Dual Knockdown of Musashi RNA-Binding Proteins MSI-1 and MSI-2 Attenuates Putative Cancer Stem Cell Characteristics and Therapy Resistance in Ovarian Cancer Cells

In ovarian cancer, therapy resistance mechanisms complicate cancer cell eradication. Targeting Musashi RNA-binding proteins (MSI) may increase therapeutic efficacy. Database analyses were performed to identify gene expression associations between MSI proteins and key therapy resistance and cancer stem cell (CSC) genes. Then, ovarian cancer cells were subjected to siRNA-based dual knockdown of MSI-1 and MSI-2. CSC and cell cycle gene expression was investigated using quantitative polymerase chain reaction (qPCR), western blots, and flow cytometry. Metabolic activity and chemoresistance were assessed by MTT assay. Clonogenic assays were used to quantify cell survival post-irradiation. Database analyses demonstrated positive associations between MSI proteins and putative CSC markers NOTCH, MYC, and ALDH4A1 and negative associations with NOTCH inhibitor NUMB. MSI-2 expression was negatively associated with the apoptosis regulator p21. MSI-1 and MSI-2 were positively correlated, informing subsequent dual knockdown experiments. After MSI silencing, CSC genes were downregulated, while cell cycle progression was reduced. Metabolic activity was decreased in some cancer cells. Both chemo- and radioresistance were reduced after dual knockdown, suggesting therapeutic potential. Dual knockdown of MSI proteins is a promising venue to impede tumor growth and sensitize ovarian cancer cells to irradiation and chemotherapy.     

Effect of ALDH1A1 Gene Knockout on Drug Resistance in Paclitaxel and Topotecan Resistant Human Ovarian Cancer Cell Lines in 2D and 3D Model

Ovarian cancer is the most common cause of gynecological cancer death. Cancer Stem Cells (CSCs) characterized by drug transporters and extracellular matrix (ECM) molecules expression are responsible for drug resistance development. The goal of our study was to examine the role of aldehyde dehydrogenase 1A1 (ALDH1A1) expression in paclitaxel (PAC) and topotecan (TOP) resistant ovarian cancer cell lines. In both cell lines, we knocked out the ALDH1A1 gene using the CRISPR/Cas9 technique. Additionally, we derived an ALDH1A1 positive TOP-resistant cell line with ALDH1A1 expression in all cells via clonal selection. The effect of ALDH1A1 gene knockout or clonal selection on the expression of ALDH1A1, drug transporters (P-gp and BCRP), and ECM (COL3A1) was determined by Q-PCR, Western blot and immunofluorescence. Using MTT assay, we compared drug resistance in two-dimensional (2D) and three-dimensional (3D) cell culture conditions. We did not observe any effect of ALDH1A1 gene knockout on MDR1/P-gp expression and drug resistance in the PAC-resistant cell line. The knockout of ALDH1A1 in the TOP-resistant cell line resulted in a moderate decrease of BCRP and COL3A1 expression and weakened TOP resistance. The clonal selection of ALDH1A1 cells resulted in very strong downregulation of BCPR and COL3A1 expression and overexpression of MDR1/P-gp. This finally resulted in decreased resistance to TOP but increased resistance to PAC. All spheroids were more resistant than cells growing as monolayers, but the resistance mechanism differs. The spheroids’ resistance may result from the presence of cell zones with different proliferation paces, the density of the spheroid, ECM expression, and drug capacity to diffuse into the spheroid.     

Butylidenephthalide Abrogates the Snail-Induced Cancer Stemness in Oral Carcinomas

Oral cancer is one of the most common cancers worldwide, especially in South Central Asia. It has been suggested that cancer stem cells (CSC) play crucial roles in tumor relapse and metastasis, and approaches to target CSC may lead to promising results. Here, aldehyde dehydrogenase 1 (ALDH1) and CD44 were utilized to isolate CSCs of oral cancer. Butylidenephthalide, a bioactive phthalide compound from Angelica sinensis, was tested for its anti-CSC effects. MTT assay showed that a lower concentration of butylidenephthalide was sufficient to inhibit the proliferation of patient-derived ALDH1⁺/CD44⁺ cells without affecting normal cells. Administration of butylidenephthalide not only reduced ALDH1 activity and CD44 expression, it also suppressed the migration, invasion, and colony formation abilities of ALDH1⁺/CD44⁺ cells using a transwell system and clonogenic assay. A patient-derived xenograft mouse model supported our in vitro findings that butylidenephthalide possessed the capacity to retard tumor development. We found that butylidenephthalide dose-dependently downregulated the gene and protein expression of Sox2 and Snail. Our results demonstrated that overexpression of Snail in ALDH1^-/CD44^- (non-CSCs) cells induced the CSC phenotypes, whereas butylidenephthalide treatment successfully diminished the enhanced self-renewal and propagating properties. In summary, this study showed that butylidenephthalide may serve as an adjunctive for oral cancer therapy.     

microRNA-21 Regulates Stemness in Pancreatic Ductal Adenocarcinoma Cells

Pancreatic ductal adenocarcinoma (PDAC) is the most common and aggressive type of pancreatic cancer (PCa) with a low survival rate. microRNAs (miRs) are endogenous, non-coding RNAs that moderate numerous biological processes. miRs have been associated with the chemoresistance and metastasis of PDAC and the presence of a subpopulation of highly plastic “stem”-like cells within the tumor, known as cancer stem cells (CSCs). In this study, we investigated the role of miR-21, which is highly expressed in Panc-1 and MiaPaCa-2 PDAC cells in association with CSCs. Following miR-21 knockouts (KO) from both MiaPaCa-2 and Panc-1 cell lines, reversed expressions of epithelial–mesenchymal transition (EMT) and CSCs markers were observed. The expression patterns of key CSC markers, including CD44, CD133, CX-C chemokine receptor type 4 (CXCR4), and aldehyde dehydrogenase-1 (ALDH1), were changed depending on miR-21 status. miR-21 (KO) suppressed cellular invasion of Panc-1 and MiaPaCa-2 cells, as well as the cellular proliferation of MiaPaCa-2 cells. Our data suggest that miR-21 is involved in the stemness of PDAC cells, may play roles in mesenchymal transition, and that miR-21 poses as a novel, functional biomarker for PDAC aggressiveness.     

Tumour Stem Cells in Breast Cancer

Tumour stem cells (CSCs) are a self-renewing population that plays important roles in tumour initiation, recurrence, and metastasis. Although the medical literature is extensive, problems with CSC identification and cancer therapy remain. This review provides the main mechanisms of CSC action in breast cancer (BC): CSC markers and signalling pathways, heterogeneity, plasticity, and ecological behaviour. The dynamic heterogeneity of CSCs and the dynamic transitions of CSC⁻ non-CSCs and their significance for metastasis are considered.     

Gallotannin from Bouea macrophylla Seed Extract Suppresses Cancer Stem-like Cells and Radiosensitizes Head and Neck Cancer

Cancer stem cells (CSCs) play a critical role in radiation resistance and recurrence. Thus, drugs targeting CSCs can be combined with radiotherapy to improve its antitumor efficacy. Here, we investigated whether a gallotannin extract from Bouea macrophylla seed (MPSE) and its main bioactive compound, pentagalloyl glucose (PGG), could suppress the stemness trait and further confer the radiosensitivity of head and neck squamous cell carcinoma (HNSCC) cell lines. In this study, we evaluate the effect of MPSE or PGG to suppress CSC-like phenotypes and radiosensitization of HNSCC cell lines using a series of in vitro experiments, tumorsphere formation assay, colony formation assay, apoptosis assay, and Western blotting analysis. We demonstrate that MPSE or PGG is able to suppress tumorsphere formation and decrease protein expression of cancer stem cell markers. MPSE or PGG also enhanced the radiosensitivity in HNSCC cells. Pretreatment of cells with MPSE or PGG increased IR-induced DNA damage (γ-H2Ax) and enhanced radiation-induced cell death. Notably, we observed that pretreatment with MPSE or PGG attenuated the IR-induced stemness-like properties characterized by tumorsphere formation and the CD44 CSC marker. Our findings describe a novel strategy for increasing therapeutic efficacy for head and neck cancer patients using the natural products MPSE and PGG.     

Repeated Irradiation with γ-Ray Induces Cancer Stemness through TGF-β-DLX2 Signaling in the A549 Human Lung Cancer Cell Line

Cancer stem cells (CSCs) play an important role in cancer recurrence and metastasis. It is suggested that the CSC properties in heterogeneous cancer cells can be induced by ionizing radiation (IR). This study investigated the role of DLX2 in the radioresistance and CSC properties induced by IR in NSCLC cancer cells. Here, A549 cells were exposed to fractionated irradiation at a cumulative dose of 52 Gy (4 Gy × 13 times) for a generation of radioresistant cells. After fractionated irradiation, surviving A549 cells exhibited resistance to IR and enhanced expression of various cancer stem cell markers. They also showed upregulation of mesenchymal molecular markers and downregulation of epithelial molecular markers, correlating with an increase in the migration and invasion. Fractionated irradiation triggered the secretion of TGF-β1 and DLX2 expression. Interestingly, the increased DLX2 following fractionated irradiation seemed to induce the expression of the gene for the EGFR-ligand betacellulin via Smad2/3 signaling. To contrast, DLX2 knockdown dramatically decreased the expression of CSC markers, migration, and proliferation. Moreover, A549 cells expressing DLX2 shRNA formed tumors with a significantly smaller volume compared to those expressing control shDNA in a mouse xenograft assay. These results suggest that DLX2 overexpression in surviving NSCLC cancer cells after fractionated IR exposure is involved in the cancer stemness, radioresistance, EMT, tumor survival, and tumorigenic capability.     

FTY720 Inhibits Expansion of Breast Cancer Stem Cells via PP2A Activation

Growing evidence suggests that breast cancer originates from a minor population of cancer cells termed cancer stem cells (CSCs), which can be identified by aldehyde dehydrogenase (ALDH) activity-based flow cytometry analysis. However, novel therapeutic drugs for the eradication of CSCs have not been discovered yet. Recently, drug repositioning, which finds new medical uses from existing drugs, has been expected to facilitate drug discovery. We have previously reported that sphingosine kinase 1 (SphK1) induced proliferation of breast CSCs. In the present study, we focused on the immunosuppressive agent FTY720 (also known as fingolimod or Gilenya), since FTY720 is known to be an inhibitor of SphK1. We found that FTY720 blocked both proliferation of ALDH-positive cells and formation of mammospheres. In addition, we showed that FTY720 reduced the expression of stem cell markers such as Oct3/4, Sox2 and Nanog via upregulation of protein phosphatase 2A (PP2A). These results suggest that FTY720 is an effective drug for breast CSCs in vitro.     

MicroRNA Regulation of Breast Cancer Stemness

Recent advances in our understanding of breast cancer have demonstrated that cancer stem-like cells (CSCs, also known as tumor-initiating cell (TICs)) are central for progression and recurrence. CSCs are a small subpopulation of cells present in breast tumors that contribute to growth, metastasis, therapy resistance, and recurrence, leading to poor clinical outcome. Data have shown that cancer cells can gain characteristics of CSCs, or stemness, through alterations in key signaling pathways. The dysregulation of miRNA expression and signaling have been well-documented in cancer, and recent studies have shown that miRNAs are associated with breast cancer initiation, progression, and recurrence through regulating CSC characteristics. More specifically, miRNAs directly target central signaling nodes within pathways that can drive the formation, maintenance, and even inhibition of the CSC population. This review aims to summarize these research findings specifically in the context of breast cancer. This review also discusses miRNAs as biomarkers and promising clinical therapeutics, and presents a comprehensive summary of currently validated targets involved in CSC-specific signaling pathways in breast cancer.     

Sasa quelpaertensis Leaf Extract Inhibits Colon Cancer by Regulating Cancer Cell Stemness in Vitro and in Vivo

A rare subpopulation of cancer cells, termed cancer stem cells (CSCs), may be responsible for tumor relapse and resistance to conventional chemotherapy. The development of a non-toxic, natural treatment for the elimination of CSCs is considered a strategy for cancer treatment with minimal side effects. In the present study, the potential for Sasa quelpaertensis leaf extract (SQE) and its two bioactive compounds, tricin and p-coumaric acid, to exert anti-CSC effects by suppressing cancer stemness characteristics were evaluated in colon cancer cells. CD133⁺CD44⁺ cells were isolated from HT29 and HCT116 cell lines using flow-activated cell sorting (FACs). SQE treatment was found to significantly suppress the self-renewal capacity of both cell lines. SQE treatment was also associated with the down-regulation of β-catenin and phosphorylated GSK3β, while significantly enhancing cell differentiation by up-regulating CK20 expression and blocking the expression of several stem cell markers, including DLK1, Notch1, and Sox-2. In vivo, SQE supplementation suppressed tumor growth in a xenograft model by down-regulating stem cell markers and β-catenin as well as HIF-1α signaling. Compared with two bioactive compounds of SQE, SQE exhibited the most effective anti-CSC properties. Taken together, these results provide evidence that SQE inhibits colon cancer by regulating the characteristics of CSCs.     

The Chick Chorioallantoic Membrane Model: A New In Vivo Tool to Evaluate Breast Cancer Stem Cell Activity

The high plasticity of cancer stem-like cells (CSCs) allows them to differentiate and proliferate, specifically when xenotransplanted subcutaneously into immunocompromised mice. CSCs are highly tumorigenic, even when inoculated in small numbers. Thus, in vivo limiting dilution assays (LDA) in mice are the current gold standard method to evaluate CSC enrichment and activity. The chick embryo chorioallantoic membrane (CAM) is a low cost, naturally immune-incompetent and reproducible model widely used to evaluate the spontaneous growth of human tumor cells. Here, we established a CAM-LDA assay able to rapidly reproduce tumor specificities—in particular, the ability of the small population of CSCs to form tumors. We used a panel of organotropic metastatic breast cancer cells, which show an enrichment in a stem cell gene signature, enhanced CD44⁺/CD24^−/low cell surface expression and increased mammosphere-forming efficiency (MFE). The size of CAM-xenografted tumors correlate with the number of inoculated cancer cells, following mice xenograft growth pattern. CAM and mice tumors are histologically comparable, displaying both breast CSC markers CD44 and CD49f. Therefore, we propose a new tool for studying CSC prevalence and function—the chick CAM-LDA—a model with easy handling, accessibility, rapid growth and the absence of ethical and regulatory constraints.     

Selective Anti-Cancer Effects of Plasma-Activated Medium and Its High Efficacy with Cisplatin on Hepatocellular Carcinoma with Cancer Stem Cell Characteristics

Hepatocellular carcinoma (HCC) is a major histological subtype of primary liver cancer. Ample evidence suggests that the pathological properties of HCC originate from hepatic cancer stem cells (CSCs), which are responsible for carcinogenesis, recurrence, and drug resistance. Cold atmospheric-pressure plasma (CAP) and plasma-activated medium (PAM) induce apoptosis in cancer cells and represent novel and powerful anti-cancer agents. This study aimed to determine the anti-cancer effect of CAP and PAM in HCC cell lines with CSC characteristics. We showed that the air-based CAP and PAM selectively induced cell death in Hep3B and Huh7 cells with CSC characteristics, but not in the normal liver cell line, MIHA. We observed both caspase-dependent and -independent cell death in the PAM-treated HCC cell lines. Moreover, we determined whether combinatorial PAM therapy with various anti-cancer agents have an additive effect on cell death in Huh7. We found that PAM highly increased the efficacy of the chemotherapeutic agent, cisplatin, while enhanced the anti-cancer effect of doxorubicin and the targeted-therapy drugs, trametinib and sorafenib to a lesser extent. These findings support the application of CAP and PAM as anti-cancer agents to induce selective cell death in cancers containing CSCs, suggesting that the combinatorial use of PAM and some specific anti-cancer agents is complemented mechanistically.     

AMBRA1 Negatively Regulates the Function of ALDH1B1, a Cancer Stem Cell Marker,by Controlling Its Ubiquitination

Activating molecule in Beclin-1-regulated autophagy (AMBRA1), a negative regulator of tumorigenesis, is a substrate receptor of the ubiquitin conjugation system. ALDH1B1, an aldehyde dehydrogenase, is a cancer stem cell (CSC) marker that is required for carcinogenesis via upregulation of the β-catenin pathway. Although accumulating evidence suggests a role for ubiquitination in the regulation of CSC markers, the ubiquitination-mediated regulation of ALDH1B1 has not been unraveled. While proteome analysis has suggested that AMBRA1 and ALDH1B1 can interact, their interaction has not been validated. Here, we show that AMBRA1 is a negative regulator of ALDH1B1. The expression of ALDH1B1-regulated genes, including PTEN, CTNNB1 (β-catenin), and CSC-related β-catenin target genes, is inversely regulated by AMBRA1, suggesting a negative regulatory role of AMBRA1 in the expression of ALDH1B1-regulated genes. We found that the K27- and K33-linked ubiquitination of ALDH1B1 is mediated via the cooperation of AMBRA1 with other E3 ligases, such as TRAF6. Importantly, ubiquitination site mapping revealed that K506, K511, and K515 are important for the K27-linked ubiquitination of ALDH1B1, while K33-linked ubiquitination occurs at K506. A ubiquitination-defective mutant of ALDH1B1 increased the self-association ability of ALDH1B1, suggesting a negative correlation between the ubiquitination and self-association of ALDH1B1. Together, our findings indicate that ALDH1B1 is negatively regulated by AMBRA1-mediated noncanonical ubiquitination.     

Gene Expression Analyses Support Fallopian Tube Epithelium as the Cell of Origin of Epithelial Ovarian Cancer

Folate receptor alpha (FOLR1/FRA) is reported to be overexpressed in epithelial ovarian cancers (EOC), especially the serous histotype. Further, while dysregulation of the folate-dependent 1-carbon cycle has been implicated in tumorogenesis, little is known relative to the potential mechanism of action of FOLR1 expression in these processes. We therefore investigated the expression of FOLR1, other folate receptors, and genes within the 1-carbon cycle in samples of EOC, normal ovary and fallopian tube on a custom TaqMan Low Density Array. Also included on this array were known markers of EOC such as MSLN, MUC16 and HE4. While few differences were observed in the expression profiles of genes in the 1-carbon cycle, genes previously considered to be overexpressed in EOC (e.g., FOLR1, MSLN, MUC16 and HE4) showed significantly increased expression when comparing EOC to normal ovary. However, when the comparator was changed to normal fallopian tube, these differences were abolished, supporting the hypothesis that EOC derives from fallopian fimbriae and, further, that markers previously considered to be upregulated or overexpressed in EOC are most likely not of ovarian origin, but fallopian in derivation. Our findings therefore support the hypothesis that the cell of origin of EOC is tubal epithelium.     

Host Mesothelin Expression Increases Ovarian Cancer Metastasis in the Peritoneal Microenvironment

Mesothelin (MSLN), a glycoprotein normally expressed by mesothelial cells, is overexpressed in ovarian cancer (OvCa) suggesting a role in tumor progression, although the biological function is not fully understood. OvCa has a high mortality rate due to diagnosis at advanced stage disease with intraperitoneal metastasis. Tumor cells detach from the primary tumor as single cells or multicellular aggregates (MCAs) and attach to the mesothelium of organs within the peritoneal cavity producing widely disseminated secondary lesions. To investigate the role of host MSLN in the peritoneal cavity we used a mouse model with a null mutation in the MSLN gene (MSLN^KO). The deletion of host MSLN expression modified the peritoneal ultrastructure resulting in abnormal mesothelial cell surface architecture and altered omental collagen fibril organization. Co-culture of murine OvCa cells with primary mesothelial cells regardless of MSLN expression formed compact MCAs. However, co-culture with MSLN^KO mesothelial cells resulted in smaller MCAs. An allograft tumor study, using wild-type mice (MSLN^WT) or MSLN^KO mice injected intraperitoneally with murine OvCa cells demonstrated a significant decrease in peritoneal metastatic tumor burden in MSLN^KO mice compared to MSLN^WT mice. Together, these data support a role for host MSLN in the progression of OvCa metastasis.     

Vitamin D Analogs Regulate the Vitamin D System and Cell Viability in Ovarian Cancer Cells

Background: Ovarian cancer (OC) is one of the most lethal cancers in women. The active form of vitamin D₃, 1,25-dihydroxyvitamin D₃ (1,25D₃, calcitriol) has anticancer activity in several cancers, including ovarian cancer, but the required pharmacological doses may cause hypercalcemia. We hypothesized that newly developed, low calcemic, vitamin D analogs (an1,25Ds) may be used as anticancer agents instead of calcitriol in ovarian cancer cells. Methods: We used two patient-derived high-grade serous ovarian cancer (HGSOC) cell lines with low (13781) and high (14433) mRNA expression levels of the gene encoding 1,25-dihydroxyvitamin D₃ 24-hydroxylase CYP24A1, one of the main target genes of calcitriol. We tested the effect of calcitriol and four structurally related series of an1,25Ds (PRI-1906, PRI-1907, PRI-5201, PRI-5202) on cell number, viability, the expression of CYP24A1, and the vitamin D receptor (VDR). Results: CYP24A1 mRNA expression increased in a concentration-dependent manner after treatment with all compounds. In both cell lines, after 4 h, PRI-5202 was the most potent analog (in 13781 cells: EC₅₀ = 2.98 ± 1.10 nmol/L, in 14433 cells: EC₅₀ = 0.92 ± 0.20 nmol/L), while PRI-1907 was the least active one (in 13781 cells: EC₅₀ = n/d, in 14433 cells: EC₅₀ = n/d). This difference among the analogs disappeared after 5 days of treatment. The 13781 cells were more sensitive to the an1,25Ds compared with 14433 cells. The an1,25Ds increased nuclear VDR levels and reduced cell viability, but only in the 13781 cell line. Conclusions: The an1,25Ds had different potencies in the HGSOC cell lines and their efficacy in increasing CYP24A1 expression was cell line- and chemical structure-dependent. Therefore, choosing sensitive cancer cell lines and further optimization of the analogs’ structure might lead to new treatment options against ovarian cancer.