Differentiation of Cancer Stem Cells by Using Synthetic Small Molecules: Toward New Therapeutic Strategies against Therapy Resistance

Despite the existing arsenal of anti-cancer drugs, 10 million people die each year worldwide due to cancers; this highlights the need to discover new therapies based on innovative modes of action against these pathologies. Current chemotherapies are based on the use of cytotoxic agents, targeted drugs, monoclonal antibodies or immunotherapies that are able to reduce or stop the proliferation of cancer cells. However, tumor eradication is often hampered by the presence of resistant cells called cancer stem-like cells or cancer stem cells (CSCs). Several strategies have been proposed to specifically target CSCs such as the use of CSC-specific antibodies, small molecules able to target CSC signaling pathways or drugs able to induce CSC differentiation rendering them sensitive to classical chemotherapy. These latter compounds are the focus of the present review, which aims to report recent advances in anticancer-differentiation strategies. This therapeutic approach was shown to be particularly promising for eradicating tumors in which CSCs are the main reason for therapeutic failure. This general view of the chemistry and mechanism of action of compounds inducing the differentiation of CSCs could be particularly useful for a broad range of researchers working in the field of anticancer therapies as the combination of compounds that induce differentiation with classical chemotherapy could represent a successful approach for future therapeutic applications.     

Keratin Profiling by Single-Cell RNA-Sequencing Identifies Human Prostate Stem Cell Lineage Hierarchy and Cancer Stem-Like Cells

Single prostate stem cells can generate stem and progenitor cells to form prostaspheres in 3D culture. Using a prostasphere-based label retention assay, we recently identified keratin 13 (KRT13)-enriched prostate stem cells at single-cell resolution, distinguishing them from daughter progenitors. Herein, we characterized the epithelial cell lineage hierarchy in prostaspheres using single-cell RNA-seq analysis. Keratin profiling revealed three clusters of label-retaining prostate stem cells; cluster I represents quiescent stem cells (PSCA, CD36, SPINK1, and KRT13/23/80/78/4 enriched), while clusters II and III represent active stem and bipotent progenitor cells (KRT16/17/6 enriched). Gene set enrichment analysis revealed enrichment of stem and cancer-related pathways in cluster I. In non-label-retaining daughter progenitor cells, three clusters were identified; cluster IV represents basal progenitors (KRT5/14/6/16 enriched), while clusters V and VI represent early and late-stage luminal progenitors, respectively (KRT8/18/10 enriched). Furthermore, MetaCore analysis showed enrichment of the “cytoskeleton remodeling–keratin filaments” pathway in cancer stem-like cells from human prostate cancer specimens. Along with common keratins (KRT13/23/80/78/4) in normal stem cells, unique keratins (KRT10/19/6C/16) were enriched in cancer stem-like cells. Clarification of these keratin profiles in human prostate stem cell lineage hierarchy and cancer stem-like cells can facilitate the identification and therapeutic targeting of prostate cancer stem-like cells.     

Analysis of Circulating Tumor and Cancer Stem Cells Provides New Opportunities in Diagnosis and Treatment of Small Cell Lung Cancer

Current methods for diagnosis and treatment of small cell lung cancer (SCLC) have only a modest efficacy. In this pilot study, we analyzed circulating tumor cells (CTCs) and cancer stem cells (CSCs) in patients with SCLC to search for new diagnostic and prognostic markers and novel approaches to improve the treatment of the disease. In other forms of lung cancer, we showed a heterogeneity of blood CTCs and CSCs populations, as well as changes in other cell populations (ALDH⁺, CD87⁺CD276⁺, and EGF⁺Axl⁺) in smokers. A number of CTCs and CSCs in patients with SCLC have been shown to be resistant to chemotherapy (CT). High cytotoxic activity and resistance to apoptosis of reprogrammed CD3⁺CD8⁺ T-lymphocytes (rTcells) in relation to naive CD3⁺CD8⁺ T-lymphocytes was demonstrated in a smoking patient with SCLC (Patient G) in vitro. The target for rTcells was patient G’s blood CSCs. Reprogramming of CD3⁺CD8⁺ T-lymphocytes was carried out with the MEK1/2 inhibitor and PD-1/PD-L1 pathway blocker nivolumab. The training procedure was performed with a suspension of dead CTCs and CSCs obtained from patient’s G blood. The presented data show a new avenue for personalized SCLC diagnosis and targeted improvement of chemotherapy based on the use of both CTCs and CSCs.     

NK Cells Lose Their Cytotoxicity Function against Cancer Stem Cell-Rich Radiotherapy-Resistant Breast Cancer Cell Populations

Cancer stem cells (CSCs) can be induced from differentiated cancer cells in the tumor microenvironment or in response to treatments and exhibit chemo- and radioresistance, leading to tumor recurrence and metastasis. We previously reported that triple negative breast cancer (TNBC) cells with acquired radioresistance exhibited more aggressive features due to an increased CSC population. Therefore, here, we isolated CSCs from radiotherapy-resistant (RT-R)-TNBC cells and investigated the effects of these CSCs on tumor progression and NK cell-mediated cytotoxicity. Compared to MDA-MB-231 and RT-R-MDA-MB-231 cells, CD24^−/low/CD44⁺ cells isolated from RT-R-MDA-MB-231 cells showed increased proliferation, migration and invasion abilities, and induced expression of tumor progression-related molecules. Moreover, similar to MDA-MB-231 cells, CD24^−/low/CD44⁺ cells recruited NK cells but suppressed NK cell cytotoxicity by regulating ligands for NK cell activation. In an in vivo model, CD24^−/low/CD44⁺ cell-injected mice showed enhanced tumor progression and lung metastasis via upregulation of tumor progression-related molecules and altered host immune responses. Specifically, NK cells were recruited into the peritumoral area tumor but lost their cytotoxicity due to the altered expression of activating and inhibitory ligands on tumors. These results suggest that CSCs may cause tumor evasion of immune cells, resulting in tumor progression.     

Cancer Stem Cells in Ovarian Cancer—A Source of Tumor Success and a Challenging Target for Novel Therapies

Ovarian cancer is the most lethal neoplasm of the female genital organs. Despite indisputable progress in the treatment of ovarian cancer, the problems of chemo-resistance and recurrent disease are the main obstacles for successful therapy. One of the main reasons for this is the presence of a specific cell population of cancer stem cells. The aim of this review is to show the most contemporary knowledge concerning the biology of ovarian cancer stem cells (OCSCs) and their impact on chemo-resistance and prognosis in ovarian cancer patients, as well as to present the treatment options targeted exclusively on the OCSCs. The review presents data concerning the role of cancer stem cells in general and then concentrates on OCSCs. The surface and intracellular OCSCs markers and their meaning both for cancer biology and clinical prognosis, signaling pathways specifically activated in OCSCs, the genetic and epigenetic regulation of OCSCs function including the recent studies on the non-coding RNA regulation, cooperation between OCSCs and the tumor microenvironment (ovarian cancer niche) including very specific environment such as ascites fluid, the role of shear stress, autophagy and metabolic changes for the function of OCSCs, and finally mechanisms of OCSCs escape from immune surveillance, are described and discussed extensively. The possibilities of anti-OCSCs therapy both in experimental settings and in clinical trials are presented, including the recent II phase clinical trials and immunotherapy. OCSCs are a unique population of cancer cells showing a great plasticity, self-renewal potential and resistance against anti-cancer treatment. They are responsible for the progression and recurrence of the tumor. Several completed and ongoing clinical trials have tested different anti-OCSCs drugs which, however, have shown unsatisfactory efficacy in most cases. We propose a novel approach to ovarian cancer diagnosis and therapy.     

Cisplatin-Resistant CD44+ Lung Cancer Cells Are Sensitive to Auger Electrons

Cancer stem cells (CSCs) are resistant to conventional therapy and present a major clinical challenge since they are responsible for the relapse of many cancers, including non-small cell lung cancer (NSCLC). Hence, future successful therapy should also eradicate CSCs. Auger electrons have demonstrated promising therapeutic potential and can induce DNA damage while sparing surrounding cells. Here, we sort primary patient-derived NSCLC cells based on their expression of the CSC-marker CD44 and investigate the effects of cisplatin and a thymidine analog (deoxyuridine) labeled with an Auger electron emitter (¹²⁵I). We show that the CD44⁺ populations are more resistant to cisplatin than the CD44⁻ populations. Interestingly, incubation with the thymidine analog 5-[¹²⁵I]iodo-2′-deoxyuridine ([¹²⁵I]I-UdR) induces equal DNA damage, G2/M cell cycle arrest, and apoptosis in the CD44⁻ and CD44⁺ populations. Our results suggest that Auger electron emitters can also eradicate resistant lung cancer CD44⁺ populations.     

G Protein-Coupled Receptor Signaling in Stem Cells and Cancer

G protein-coupled receptors (GPCRs) are a large superfamily of cell-surface signaling proteins that bind extracellular ligands and transduce signals into cells via heterotrimeric G proteins. GPCRs are highly tractable drug targets. Aberrant expression of GPCRs and G proteins has been observed in various cancers and their importance in cancer stem cells has begun to be appreciated. We have recently reported essential roles for G protein-coupled receptor 84 (GPR84) and G protein subunit Gα_q in the maintenance of cancer stem cells in acute myeloid leukemia. This review will discuss how GPCRs and G proteins regulate stem cells with a focus on cancer stem cells, as well as their implications for the development of novel targeted cancer therapies.     

Molecular Effectors of Photodynamic Therapy-Mediated Resistance to Cancer Cells

Photodynamic therapy (PDT) is currently enjoying considerable attention as the subject of experimental research to treat resistant cancers. The preferential accumulation of a non-toxic photosensitizer (PS) in different cellular organelles that causes oxidative damage by combining light and molecular oxygen leads to selective cell killing. However, one major setback, common among other treatment approaches, is tumor relapse and the development of resistance causing treatment failure. PDT-mediated resistance could result from increased drug efflux and decreased localization of PS, reduced light exposure, increased DNA damage repair, and altered expression of survival genes. This review highlights the essential insights of PDT reports in which PDT resistance was observed and which identified some of the molecular effectors that facilitate the development of PDT resistance. We also discuss different perceptions of PDT and how its current limitations can be overturned to design improved cancer resistant treatments.     

Epigenetic Regulation of Breast Cancer Stem Cells Contributing to Carcinogenesis and Therapeutic Implications

Globally, breast cancer has remained the most commonly diagnosed cancer and the leading cause of cancer death among women. Breast cancer is a highly heterogeneous and phenotypically diverse group of diseases, which require different selection of treatments. Breast cancer stem cells (BCSCs), a small subset of cancer cells with stem cell-like properties, play essential roles in breast cancer progression, recurrence, metastasis, chemoresistance and treatments. Epigenetics is defined as inheritable changes in gene expression without alteration in DNA sequence. Epigenetic regulation includes DNA methylation and demethylation, as well as histone modifications. Aberrant epigenetic regulation results in carcinogenesis. In this review, the mechanism of epigenetic regulation involved in carcinogenesis, therapeutic resistance and metastasis of BCSCs will be discussed, and finally, the therapies targeting these biomarkers will be presented.     

Phytochemicals as Innovative Therapeutic Tools against Cancer Stem Cells

The theory that several carcinogenetic processes are initiated and sustained by cancer stem cells (CSCs) has been validated, and specific methods to identify the CSCs in the entire population of cancer cells have also proven to be effective. This review aims to provide an overview of recently acquired scientific knowledge regarding phytochemicals and herbal extracts, which have been shown to be able to target and kill CSCs. Many genes and proteins that sustain the CSCs’ self-renewal capacity and drug resistance have been described and applications of phytochemicals able to interfere with these signaling systems have been shown to be operatively efficient both in vitro and in vivo. Identification of specific surface antigens, mammosphere formation assays, serial colony-forming unit assays, xenograft transplantation and label-retention assays coupled with Aldehyde dehydrogenase 1 (ALDH1) activity evaluation are the most frequently used techniques for measuring phytochemical efficiency in killing CSCs. Moreover, it has been demonstrated that EGCG, curcumin, piperine, sulforaphane, β-carotene, genistein and the whole extract of some plants are able to kill CSCs. Most of these phytochemicals act by interfering with the canonical Wnt (β-catenin/T cell factor-lymphoid enhancer factor (TCF-LEF)) pathway implicated in the pathogenesis of several cancers. Therefore, the use of phytochemicals may be a true therapeutic strategy for eradicating cancer through the elimination of CSCs.     

Therapeutic Targeting of Glutaminolysis as a Novel Strategy to Combat Cancer Stem Cells

Rare subpopulations of cancer stem cells (CSCs) have the ability to self-renew and are the primary driving force behind cancer metastatic dissemination and the preeminent hurdle to cancer treatment. As opposed to differentiated, non-malignant tumor offspring, CSCs have sophisticated metabolic patterns that, depending on the kind of cancer, rely mostly on the oxidation of major fuel substrates such as glucose, glutamine, and fatty acids for survival. Glutaminolysis is a series of metabolic reactions that convert glutamine to glutamate and, eventually, α-ketoglutarate, an intermediate in the tricarboxylic acid (TCA) cycle that provides biosynthetic building blocks. These building blocks are mostly utilized in the synthesis of macromolecules and antioxidants for redox homeostasis. A recent study revealed the cellular and molecular interconnections between glutamine and cancer stemness in the cell. Researchers have increasingly focused on glutamine catabolism in their attempt to discover an effective therapy for cancer stem cells. Targeting catalytic enzymes in glutaminolysis, such as glutaminase (GLS), is achievable with small molecule inhibitors, some of which are in early-phase clinical trials and have promising safety profiles. This review summarizes the current findings in glutaminolysis of CSCs and focuses on novel cancer therapies that target glutaminolysis in CSCs.     

Targeting Lipocalin-2 in Inflammatory Breast Cancer Cells with Small Interference RNA and Small Molecule Inhibitors

Inflammatory Breast Cancer (IBC) is an aggressive form of invasive breast cancer, highly metastatic, representing 2–4% of all breast cancer cases in the United States. Despite its rare nature, IBC is responsible for 7–10% of all breast cancer deaths, with a 5-year survival rate of 40%. Thus, targeted and effective therapies against IBC are needed. Here, we proposed Lipocalin-2 (LCN2)—a secreted glycoprotein aberrantly abundant in different cancers—as a plausible target for IBC. In immunoblotting, we observed higher LCN2 protein levels in IBC cells than non-IBC cells, where the LCN2 levels were almost undetectable. We assessed the biological effects of targeting LCN2 in IBC cells with small interference RNAs (siRNAs) and small molecule inhibitors. siRNA-mediated LCN2 silencing in IBC cells significantly reduced cell proliferation, viability, migration, and invasion. Furthermore, LCN2 silencing promoted apoptosis and arrested the cell cycle progression in the G0/G1 to S phase transition. We used in silico analysis with a library of 25,000 compounds to identify potential LCN2 inhibitors, and four out of sixteen selected compounds significantly decreased cell proliferation, cell viability, and the AKT phosphorylation levels in SUM149 cells. Moreover, ectopically expressing LCN2 MCF7 cells, treated with two potential LCN2 inhibitors (ZINC00784494 and ZINC00640089) showed a significant decrease in cell proliferation. Our findings suggest LCN2 as a promising target for IBC treatment using siRNA and small molecule inhibitors.     

TRAIL and Paclitaxel Synergize to Kill U87 Cells and U87-Derived Stem-Like Cells in Vitro

U87-derived stem-like cells (U87-SLCs) were cultured using serum-free stem cell media and identified by both biological behaviors and markers. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and paclitaxel (PX), in combination or alone, was used to treat U87-MG human glioma cells (U87 cells) or U87-SLCs. The results showed that TRAIL/PX cannot only synergistically inhibit U87 cells but also U87-SLCs. We observed a significantly higher apoptotic rate in U87 cells simultaneously treated with TRAIL/PX for 24 h compared to cells treated with either drug alone. Furthermore, there was a remarkably higher apoptosis rate in U87-SLCs induced by the TRAIL/PX combination compared with either drug alone. Unlike the simultaneous treatment in U87 cells, U87-SLCs were pretreated for 24 h with 1 μmol/L of PX followed by 1000 ng/mL of TRAIL. Protein assays revealed that TRAIL/PX synergy was related to DR4, cleaved caspase-8 and cleaved caspase-3 upregulation, whereas the mitochondrial pathway was not involved in TRAIL-induced apoptosis. The present study indicates that PX can sensitize U87 cells and U87-SLCs to TRAIL treatment through an extrinsic pathway of cell apoptosis. The combined treatment of TRAIL and PX may be a promising glioma chemotherapy because of its successful inhibition of U87-SLCs, which are hypothesized to influence chemotherapeutic outcomes of gliomas.     

Cancer Stem Cells and Their Vesicles,Together with Other Stem and Non-Stem Cells,Govern Critical Cancer Processes: Perspectives for Medical Development

Stem cells, identified several decades ago, started to attract interest at the end of the nineties when families of mesenchymal stem cells (MSCs), concentrated in the stroma of most organs, were found to participate in the therapy of many diseases. In cancer, however, stem cells of high importance are specific to another family, the cancer stem cells (CSCs). This comprehensive review is focused on the role and the mechanisms of CSCs and of their specific extracellular vesicles (EVs), which are composed of both exosomes and ectosomes. Compared to non-stem (normal) cancer cells, CSCs exist in small populations that are preferentially distributed to the niches, such as minor specific tissue sites corresponding to the stroma of non-cancer tissues. At niches and marginal sites of other cancer masses, the tissue exhibits peculiar properties that are typical of the tumor microenvironment (TME) of cancers. The extracellular matrix (ECM) includes components different from non-cancer tissues. CSCs and their EVs, in addition to effects analogous to those of MSCs/EVs, participate in processes of key importance, specific to cancer: generation of distinct cell subtypes, proliferation, differentiation, progression, formation of metastases, immune and therapy resistance, cancer relapse. Many of these, and other, effects require CSC cooperation with surrounding cells, especially MSCs. Filtered non-cancer cells, especially macrophages and fibroblasts, contribute to collaborative cancer transition/integration processes. Therapy developments are mentioned as ongoing preclinical initiatives. The preliminary state of clinical medicine is presented in terms of both industrial development and future treatments. The latter will be administered to specific patients together with known drugs, with the aim of eradicating their tumor growth and metastases.     

In Ovarian Cancer Multicellular Spheroids,Platelet Releasate Promotes Growth,Expansion of ALDH+ and CD133+ Cancer Stem Cells,and Protection against the Cytotoxic Effects of Cisplatin,Carboplatin and Paclitaxel

A high platelet count is associated with a poor prognosis in ovarian cancer (OvCa). Despite good clinical responses with platinating agents in combination with taxanes, numerous OvCa patients relapse due to chemotherapy resistance. Here, we report that treatment of OvCa cells A2780, OVCAR5 and MDAH with releasate from activated platelets (PR) promoted multicellular tumor spheroid (MCTS) formation. These OvCa-MCTSs had increased percentages of CD133+ and aldehyde dehydrogenase (ALDH)+ cells, bona fide markers of OvCa cancer stem cells (CSCs). PR increased OVCAR5- and MDAH-MCTS viability and decreased the cytotoxic and pro-apoptotic effects of paclitaxel, cisplatin and carboplatin. PR increased the volume of spontaneously formed OVCAR8-MCTSs and counteracted their size reduction due to cisplatin, carboplatin and paclitaxel treatment. PR promoted the survival of ALDH+ and CD133+ OvCa cells during cisplatin, carboplatin and paclitaxel treatment. In conclusion, molecules and growth factors released by activated platelets (EGF, PDGF, TGF-β, IGF and CCL5) may protect tumor cells from chemotherapy by promoting the expansion of ALDH+ and CD133+ OvCa-CSCs, favoring drug resistance and tumor relapse.     

Cell Fusion of Mesenchymal Stem/Stromal Cells and Breast Cancer Cells Leads to the Formation of Hybrid Cells Exhibiting Diverse and Individual (Stem Cell) Characteristics

Cancer is one of the most common diseases worldwide, and treatment bears many challenges such as drug and radioresistance and formation of metastases. These difficulties are due to tumor heterogeneity, which has many origins. One may be cell fusion, a process that is relevant in both physiological (e.g., wound healing) and pathophysiological (cancer and viral infection) processes. In this study, we examined if cell fusion between mesenchymal stem/stromal cells (MSCs) and breast cancer (BC) cells occurs and if newly generated hybrid cells may exhibit cancer stem/initiating cell (CS/IC) characteristics. Therefore, several methods such as mammosphere assay, AldeRed assay, flow cytometry (CD24, CD44, CD104) and Western blot analysis (of epithelial to mesenchymal transition markers such as SNAIL, SLUG and Twist) were applied. In short, four different hybrid clones, verified by short tandem repeat (STR) analysis, were analyzed; each expressed an individual phenotype that seemed not to be explicitly related to either a more stem cell or cancer cell phenotype. These results show that cancer cells and MSCs are able to fuse spontaneously in vitro, thereby giving rise to hybrid cells with new properties, which likely indicate that cell fusion may be a trigger for tumor heterogeneity.