Melatonin regulates cancer migration and stemness and enhances the anti-tumour effect of cisplatin

Melatonin, a lipophilic hormone released from the pineal gland, has oncostatic effects on various types of cancers. However, its cancer treatment potential needs to be improved by deciphering its corresponding mechanisms of action and optimising therapeutic strategy. In the present study, melatonin inhibited gastric cancer cell migration and soft agar colony formation. Magnetic-activated cell sorting was applied to isolate CD133⁺ cancer stem cells. Gene expression analysis showed that melatonin lowered the upregulation of LC3-II expression in CD133⁺ cells compared to CD133⁻ cells. Several long non-coding RNAs and many components in the canonical Wnt signalling pathway were altered in melatonin-treated cells. In addition, knockdown of long non-coding RNA H19 enhanced the expression of pro-apoptotic genes, Bax and Bak, induced by melatonin treatment. Combinatorial treatment with melatonin and cisplatin was investigated to improve the applicability of melatonin as an anticancer therapy. Combinatorial treatment increased the apoptosis rate and induced G0/G1 cell cycle arrest. Melatonin can regulate migration and stemness in gastric cancer cells by modifying many signalling pathways. Combinatorial treatment with melatonin and cisplatin has the potential to improve the therapeutic efficacy of both.     

Uncoupling Warburg effect and stemness in CD133<Superscript>+ve</Superscript> cancer stem cells from Saos-2 (osteosarcoma) cell line under hypoxia

Cancer stem cells (CSCs) which are known to be residing deep inside the core of the tumor in its hypoxia niche is responsible for relapse of cancers. Owing to this hypoxic niche, the residing CSCs simultaneously fuel their stemness, cancerous and drug resistance properties. Attributes of CSCs are still not properly understood in its hypoxia niche. Addressing this, we sorted CSCs from Saos-2 (osteosarcoma) cell line using CD133 antibody. The CD133^+ve CSCs exhibited quiescent cell proliferation in DNA doubling, Ca²⁺ signaling and cell cycle analysis. CD133^+ve CSCs exhibited increased production of ATP and lactate dehydrogenase (LDH) activity under hypoxia. CD133^+ve cells exhibited decreased glucose uptake compared to ATP levels under hypoxia. Moreover, there was only negligible LDH activity in CD133^+ve cells under normoxia which do not rely on Warburg effect. Stemness markers (such as c-Myc, SOX2, Oct4 and TERT), metastasis marker (CD44) and drug resistance marker (ABCG2) were highly expressed in CD133^+ve cells. In summary, both CD133^+ve/?ve cells of Saos-2 (osteosarcoma) cell line did not exhibit Warburg effect under normoxic condition. Moreover, this significantly indicates an uncoupling between stemness and Warburg effect in CD133^+ve. This work provides a novel insight into the metabolic and functional features of CSCs in a hypoxic environment which could open new avenues for therapeutic strategies aimed to target CSCs.     

Development and characterization of cancer stem cell-based tumoroids as an osteosarcoma model

Three-dimensional (3D) cancer tumor models are becoming vital approaches for high-throughput drug screening, drug targeting, development of novel theranostic systems, and personalized medicine. Yet, it is becoming more evident that the tumor progression and metastasis is fueled by a subpopulation of stem-like cells within the tumor that are also called cancer stem cells (CSCs). This study aimed to develop a tumoroid model using CSCs. For this purpose CD133⁺ cells were isolated from SaOS-2 osteosarcoma cell line with magnetic-activated cell sorting. To evaluate tumoroid formation ability, the cells were incubated in different cell numbers in agar gels produced by 3D Petri Dish® method. Subsequently, CD133⁺ cells and CD133⁻ cells were co-cultured to investigate CD133⁺ cell localization in tumoroids. The characterization of tumoroids was performed using Live&Dead staining, immunohistochemistry, and quantitative polymerase chain reaction analysis. The results showed that, CD133⁺, CD133⁻ and SaOS-2 cells were all able to form 3D tumoroids regardless of the initial cell number, but, while 72 hr were needed for CD133⁺ cells to self-assemble, 24 hr were enough for CD133⁻ and SaOS-2 cells. CD133⁺ cells were located within tumoroids randomly with high cell viability. Finally, when compared to two-dimensional (2D) cultures, there were 5.88, 4.14, 6.95, and 1.68-fold higher messenger RNA expressions for Sox2, OCT3/4, Nanog, and Nestin, respectively, in CD133⁺ cells that were cultured within 3D tumoroids, showing longer maintenance of stem cell phenotype in 3D, that can allow more relevant screening and targeting efficiency in pharmaceutical testing. It was concluded that CSC-based tumoroids are propitious as 3D tumor models to fill the gap between conventional 2D in vitro culture and in vivo animal experiments for cancer research.     

Self-renewal and circulating capacities of metastatic hepatocarcinoma cells required for collaboration between TM4SF5 and CD44

Tumor metastasis involves circulating and tumor-initiating capacities of metastatic cancer cells. Hepatic TM4SF5 promotes EMT for malignant growth and migration. Hepatocellular carcinoma (HCC) biomarkers remain unexplored for metastatic potential throughout metastasis. Here, novel TM4SF5/CD44 interaction-mediated self-renewal and circulating tumor cell (CTC) capacities were mechanistically explored. TM4SF5-dependent sphere growth was correlated with CD133⁺, CD24^-, ALDH activity, and a physical association between CD44 and TM4SF5. The TM4SF5/CD44 interaction activated c-Src/STAT3/ Twist1/ B mi1 signaling for spheroid formation, while disturbing the interaction, expression, or activity of any component in this signaling pathway inhibited spheroid formation. In serial xenografts of less than 5,000 cells/injection, TM4SF5-positive tumors exhibited locally-increased CD44 expression, suggesting tumor cell differentiation. TM4SF5-positive cells were identified circulating in blood 4 to 6 weeks after orthotopic liver-injection. Anti-TM4SF reagents blocked their metastasis to distal intestinal organs. Altogether, our results provide evidence that TM4SF5 promotes self-renewal and CTC properties supported by CD133⁺/TM4SF5⁺/CD44^{+(TM4SF5-bound)}/ALDH⁺/ CD24^- markers during HCC metastasis. [BMB Reports 2015; 48(3): 127-128]     

The mechanisms underlying the enrichment and action of glypican-1-positive exosomes in colorectal cancer cells

BackgroundGlypican-1 (GPC1) is overexpressed in several tumors, and GPC1⁺ exosomes have shown the potential to predict early colorectal cancer (CRC). However, the mechanisms underlying the enrichment and action of GPC1⁺ exosomes in CRC remain unknown.MethodsThe expression of slit guidance ligand 2 (SLIT2), hypoxia-inducible factor (HIF)-1α/2α, and GPC1 in clinical CRC tissues was detected using immunohistochemistry and western blot. Exosomes were isolated from the supernatants of CRC cell cultures. The effects of SLIT2, hypoxia, heparin, and phospholipase C (PLC) on exosomal GPC1 expression and GPC1⁺ exosome enrichment in CRC cells were analyzed with western blot and flow cytometry. CRC cell proliferation was assessed with MTT and colony formation assays. Co-immunoprecipitation was used to detect the binding of GPC1 and SLIT2 in SW480 cells. Nude mice were subcutaneously inoculated with SW480 cells with different treatments. The Wnt signaling was detected.ResultsSLIT2 was poorly expressed and GPC1, HIF-1α, and HIF-2α were highly expressed in human CRC tissues. SLIT2 in CRC cells inhibited GPC1⁺ exosome enrichment and exosomal GPC1 expression. PLC and heparin increased GPC1⁺ exosome enrichment in CRC cells in a concentration-dependent manner. Hypoxia increased the enrichment of GPC1⁺ exosomes in CRC cells depending on HIF-2α expression. GPC1⁺ exosomes stimulated CRC cell proliferation and xenograft tumor growth through activation of Wnt signaling.ConclusionsGPC1⁺ exosome enrichment is related to PLC and heparin. Hypoxia increases the enrichment of GPC1⁺ exosomes in CRC cells by activating HIF-2α and downregulating SLIT2. GPC1⁺ exosomes further drive CRC progression by activating Wnt signaling.     

MicroRNA expression profile of colon cancer stem-like cells in HT29 adenocarcinoma cell line

Increasing evidence has suggested cancer stem cells (CSCs) are considered to be responsible for cancer formation, recurrence, and metastasis. Recently, many studies have also revealed that microRNAs (miRNAs) strongly implicate in regulating self renewal and tumorigenicity of CSCs in human cancers. However, with respect to colon cancer, the role of miRNAs in stemness maintenance and tumorigenicity of CSCs still remains to be unknown. In the present study, we isolated a population of colon CSCs expressing a CD133 surface phenotype from human HT29 colonic adenocarcinoma cell line by Flow Cytometry Cell Sorting. The CD133⁺ cells possess a greater tumor sphere-forming efficiency in vitro and higher tumorigenic potential in vivo. Furthermore, the CD133⁺ cells are endowed with stem/progenitor cells-like property including expression of “stemness” genes involved in Wnt2, BMI1, Oct3/4, Notch1, C-myc and other genes as well as self-renewal and differentiation capacity. Moreover, we investigated the miRNA expression profile of colon CSCs using miRNA array. Consequently, we identified a colon CSCs miRNA signature comprising 11 overexpressed and 8 underexpressed miRNAs, such as miR-429, miR-155, and miR-320d, some of which may be involved in regulation of stem cell differentiation. Our results suggest that miRNAs might play important roles in stemness maintenance of colon CSCs, and analysis of specific miRNA expression signatures may contribute to potential cancer therapy.     

The role of A-kinase interacting protein 1 in regulating progression and stemness as well as indicating the prognosis in glioblastoma

BackgroundA-kinase interacting protein 1 (AKIP1) is recently implicated in the pathogenesis of several solid tumors, while its role in glioblastoma multiforme (GBM) is largely unknown. Therefore, the current study aimed to investigate the effect of AKIP1 on GBM cell malignant behaviors, stemness, and its underlying molecular mechanisms.MethodsU-87 MG and A172 cells were transfected with control or AKIP1 overexpression plasmid; control or AKIP1 siRNA plasmid. Then cell proliferation, apoptosis, invasion, CD133⁺ cell proportion, and sphere formation assays were performed. Furthermore, RNA-Seq was performed in U-87 MG cells. Besides, AKIP1 expression was detected in 25 GBM and 25 low-grade glioma (LGG) tumor samples.ResultsAKIP1 was increased in several GBM cell lines compared to the control cell line. After transfections, it was found that AKIP1 overexpression increased cell invasion, CD133⁺ cell proportion, and sphere formation ability while less affecting cell proliferation or cell apoptosis in U-87 MG and A172 cells. Moreover, AKIP1 siRNA achieved the opposite effect in these cells, except that it inhibited cell proliferation but induced cell apoptosis to some extent. Subsequent RNA-Seq assay showed several critical carcinogenetic pathways, such as PI3K/AKT, Notch, EGFR tyrosine kinase inhibitor resistance, Ras, ErbB, mTOR pathways, etc. were potentially related to the function of AKIP1 in U-87 MG cells. Clinically, AKIP1 expression was higher in GBM tissues than in LGG tissues, which was also correlated with the poor prognosis of GBM to some degree.ConclusionsAKIP1 regulates the malignant behaviors and stemness of GBM via regulating multiple carcinogenetic pathways.     

MiR‐30‐5p suppresses cell chemoresistance and stemness in colorectal cancer through USP22/Wnt/β‐catenin signaling axis

Colorectal cancer (CRC) remains both common and fatal, and its successful treatment is greatly limited by the development of stem cell‐like characteristics (stemness) and chemoresistance. MiR‐30‐5p has been shown to function as a tumor suppressor by targeting the Wnt/β‐catenin signaling pathway, but its activity in CRC has never been assessed. We hypothesized that miR‐30‐5p exerts anti‐oncogenic effects in CRC by regulating the USP22/Wnt/β‐catenin signaling axis. In the present study, we demonstrate that tissues from CRC patients and human CRC cell lines show significantly decreased miR‐30‐5p family expression. After identifying the 3’UTR of USP22 as a potential binding site of miR‐30‐5p, we constructed a luciferase reporter containing the potential miR‐30‐5p binding site and measured the effects on USP22 expression. Western blot assays showed that miR‐30‐5p decreased USP22 protein expression in HEK293 and Caco2 CRC cells. To evaluate the effects of miR‐30‐5p on CRC cell stemness, we isolated CD133 + CRC cells (Caco2 and HCT15). We then determined that, while miR‐30‐5p is normally decreased in CD133 + CRC cells, miR‐30‐5p overexpression significantly reduces expression of stem cell markers CD133 and Sox2, sphere formation, and cell proliferation. Similarly, we found that miR‐30‐5p expression is normally reduced in 5‐fluorouracil (5‐FU) resistant CRC cells, whereas miR‐30‐5p overexpression in 5‐FU resistant cells reduces sphere formation and cell viability. Inhibition of miR‐30‐5p reversed the process. Finally, we determined that miR‐30‐5p attenuates the expression of Wnt/β‐catenin signaling target genes (Axin2 and MYC), Wnt luciferase activity, and β‐catenin protein levels in CRC stem cells.     

REC8 enhances stemness and promotes metastasis of colorectal cancer through BTK/Akt/β-catenin signaling pathway

Cancer/testis antigens (CTAs) are often aberrantly expressed in cancer stem cells (CSCs) which are responsible for tumor metastasis. Rec8 meiotic recombination protein (REC8), a member of CTAs, shares distinct roles in various cancers, while its contribution to CSCs and colorectal cancer (CRC) remains unclear. We found that overexpression of REC8 facilitated the migration and invasion of CRC cells (DLD-1 and SW480 cells) in vitro and promoted the liver metastasis of CRC in vivo. Moreover, REC8 is highly expressed in CRC stem-like cells and is required for the maintenance of CSC stemness. Mechanistic studies suggested that REC8 mediated through the activation of Bruton tyrosine kinase (BTK). Inhibition of BTK by ibrutinib not only suppressed the migration and invasion-promoting ability, but also declined the increased expression of p-BTK, p-Akt, β-catenin, and CSC markers upon REC8 overexpression. Importantly, high expression of REC8 in cancerous tissues was related to advanced clinical stage and lymph node metastasis of 62 CRC patients, and REC8 was enriched in the cancerous cells positive for CSC markers. Collectively, our results indicate that REC8 promotes CRC metastasis by increasing cell stemness through BTK/Akt/β-catenin pathway.     

microRNA-448 inhibits stemness maintenance and self-renewal of hepatocellular carcinoma stem cells through the MAGEA6-mediated AMPK signaling pathway

Hepatocellular carcinoma (HCC) occurs mainly in patients with chronic liver disease and cirrhosis. Increasing evidence has identified the involvement of microRNAs (miRNAs) acting as essential regulators in the progression of HCC. As predicted by microarray analysis, miR-448 might potentially affect HCC progression by regulating the melanoma-associated antigen (MAGEA). Therefore, the present investigation focused on exploring whether or not miR-448 and MAGEA6 were involved in the self-renewal and stemness maintenance of HCC stem cells. The interaction among miR-448, MAGEA6, and the AMPK signaling pathway was evaluated. It was noted that miR-448 targeted and downregulated MAGEA6, thus activating the AMP-activated protein kinase (AMPK) signaling pathway in HCC. Furthermore, for the purpose of exploring the functional relevance of MAGEA6 and miR-448 on the sphere formation, colony formation, and invasion and migration of HCC stem cells, the CD133⁺CD44 ⁺ HCC stem cells were sorted and treated with the mimic or inhibitor of miR-448, small interfering RNA (siRNA) against MAGEA6 or an AMPK activator AICAR. MAGEA6 silencing or miR-448 overexpression was demonstrated to inhibit the abilities of sphere formation, colony formation, cell migration, and invasion of HCC cells. Afterwards, a rescue experiment was conducted and revealed that MAGEA6 silencing reversed the effects of miR-448 inhibitor on stemness maintenance and self-renewal of HCC stem cells. Finally, after the in vivo experiment was carried out, miR-448 was observed to restrain the tumor formation and stemness in vivo. Altogether, miR-448 activates the AMPK signaling pathway by downregulating MAGEA6, thus inhibiting the stemness maintenance and self-renewal of HCC stem cells, which identifies miR-448 as a new therapeutic strategy for HCC.     

hsa_circ_0003222 accelerates stemness and progression of non-small cell lung cancer by sponging miR-527

The relationship between circular RNA (circRNA) and cancer stem cells (CSCs) is uncertain. We have investigated the combined influence of CSCs, circRNA (hsa_circ_0003222), and immune checkpoint inhibitors in NSCLC progression and therapy resistance. We constructed lung CSCs (LCSCs; PC9 and A549). The effects of hsa_circ_0003222 in vitro were determined by cell counting, colony and sphere formation, and Transwell assays. A tumor xenograft model of metastasis and orthotopic model were built for in vivo analysis. We found that hsa_circ_0003222 was highly expressed in NSCLC tissues and LCSCs. Higher levels of hsa_circ_0003222 were associated with the stage, metastasis, and survival rate of patients with NSCLC. Reduced levels of hsa_circ_0003222 decreased tumor cell proliferation, migration, invasion, stemness-like properties, and chemoresistance. The silencing of hsa_circ_0003222 was found to downregulate PHF21B expression and its downstream, β-catenin by relieving the sponging effect of miR-527. Moreover, silencing hsa_circ_0003222 alleviated NSCLC resistance to anti-programmed cell death-ligand 1 (PD-L1)-based therapy in vivo. Our data demonstrate the significant role of hsa_circ_0003222 in NSCLC cell stemness-like properties. The manipulation of circRNAs in combination with anti-PD-L1 therapy may alleviate NSCLC stemness and progression.Subject terms: Cancer microenvironment, Cancer stem cells     

Tumor exosome promotes Th17 cell differentiation by transmitting the lncRNA CRNDE-h in colorectal cancer

The T helper 17 (Th17) cells in tumor microenvironment play an important role in colorectal cancer (CRC) progression. This study investigated the mechanism of Th17 cell differentiation in CRC with a focus on the role of tumor exosome-transmitted long noncoding RNA (lncRNA). Exosomes were isolated from the CRC cells and serum of CRC patients. The role and mechanism of the lncRNA CRNDE-h transmitted by CRC exosomes in Th17 cell differentiation were assessed by using various molecular biological methods. The serum exosomal CRNDE-h level was positively correlated with the proportion of Th17 cells in the tumor-infiltrating T cells in CRC patients. CRC exosomes contained abundant CRNDE-h and transmitted them to CD4⁺ T cells to increase the Th17 cell proportion, RORγt expression, and IL-17 promoter activity. The underlying mechanism is that, CRNDE-h bound to the PPXY motif of RORγt and impeded the ubiquitination and degradation of RORγt by inhibiting its binding with the E3 ubiquitin ligase Itch. The in vivo experiments confirmed that the targeted silence of CRNDE-h in CD4⁺ T cells attenuated the CRC tumor growth in mice. The present findings demonstrated that the tumor exosome transmitted CRNDE-h promoted Th17 cell differentiation by inhibiting the Itch-mediated ubiquitination and degradation of RORγt in CRC, expanding our understanding of Th17 cell differentiation in CRC.Subject terms: Cancer, Cell biology     

Wnt/β-catenin Signaling Inhibitors suppress the Tumor-initiating properties of a CD44+CD133+ subpopulation of Caco-2 cells

Tumor-initiating cells or cancer stem cells are a subset of cancer cells that have tumorigenic potential in human cancer. Although several markers have been proposed to distinguish tumor-initiating cells from colorectal cancer cells, little is known about how this subpopulation contributes to tumorigenesis. Here, we characterized a tumor-initiating cell subpopulation from Caco-2 colorectal cancer cells. Based on the findings that Caco-2 cell subpopulations express different cell surface markers, we were able to discriminate three main fractions, CD44^-CD133^-, CD44^-CD133⁺, and CD44⁺CD133⁺ subsets, and characterized their biochemical and tumorigenic properties. Our results show that CD44⁺CD133⁺ cells possessed an unusual capacity to proliferate and could form tumors when transplanted into NSG mice. Additionally, primary tumors grown from CD44⁺CD133⁺ Caco-2 cells contained mixed populations of CD44⁺CD133⁺ and non-CD44⁺CD133⁺ Caco-2 cells, indicating that the full phenotypic heterogeneity of the parental Caco-2 cells was re-created. Notably, only the CD44⁺CD133⁺ subset of Caco-2-derived primary tumors had tumorigenic potential in NSG mice, and the tumor growth of CD44⁺CD133⁺ cells was faster in secondary xenografts than in primary transplants. Gene expression analysis revealed that the Wnt/β-catenin pathway was over-activated in CD44⁺CD133⁺ cells, and the growth and tumorigenic potential of this subpopulation were significantly suppressed by small-molecule Wnt/β-catenin signaling inhibitors. Our findings suggest that the CD44⁺CD133⁺ subpopulation from Caco-2 cells was highly enriched in tumorigenic cells and will be useful for investigating the mechanisms leading to human colorectal cancer development.     

The most reliable surface marker for the identification of colorectal cancer stem-like cells: A systematic review and meta-analysis

Several surface markers have been proposed for the identification and characterization of colorectal cancer stem-like cells (CR-CSLCs). However, their reliability in CR-CSLCs identification remains controversial. This study evaluated the correlation between all candidate surface marker's expression and CSLCs properties (tumorigenicity) through monitoring in vivo tumor incidence and final tumor volume. PubMed, Web of Science, and Scopus databases were systematically searched until November 2017. A total of 27 studies were found that met the inclusion criteria for cluster of differentiation 133 (CD133) and CD44 markers. Results indicated that either CD133 or CD44 positive cells caused about twofold increase in tumor volume compared with the negative cells (p < 0.05). In two groups of cells derived from primary tumors and cell lines, CD133 ⁺ cells had 25 and 1.45 times higher tumor incidence potential than CD133 ⁻ cells, respectively ( p < 0.05). Also, cohort evaluation showed that CD133 overexpression at protein level is a marker of poor overall survival in colorectal cancer (CRC) patients. While CD44 ⁺ cells displayed twofold tumorigenicity compared with the negative cells ( p < 0.05), combination of CD44 and CD133 showed about sevenfold tumorigenicity potential ( p < 0.05). In conclusion, the present meta-analysis suggests that CD133 is a robust biomarker to identify primary tumor CSLCs and can be proposed as a prognostic marker of CRC patient whereas it should be used with caution in cell lines. It seems to be more reliable to use CD133 in combination with CD44 as target biomarkers for the isolation of CR-CSLCs in both cell line and primary tumor cells populations.     

LncRNA DLGAP1-AS2 promotes the radioresistance of rectal cancer stem cells by upregulating CD151 expression via E2F1

BackgroundRadiotherapy resistance is one of the major causes of rectal cancer treatment failure. LncRNA DLGAP1-AS2 participates in the progression of several cancers. We explored the role and potential mechanism of DLGAP1-AS2 in the radioresistance of rectal cancer stem cells.MethodsHR8348-R cells, radioresistant cells from HR8348 after irradiation, were isolated into CD133 negative (CD133⁻) and positive (CD133⁺) cells. Cell proliferation, apoptosis, migration and tumorsphere formation were determined by CCK-8, flow cytometry, wound healing assay and tumorsphere formation assay, respectively. CD133, tumor stem cell drug resistance gene (MDR1 and BCRP1), DNA repair marker (γ-H2AX) and AKT/mTOR/cyclinD1 signaling were measured by Western blot. The relationship between DLGAP1-AS2 and E2F1 was verified using RIP. The interaction between E2F1 and CD151 promoter was confirmed using dual-luciferase reporter gene assay and ChIP. AKT inhibitor API-2 was employed for validating the effect of AKT/mTOR/cyclinD1 signaling in the radioresistance of rectal cancer cells.ResultsThe DLGAP1-AS2 level was increased in CD133⁺ cells after irradiation. DLGAP1-AS2 knockdown inhibited the proliferation, migration and tumorsphere formation while stimulating apoptosis in CD133⁺ cells. DLGAP1-AS2 inhibition downregulated the expression of CD133, MDR1, BCRP1 and γ-H2AX and suppressed AKT/mTOR/cyclinD1 activation. DLGAP1-AS2 upregulated the expression of CD151 by interacting with E2F1. API-2 neutralized the promotive effects of overexpressed CD151 on radioresistance.ConclusionDLGAP1-AS2 accelerates the radioresistance of rectal cancer cells through interactions with E2F1 to upregulate CD151 expression via the activation of the AKT/mTOR/cyclinD1 pathway.