Margaret L. Thomas

Chemoresistance in Cancer Stem Cells and Strategies to Overcome Resistance

In cancers, there exists a subpopulation of cells which are referred to as cancer stem cells (CSCs) or tumor initiating cells that have enhanced tumor-initiating capacity and metastatic potential, and drive tumor progression. Since the initial identification of acute myeloid leukemia CSCs in 1997, CSCs have been found in many types of cancer and have intrinsic resistance to the current chemotherapeutic strategies. With increased levels of detoxifying enzymes, enhanced DNA repair abilities, impressive efflux capacity, and a slower cell-cycle; CSCs present a formidable obstacle against effective chemotherapy. Several methods of specifically targeting CSCs have been developed in recent years, and these compounds have potential as adjuvant therapies. The following is a review of the mechanisms responsible for chemoresistance in CSCs, with an emphasis on potential strategies to overcome this resistance.

Retinoid Signaling in Cancer and its Promise for Therapy

Deregulated signal transduction is a major facet of cancer development and progression. Herein, we review the current paradigm for retinoic acid signaling, its role in cancer and potential therapeutic applications and challenges. Retinoic acid is used with remarkable success in the treatment of one of the most high-risk leukemias, acute promyelocytic leukemia; however, extending its use in the treatment of other cancers has had limited success at best. Functional studies provide clues for the poor performance of retinoic acid as a general cancer therapeutic, connecting retinoic acid signaling to both cell growth arrest and proliferation with tumor suppression and cancer progression consequences. The dualistic role of the retinoic acid signaling pathway in cancer is revealed in its gene transcription targets, cross-talk with other transcription factors, mediation of apoptotic pathways, and influence in the immune system. If the greatest potential benefit of retinoid-based cancer therapeutics is to be achieved, the many physiological roles of retinoic acid need to be considered.

Breast cancer subtype dictates DNA methylation and ALDH1A3-mediated expression of tumor suppressor RARRES1

Breast cancer subtyping, based on the expression of hormone receptors and other genes, can determine patient prognosis and potential options for targeted therapy. Among breast cancer subtypes, tumors of basal-like and claudin-low subtypes are typically associated with worse patient outcomes, are primarily classified as triple-negative breast cancers (TNBC), and cannot be treated with existing hormone-receptor-targeted therapies. Understanding the molecular basis of these subtypes will lead to the development of more effective treatment options for TNBC. In this study, we focus on retinoic acid receptor responder 1 (RARRES1) as a paradigm to determine if breast cancer subtype dictates protein function and gene expression regulation. Patient tumor dataset analysis and gene expression studies of a 26 cell-line panel, representing the five breast cancer subtypes, demonstrate that RARRES1 expression is greatest in basal-like TNBCs. Cell proliferation and tumor growth assays reveal that RARRES1 is a tumor suppressor in TNBC. Furthermore, gene expression studies, Illumina HumanMethylation450 arrays, and chromatin immunoprecipitation demonstrate that expression of RARRES1 is retained in basal-like breast cancers due to hypomethylation of the promoter. Additionally, expression of the cancer stem cell marker, aldehyde dehydrogenase 1A3, which provides the required ligand (retinoic acid) for RARRES1 transcription, is also specific to the basal-like subtype. We functionally demonstrate that the combination of promoter methylation and retinoic acid signaling dictates expression of tumor suppressor RARRES1 in a subtype-specific manner. These findings provide a precedent for a therapeutically-inducible tumor suppressor and suggest novel avenues of therapeutic intervention for patients with basal-like breast cancer.

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.

Citral reduces breast tumor growth by inhibiting the cancer stem cell marker ALDH1A3.

Breast cancer stem cells (CSCs) can be identified by increased Aldefluor fluorescence caused by increased expression of aldehyde dehydrogenase 1A3 (ALDH1A3), as well as ALDH1A1 and ALDH2. In addition to being a CSC marker, ALDH1A3 regulates gene expression via retinoic acid (RA) signaling and plays a key role in the progression and chemotherapy resistance of cancer. Therefore, ALDH1A3 represents a druggable anti‐cancer target of interest. Since to date, there are no characterized ALDH1A3 isoform inhibitors, drugs that were previously described as inhibiting the activity of other ALDH isoforms were tested for anti‐ALDH1A3 activity. Twelve drugs (3‐hydroxy‐dl‐kynurenine, benomyl, citral, chloral hydrate, cyanamide, daidzin, DEAB, disulfiram, gossypol, kynurenic acid, molinate, and pargyline) were compared for their efficacy in inducing apoptosis and reducing ALDH1A3, ALDH1A1 and ALDH2‐associated Aldefluor fluorescence in breast cancer cells. Citral was identified as the best inhibitor of ALDH1A3, reducing the Aldefluor fluorescence in breast cancer cell lines and in a patient‐derived tumor xenograft. Nanoparticle encapsulated citral specifically reduced the enhanced tumor growth of MDA‐MB‐231 cells overexpressing ALDH1A3. To determine the potential mechanisms of citral‐mediated tumor growth inhibition, we performed cell proliferation, clonogenic, and gene expression assays. Citral reduced ALDH1A3‐mediated colony formation and expression of ALDH1A3‐inducible genes. In conclusion, citral is an effective ALDH1A3 inhibitor and is able to block ALDH1A3‐mediated breast tumor growth, potentially via blocking its colony forming and gene expression regulation activity. The promise of ALDH1A3 inhibitors as adjuvant therapies for patients with tumors that have a large population of high‐ALDH1A3 CSCs is discussed.

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

Profiling of the transcriptional response to all-trans retinoic acid in breast cancer cells reveals RARE-independent mechanisms of gene expression

Retinoids, derivatives of vitamin A, are key physiological molecules with regulatory effects on cell differentiation, proliferation and apoptosis. As a result, they are of interest for cancer therapy. Specifically, models of breast cancer have varied responses to manipulations of retinoid signaling. This study characterizes the transcriptional response of MDA-MB-231 and MDA-MB-468 breast cancer cells to retinaldehyde dehydrogenase 1A3 (ALDH1A3) and all-trans retinoic acid (atRA). We demonstrate limited overlap between ALDH1A3-induced gene expression and atRA-induced gene expression in both cell lines, suggesting that the function of ALDH1A3 in breast cancer progression extends beyond its role as a retinaldehyde dehydrogenase. Our data reveals divergent transcriptional responses to atRA, which are largely independent of genomic retinoic acid response elements (RAREs) and consistent with the opposing responses of MDA-MB-231 and MDA-MB-468 to in vivo atRA treatment. We identify transcription factors associated with each gene set. Manipulation of the IRF1 transcription factor demonstrates that it is the level of atRA-inducible and epigenetically regulated transcription factors that determine expression of target genes (e.g. CTSS, cathepsin S). This study provides a paradigm for complex responses of breast cancer models to atRA treatment, and illustrates the need to characterize RARE-independent responses to atRA in a variety of models.

Cancer Stem Cells and Chemoresistance: Strategies to Overcome Therapeutic Resistance

Cancer stem cells (CSCs) are hypothesized to initiate cancer and give rise to heterogeneous tumors made up of self-renewing CSCs and the differentiated, less tumorigenic non-CSCs, which make up the bulk of the tumor. Importantly, in terms of successful patient treatment, CSCs are also more resistant to commonly used chemotherapeutics. Multiple mechanisms have been identified for CSC-associated chemoresistance. These mechanisms include increased expression of ABC transporter efflux pumps, aldehyde dehydrogenase (ALDH) detoxification enzymes, anti-apoptosis proteins, enhanced DNA repair mechanisms, increased activation of the embryonic signaling pathways (Notch, Wnt and Hedgehog), and quiescence. Identification of these mechanisms has led to development of specific strategies to circumvent CSC-associated chemoresistance (e.g. inhibitors of ABC transporters, ALDH enzymes, and Notch, Wnt, and Hedgehog pathways, and epigenetic modifying drugs). Future clinical evidence will reveal if employing these adjuvant therapies will eradicate CSCs along with the bulk of the tumor, and lead to improved patient outcomes with decreased cancer recurrence.

Regulation of cell surface protease receptor S100A10 by retinoic acid therapy in acute promyelocytic leukemia (APL)

S100A10 (p11), a member of the S100 family of small dimeric EF-hand-type Ca2+-binding proteins, plays a role in a variety of both intracellular and extracellular processes. Previous studies have suggested that p11 is intrinsically unstable and requires binding to annexin A2 (p36) to prevent its rapid ubiquitylation and degradation. Our laboratory has shown that p11 levels are stimulated by the expression of the oncoprotein, PML/RARα. Furthermore, treatment of the APL cell line, NB4 with all-trans retinoic acid (ATRA) causes the rapid loss of p36 and p11 protein. However, the mechanism by which ATRA regulates p11 levels has not been established. Here, we show that the proteasomal inhibitor, lactacystin reversed the ATRA-dependent loss of p11, but did not cause an accumulation of ubiquitylated forms of p11, suggesting that ATRA promotes the proteasomal degradation of p11 in an ubiquitin-independent manner. ATRA treatment of MCF-7 breast cancer cells reduced p11 but not p36 transcript and protein levels, thus indicating that ATRA can regulate p11 levels independently of PML/RARα and p36. Overexpression of p36 upregulated p11 protein but not mRNA levels, indicating that p36 affects p11 post translationally. The forced expression of ubiquitin and p11 in 293 T cells resulted in ubiquitylation of p11 that was blocked by mutagenesis of lysine 57. This study highlights the complex regulation of p11 by retinoid signaling and challenges the hypothesis that ubiquitin-mediated proteasomal degradation of p11 represents a universal mechanism of regulation of this protein.

DNA Methylation Predicts the Response of Triple-Negative Breast Cancers to All-Trans Retinoic Acid

All-trans retinoic acid (atRA) regulates gene expression and is used to treat acute promyelocytic leukemia. Attempts to use atRA in breast cancer without a stratification strategy have resulted in limited overall effectiveness. To identify biomarkers for the treatment of triple-negative breast cancer (TNBC) with atRA, we characterized the effects of atRA on the tumor growth of 13 TNBC cell lines. This resulted in a range of effects that was not predictable based on previously hypothesized predictors of response, such as the levels of atRA nuclear shuttling proteins fatty acid binding protein 5 (FABP5) and cellular retinoic acid binding protein 2 (CRABP2). Transcriptional profiling revealed that atRA induced distinct gene expression changes in the sensitive versus resistant cell lines that were mostly independent of the presence of retinoic acid response elements (RAREs) or peroxisome proliferator response elements (PPREs). Given the importance of DNA methylation in regulating gene expression, we hypothesized that differential DNA methylation could predict the response of TNBCs to atRA. We identified over 1400 sites that were differentially methylated between atRA resistant and sensitive cell lines. These CpG sites predicted the response of four TNBC patient-derived xenografts to atRA, and we utilized these xenografts to refine the profile and identified that as many as 17% of TNBC patients could benefit from atRA treatment. These data illustrate that differential methylation of specific CpGs may be useful biomarkers for predicting the response of patient tumors to atRA treatment.