. Our data show that reovirus induces preferential differentiation of highly suppressive CD11b(+), Gr-1(+), Ly6C(high) myeloid cells from bone marrow hematopoietic progenitor cells. Furthermore, reovirus administration in tumor-bearing hosts drives time-dependent recruitment of CD11b(+), Gr-1(+), Ly6C(high) myeloid cells in the tumor milieu, which is further supported by virus-induced increased expression of numerous immune factors involved in myeloid-derived suppressor cell survival and trafficking. Most importantly, CD11b(+), Gr-1(+), Ly6C(high) myeloid cells specifically potentiate the suppression of T cell proliferation and are associated with the absence of IFN-γ response in the tumor microenvironment early during oncotherapy. Considering that the qualitative traits of a specific antitumor immunity are largely dictated by the immunological events that precede its development, our findings are of critical importance and must be considered while devising complementary interventions aimed at promoting the optimum efficacy of oncolytic virus-based anticancer immunotherapies.

Multifaceted therapeutic targeting of ovarian peritoneal carcinomatosis through virus-induced immunomodulation.

Immunosuppression associated with ovarian cancer (OC) and resultant peritoneal carcinomatosis (PC) hampers the efficacy of many promising treatment options, including immunotherapies. It is hypothesized that oncolytic virus-based therapies can simultaneously kill OC and mitigate immunosuppression. Currently, reovirus-based anticancer therapy is undergoing phase I/II clinical trials for the treatment of OC. Hence, this study was focused on characterizing the effects of reovirus therapy on OC and associated immune microenvironment. Our data shows that reovirus efficiently killed OC cells and induced higher expression of the molecules involved in antigen presentation including major histocompatibility complex (MHC) class I, ß2-microglobulin (ß2M), TAP-1, and TAP-2. In addition, in the presence of reovirus, dendritic cells (DCs) overcame the OC-mediated phenotypic suppression and successfully stimulated tumor-specific CD8+ T cells. In animal studies, reovirus targeted local and distal OC, alleviated the severity of PC and significantly prolonged survival. These therapeutic effects were accompanied by decreased frequency of suppressive cells, e.g., Gr1.1+, CD11b+ myeloid derived suppressor cells (MDSCs), and CD4+, CD25+, FOXP3+ Tregs, tumor-infiltration of CD3+ cells and higher expression of Th1 cytokines. Finally, reovirus therapy during early stages of OC also resulted in the postponement of PC development. This report elucidates timely information on a therapeutic approach that can target OC through clinically desired multifaceted mechanisms to better the outcomes.

LncRNAs exhibit subtype-specific expression, survival associations, and cancer-promoting effects in breast cancer.

Long non-coding RNAs (lncRNAs) play important roles in cancer progression, influencing processes such as invasion, metastasis, and drug resistance. Their reported cell type-dependent expression patterns suggest the potential for specialized functions in specific contexts. In breast cancer, lncRNA expression has been associated with different subtypes, highlighting their relevance in disease heterogeneity. However, our understanding of lncRNA function within breast cancer subtypes remains limited, warranting further investigation. We conducted a comprehensive analysis using the TANRIC dataset derived from the TCGA-BRCA cohort, profiling the expression, patient survival associations and immune cell type correlations of 12,727 lncRNAs across subtypes. Our findings revealed subtype-specific associations of lncRNAs with patient survival, tumor infiltrating lymphocytes and other immune cells. Targeting of lncRNAs exhibiting subtype-specific survival associations and expression in a panel of breast cancer cells demonstrated a selective reduction in cell proliferation within their associated subtype, supporting subtype-specific functions of certain lncRNAs. Characterization of HER2 + -specific lncRNA LINC01269 and TNBC-specific lncRNA AL078604.2 showed nuclear localization and altered expression of hundreds of genes enriched in cancer-promoting processes, including apoptosis, cell proliferation and immune cell regulation. This work emphasizes the importance of considering the heterogeneity of breast cancer subtypes and the need for subtype-specific analyses to fully uncover the relevance and potential impact of lncRNAs. Collectively, these findings demonstrate the contribution of lncRNAs to the distinct molecular, prognostic, and cellular composition of breast cancer subtypes.

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.

Three-dimensional growth sensitizes breast cancer cells to treatment with ferroptosis-promoting drugs.

Drugs causing ferroptosis, iron-mediated cell death, represent promising tools for cancer treatment. While exploring the effect of these drugs on breast cancer (BC), we found that a ferroptosis-inducing drug erastin dramatically inhibits tumorigenicity of human BC cells in mice but when used at a concentration known to effectively kill other cell types only modestly reduces such growth in 2D monolayer culture. BCs grow in vivo as 3D masses, and we found that ferroptosis inducers erastin and sulfasalazine inhibit growth of multiple human BC cell lines in 3D culture significantly stronger than in 2D culture. To understand the mechanism of this differential effect, we found that ferroptosis inducers upregulate mRNAs encoding multiple direct and indirect autophagy stimulators, such as ATG16L2, ATG9A, ATG4D, GABARAP, SQSTM/p62, SEC23A and BAX, in tumor cells growing in 2D but not in 3D culture. Furthermore, these drugs promoted autophagy of tumor cells growing in a 2D but not in a 3D manner. We observed that pharmacological inhibition of autophagy-stimulating protein kinase ULK1 or RNA interference-mediated knockdown of autophagy mediator ATG12 significantly sensitized tumor cells to erastin treatment in 2D culture. We also found that ferroptosis-promoting treatments upregulate heme oxygenase-1 (HO-1) in BC cells. HO-1 increases cellular free iron pool and can potentially promote ferroptosis. Indeed, we observed that HO-1 knockdown by RNA interference reversed the effect of ferroptosis inducers on BC cell 3D growth. Hence, the effect of these drugs on such growth is mediated by HO-1. In summary, autophagy triggered by ferroptosis-promoting drugs reduces their ability to kill BC growing in a 2D manner. This protection mechanism is inhibited in BC cells growing as a 3D mass, and ferroptosis-promoting drugs kill such cells more effectively. Moreover, this death is mediated by HO-1. Thus, ferroptosis induction represents a promising strategy for blocking 3D BC growth.

The Expanding Role of Cancer Stem Cell Marker ALDH1A3 in Cancer and Beyond.

Aldehyde dehydrogenase 1A3 (ALDH1A3) is one of 19 ALDH enzymes expressed in humans, and it is critical in the production of hormone receptor ligand retinoic acid (RA). We review the role of ALDH1A3 in normal physiology, its identification as a cancer stem cell marker, and its modes of action in cancer and other diseases. ALDH1A3 is often over-expressed in cancer and promotes tumor growth, metastasis, and chemoresistance by altering gene expression, cell signaling pathways, and glycometabolism. The increased levels of ALDH1A3 in cancer occur due to genetic amplification, epigenetic modifications, post-transcriptional regulation, and post-translational modification. Finally, we review the potential of targeting ALDH1A3, with both general ALDH inhibitors and small molecules specifically designed to inhibit ALDH1A3 activity.

Aldehyde dehydrogenase activity of breast cancer stem cells is primarily due to isoform ALDH1A3 and its expression is predictive of metastasis.

Cancer stem cells (CSCs) are proposed to initiate cancer and propagate metastasis. Breast CSCs identified by aldehyde dehydrogenase (ALDH) activity are highly tumorigenic in xenograft models. However, in patient breast tumor immunohistological studies, where CSCs are identified by expression of ALDH isoform ALDH1A1, CSC prevalence is not correlative with metastasis, raising some doubt as to the role of CSCs in cancer. We characterized the expression of all 19 ALDH isoforms in patient breast tumor CSCs and breast cancer cell lines by total genome microarray expression analysis, immunofluorescence protein expression studies, and quantitative polymerase chain reaction. These studies revealed that ALDH activity of patient breast tumor CSCs and cell lines correlates best with expression of another isoform, ALDH1A3, not ALDH1A1. We performed shRNA knockdown experiments of the various ALDH isoforms and found that only ALDH1A3 knockdown uniformly reduced ALDH activity of breast cancer cells. Immunohistological studies with fixed patient breast tumor samples revealed that ALDH1A3 expression in patient breast tumors correlates significantly with tumor grade, metastasis, and cancer stage. Our results, therefore, identify ALDH1A3 as a novel CSC marker with potential clinical prognostic applicability, and demonstrate a clear correlation between CSC prevalence and the development of metastatic breast cancer.

Oncolytic virus-initiated protective immunity against prostate cancer.

Recently reovirus-based oncotherapy has been successfully implemented for the treatment of prostate cancer. In this report, we show that apart from its primary direct cancer-killing activity, reovirus oncotherapy overrides tumor-associated immune evasion strategies and confers protective antiprostate cancer immunity. Prostate cancer represents an ideal target for immunotherapies. However, currently available immune interventions fail to induce clinically significant antiprostate cancer immune responses, owing to the immunosuppressive microenvironment associated with this disease. We show here that during the process of oncolysis, reovirus acts upon prostate cancer cells and initiates proinflammatory cytokines and major histocompatibility complex (MHC) class I molecule expression. In an immunocompetent transgenic adenocarcinoma of mouse prostate (TRAMP) model, reovirus oncotherapy induces the homing of CD8(+) T and NK cells in tumors and the display of tumor-associated antigens (TAAs) on antigen-presenting cells (APCs), and endows dendritic cells (DCs) with a capacity to successfully present TAAs to tumor-specific CD8(+) T cells. These newly generated immunological events lead to the development of strong antiprostate cancer T cell responses, which restrict the growth of subsequently, implanted syngeneic tumor in an antigen-specific, but reovirus-independent manner. Such reovirus-initiated antiprostate cancer immunity represents a clinically valuable entity that can promote long-term cancer-free health even after discontinuation of the primary oncotherapy.

Breast Cancer Xenograft Murine Models.

Mice are used as model organisms to understand the pathological basis of a variety of human diseases, including breast cancer. Both immunocompetent and immunocompromised mouse models are used depending on the scope of the study. Immunocompetent models allow the study of the impact of the immune system in murine models of mammary cancer, while immunodeficient mice serve as ideal host organisms to understand the behavior of human breast cancers within a biological system. Xenografting of human breast cancer cells into immunocompromised mouse models continues to be the most used fundamental animal model in preclinical breast cancer research. These in vivo models allow critical understanding of tumor biology and assessment of novel treatments, a necessary prelude to testing new drugs in the clinic. In this chapter, we provide detailed methodology for the use of non-obese diabetic (NOD) severe combined immunodeficient (SCID) mice in several breast cancer xenografting procedures, including established cell lines and patient-derived xenografts (PDXs).

Breast Cancer Subtype-Specific miRNAs: Networks, Impacts, and the Potential for Intervention.

The regulatory and functional roles of non-coding RNAs are increasingly demonstrated as critical in cancer. Among non-coding RNAs, microRNAs (miRNAs) are the most well-studied with direct regulation of biological signals through post-transcriptional repression of mRNAs. Like the transcriptome, which varies between tissue type and disease condition, the miRNA landscape is also similarly altered and shows disease-specific changes. The importance of individual tumor-promoting or suppressing miRNAs is well documented in breast cancer; however, the implications of miRNA networks is less defined. Some evidence suggests that breast cancer subtype-specific cellular effects are influenced by distinct miRNAs and a comprehensive network of subtype-specific miRNAs and mRNAs would allow us to better understand breast cancer signaling. In this review, we discuss the altered miRNA landscape in the context of breast cancer and propose that breast cancer subtypes have distinct miRNA dysregulation. Further, given that miRNAs can be used as diagnostic and/or prognostic biomarkers, their impact as novel targets for subtype-specific therapy is also possible and suggest important implications for subtype-specific miRNAs.

Isolation of cancer stem cells from cultured breast cancer cells and xenografted breast tumors based on aldehyde dehydrogenase activity.

The heterogeneity of breast tumors is a major factor in the development, progression, and therapeutic response of breast cancer. In terms of therapy resistance, a subset of tumor cells commonly referred to as cancer stem cells (CSCs) or tumor initiating cells (TICs) have a prominent role. These cells have inherent increased tumorigenicity, self-renewal and differentiation capacity, and mechanisms for chemotherapy and radiation resistance. The importance of CSCs/TICs in cancer makes isolating and studying these cells via reliable methods critical. CSCs/TICs can be enriched for by discrete markers. Increased aldehyde dehydrogenase (ALDH) activity as detected by the AldefluorTM assay is a commonly used method. In this chapter, we describe the detailed methods for identification and isolation of putative CSCs/TICs from cultured cells and xenografted breast tumors using the AldefluorTM assay and describe the importance of the ALDH isoforms in breast cancer.

In Vivo Genome-Wide Pooled RNAi Screens in Cancer Cells to Identify Determinants of Chemotherapy/Drug Response.

Large-scale RNAi screens (i.e., genome-wide arrays and pools) can reveal the essential biological functions of previously uncharacterized genes. Due to the nature of the selection process involved in screens, RNAi screens are also very useful for identifying genes involved in drug responses. The information gained from these screens could be used to predict a cancer patient's response to a specific drug (i.e., precision medicine) or identify anti-cancer drug resistance genes, which could be targeted to improve treatment outcomes. In this capacity, screens have been most often performed in vitro. However, there is limitation to performing these screens in vitro: genes which are required in only an in vivo setting (e.g., rely on the tumor microenvironment for function) will not be identified. As such, it can be desirable to perform RNAi screens in vivo. Here we outline the additional technical details that should be considered for performing genome-wide RNAi drug screens of cancer cells under in vivo conditions (i.e., tumor xenografts).

Triple-Negative Breast Cancer and the COVID-19 Pandemic: Clinical Management Perspectives and Potential Consequences of Infection.

The COVID-19 pandemic has caused the need for prioritization strategies for breast cancer treatment, where patients with aggressive disease, such as triple-negative breast cancer (TNBC) are a high priority for clinical intervention. In this review, we summarize how COVID-19 has thus far impacted the management of TNBC and highlighted where more information is needed to hone shifting guidelines. Due to the immunocompromised state of most TNBC patients receiving treatment, TNBC management during the pandemic presents challenges beyond the constraints of overburdened healthcare systems. We conducted a literature search of treatment recommendations for both primary and targeted TNBC therapeutic strategies during the COVID-19 outbreak and noted changes to treatment timing and drugs of choice. Further, given that SARS-CoV-2 is a respiratory virus, which has systemic consequences, management of TNBC patients with metastatic versus localized disease has additional considerations during the COVID-19 pandemic. Published dataset gene expression analysis of critical SARS-CoV-2 cell entry proteins in TNBCs suggests that the virus could in theory infect metastasized TNBC cells it contacts. This may have unforeseen consequences in terms of both the dynamics of the resulting acute viral infection and the progression of the chronic metastatic disease. Undoubtedly, the results thus far suggest that more research is required to attain a full understanding of the direct and indirect clinical impacts of COVID-19 on TNBC patients.

LncRNA-miRNA axes in breast cancer: Novel points of interaction for strategic attack.

Therapeutic effectiveness in breast cancer can be limited by the underlying mechanisms of pathogenesis, including epithelial-mesenchymal transition (EMT), cancer stem cells (CSCs) and drug resistance. Long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) are master regulators of gene expression and are functionally important mediators in these mechanisms of pathogenesis. Intricate crosstalks between these non-coding RNAs form complex regulatory networks of post-transcriptional gene regulation. Depending on the specific lncRNA/miRNA interaction, the lncRNA-miRNA axis can have tumor suppressor or oncogenic effects, thus defining the lncRNA-miRNA axis is important for determining targetability. Herein, we summarize the current literature describing lncRNA-miRNA interactions that are critical in the molecular mechanisms that regulate EMT, CSCs and drug resistance in breast cancer. Further, we review both the well-studied and potential novel mechanisms of lncRNA-miRNA interactions in breast cancer.

Datasets exploring putative lncRNA-miRNA-mRNA axes in breast cancer cell lines.

Long non-coding RNA (lncRNA)/microRNA (miRNA)/messenger RNA (mRNA) interactions regulate oncogenesis and tumour suppression in breast cancer. Oncogenic lncRNA/miRNA/mRNA axes may offer novel therapeutic targets; therefore, identifying such axes is a clinically relevant undertaking. To explore miRNAs regulated by oncogenic lncRNAs, we queried the NCBI Gene Expression Omnibus (GEO) database to find datasets that profiled gene expression changes upon lncRNA knockdown in breast cancer. We identified four microarray datasets that permitted our interrogation of genes regulated by lncRNAs LincK, LincIN, SPRY4-IT1 and AC009283.1. We specifically analysed changes in miRNA transcripts within these datasets to study miRNAs regulated by each of the four lncRNAs. We subsequently identified the predicted mRNA targets for these miRNAs to uncover possible lncRNA/miRNA/mRNAs axes in breast cancer. These axes may be candidates for future investigation of gene regulation in breast cancer.

The Prostaglandin E2 Pathway and Breast Cancer Stem Cells: Evidence of Increased Signaling and Potential Targeting.

Culprits of cancer development, metastasis, and drug resistance, cancer stem cells (CSCs) are characterized by specific markers, active developmental signaling pathways, metabolic plasticity, increased motility, invasiveness, and epithelial-mesenchymal transition. In breast cancer, these cells are often more prominent in aggressive disease, are amplified in drug-resistant tumors, and contribute to recurrence. For breast cancer, two distinct CSC populations exist and are typically defined by CD44+/CD24- cell surface marker expression or increased aldehyde dehydrogenase (ALDH) activity. These CSC populations share many of the same properties but also exhibit signaling pathways that are more active in CD44+/CD24- or ALDH+ populations. Understanding these CSC populations and their shared or specific signaling pathways may lead to the development of novel therapeutic strategies that will improve breast cancer patient outcomes. Herein, we review the current evidence and assess published patient tumor datasets of sorted breast CSC populations for evidence of heightened prostaglandin E2 (PGE2) signaling and activity in these breast CSC populations. PGE2 is a biologically active lipid mediator and in cancer PGE2 promotes tumor progression and poor patient prognosis. Overall, the data suggests that PGE2 signaling is important in propagating breast CSCs by enhancing inherent tumor-initiating capacities. Development of anti-PGE2 signaling therapeutics may be beneficial in inhibiting tumor growth and limiting breast CSC populations.