Melanoma Stem Cells Research Articles

Melanoma Stem Cells Educate Neutrophils to Support Cancer Progression.

Simple SummaryIn melanoma patients, poor prognosis often correlates with high presence of cancer-associated neutrophils, indicating that tumors can recruit these immune cells to specifically sustain their own development and progression. However, the role of cancer stem cells (CSCs) in this dialogue has not been elucidated yet. Our results revealed that melanoma SCs can reshape the immune microenvironment by triggering a pro-tumor N2 phenotype in neutrophils, which in turn are able to confer stemness properties to melanoma cells.Background: It is now well-established that cancer stem cells (CSCs) can support melanoma progression by reshaping the tumor immune microenvironment. However, the molecular mechanisms underlying the crosstalk between melanoma SCs and cancer-associated neutrophils have not been elucidated yet. Methods: The aim of the present study was to unravel the role of melanoma SCs in neutrophil polarization. HL60 neutrophil-like (dHL60) cells were treated with conditioned medium from A375 melanoma SCs (CSC-CM), and their phenotype was investigated. Results: We demonstrated that CSC-CM could specifically activate immune cells by increasing CD66b and CD11b expression. In particular, we revealed that A375 CSCs could release various soluble factors, namely TGF-β, IL-6, and IL-8, able to promote the recruitment of neutrophils and their switch toward an N2 phenotype characterized by the activation of ERK, STAT3, and P38 pathways and the overexpression of CXCR2 and NF-kB. Moreover, after exposure to CSC-CM, dHL60 cells exhibited enhanced ROS production and NET release, without undergoing cell death; increased secretion of MMP-9 and pro-inflammatory cytokines was also observed. Finally, CSC-CM-activated neutrophils endowed A375 cells with stemness traits, stimulating both sphere formation and ABCG2 expression. Conclusion: Collectively, our results suggest that melanoma SCs can prime neutrophils to support cancer progression.

Dexamethasone Promotes a Stem-Like Phenotype in Human Melanoma Cells via Tryptophan 2,3 Dioxygenase

In addition to its well-established immunosuppressive actions, tryptophan 2,3-dioxygenase (TDO) appears to elicit direct effects on tumor cell function. Although TDO has been associated with cancer stemness, its involvement in melanoma stem cell biology remains largely unknown. Since we showed that by upregulating TDO, dexamethasone (dex) promotes proliferation and migration of SK-Mel-28 human melanoma cells, we sought to investigate dex effects on melanoma spherogenesis and stemness, and whether these events are mediated by TDO. We demonstrate here that dex significantly upregulates TDO in A375, a more aggressive melanoma cell line, confirming that dex effects are not limited to SK-Mel-28 cells. Moreover, dex stimulates spherogenesis of both cell lines, which is mediated by TDO, evident by its suppression with 680C91, a TDO inhibitor. The formed melanospheres appear to be enriched with embryonic stem cell marker mRNAs, the expression of which is potentiated by dex. Expression of cancer stem cell markers (CD133, CD44, ganglioside GD2) was significantly increased in A375 spheres, as detected by flow cytometry. Taken together, our results suggest that TDO could represent a promising target in the management of melanoma and that dex, routinely used as a co-medication also in advanced melanoma, may stimulate melanoma cell function/tumor-supporting properties, a rather debilitating and undesired side effect.

Exosomal microRNA-4535 of Melanoma Stem Cells Promotes Metastasis by Inhibiting Autophagy Pathway.

High mortality rate and poor survival in melanoma are associated with efficient metastatic colonization. The underlying mechanisms remain elusive. Elucidating the role of exosomes in mediating the interactions between cancer cells and the metastatic microenvironment has been focused on cancer cell derived exosomes in modulating the functions of stromal cells. Whether cancer stem cells (CSCs) can modify the metastatic properties of non-CSC cells, and whether exosomal crosstalk plays a role have not been demonstrated prior to this report. In this study, a paired M14 melanoma derivative cell line, i.e., melanoma parental cell (MPC) and its CSC derivative cell line melanoma stem cell (MSC) were employed. We demonstrated that exosomal crosstalk betwen MSCs and non-CSC MPCs is a new mechanism that underlies melanoma metastasis. Low metastatic melanoma cells (MPCs) can acquire the "metastatic power" from highly metastatic melanoma CSCs (MSCs). We illustrated an uncharacterized microRNA, miR-4535 in mediating such exosomal crosstalk. MSCs deliver its exosomal miR-4535 to the targeted MPCs. Upon entering MPCs, miR-4535 augments metastatic colonization of MPCs by inactivating the autophagy pathway.

Employing CRISPR-Cas9 to Generate CD133 Synthetic Lethal Melanoma Stem Cells.

Malignant melanoma is a lethal skin cancer containing melanoma-initiating cells (MIC) implicated in tumorigenesis, invasion, and drug resistance, and is characterized by the elevated expression of stem cell markers, including CD133. The siRNA knockdown of CD133 enhances apoptosis induced by the MEK inhibitor trametinib in melanoma cells. This study investigates the underlying mechanisms of CD133’s anti-apoptotic activity in patient-derived BAKP and POT cells, harboring difficult-to-treat NRASQ61K and NRASQ61R drivers, after CRISPR-Cas9 CD133 knockout or Dox-inducible expression of CD133. MACS-sorted CD133(+) BAKP cells were conditionally reprogrammed to derive BAKR cells with sustained CD133 expression and MIC features. Compared to BAKP, CD133(+) BAKR exhibit increased cell survival and reduced apoptosis in response to trametinib or the chemotherapeutic dacarbazine (DTIC). CRISPR-Cas9-mediated CD133 knockout in BAKR cells (BAKR-KO) re-sensitized cells to trametinib. CD133 knockout in BAKP and POT cells increased trametinib-induced apoptosis by reducing anti-apoptotic BCL-xL, p-AKT, and p-BAD and increasing pro-apoptotic BAX. Conversely, Dox-induced CD133 expression diminished apoptosis in both trametinib-treated cell lines, coincident with elevated p-AKT, p-BAD, BCL-2, and BCL-xL and decreased activation of BAX and caspases-3 and -9. AKT1/2 siRNA knockdown or inhibition of BCL-2 family members with navitoclax (ABT-263) in BAKP-KO cells further enhanced caspase-mediated apoptotic PARP cleavage. CD133 may therefore activate a survival pathway where (1) increased AKT phosphorylation and activation induces (2) BAD phosphorylation and inactivation, (3) decreases BAX activation, and (4) reduces caspases-3 and -9 activity and caspase-mediated PARP cleavage, leading to apoptosis suppression and drug resistance in melanoma. Targeting nodes of the CD133, AKT, or BCL-2 survival pathways with trametinib highlights the potential for combination therapies for NRAS-mutant melanoma stem cells for the development of more effective treatments for patients with high-risk melanoma.

Suppression of RNA editing by miR-17 inhibits the stemness of melanoma stem cells

More and more evidence suggests that microRNA (miRNA) and RNA editing play key roles in the development and progression of tumor. However, the influence of miRNA-mediated RNA editing on tumor stem cells remains unclear. In this study, the results demonstrated that miR-17, which was downregulated in melanoma stem cells, acted as a tumor inhibitor by suppressing the stemness of melanoma stem cells and promoting cell differentiation. MiR-17 targeted ADAR2 (adenosine deaminase acting on RNA 2), a gene encoding an editing enzyme required for the maintenance of melanoma stem cell stemness. In melanoma stem cells, ADAR2 was responsible for DOCK2 mRNA editing, which was able to increase the stability of DOCK2 mRNA. The in vitro and in vivo data demonstrated that DOCK2 mRNA editing upregulated the expressions of stemness and anti-apoptotic genes by activating Rac1 and then phosphorylating Akt and NF-κB, thus leading to oncogenesis of melanoma stem cells. Our findings contribute new perspectives to miRNA-regulated RNA editing in tumor progression.

An Integrative Analysis of The Micro-RNAs Contributing in Stemness, Metastasis and B-Raf Pathways in Malignant Melanoma and Melanoma Stem Cell.

Objective Epithelial-mesenchymal transition (EMT) and the stemness potency in association with BRAF mutation are in dispensable to the progression of melanoma. Recently, microRNAs (miRNAs) have been introduced as the regulator of a multitude of oncogenic functions in most of tumors. Therefore identifying and interpreting the expression patterns of these miRNAs is essential. The present study sought to find common miRNAs regulating all three important pathways in melanoma development. Materials and Methods In this experimental study, 18 miRNAs that importantly contribute to EMT and have a role in regulating self-renewal and the BRAF pathway were selected based on current literature and cross-analysis with available databases. Subsequently, their expression patterns were evaluated in 20 melanoma patients, normal tissues, serum from patients and control subjects, and melanospheres. Pattern discovery and integrative regulatory network analysis were used to find the most important miRNAs in melanoma progression. Results Among 18 selected miRNAs, miR-205, -141, -203, -15b, and -9 were differentially expressed in tumor samples than normal tissues. Among them, miR-205, -15b, and -9 significantly expressed in serum samples and healthy donors. Attribute Weighting and decision trees (DT) analysis presented evidence that the combination of miR-205, -203, -9, and -15b can regulate self-renewal and EMT process, by affecting CDH1, CCND1, and VEGF expression. ConclusionWe suggested here that miR-205, -15b, -203, -9 pattern as the key miRNAs linked to melanoma status, the pluripotency, proliferation, and motility of malignant cells. However, further investigations are required to find the mechanisms underlying the combinatory effects of the above mentioned miRNAs.

Effect of melanoma stem cells on melanoma metastasis.

Cancer stem cells (CSCs) are involved in the metastatic process, the resistance of many types of cancer to therapeutic treatments and consequently the onset of recurrences. The CSC concept therefore significantly extends our understanding of melanoma biology. More recently, melanoma stem cells (MSCs) have been described in melanoma as expressing specific biomarkers. These primitive melanoma cells are not only capable of self-renewal and differentiation plasticity, but may also confer virulence via immune evasion and multidrug resistance, and potentially, via vasculogenic mimicry and transition to migratory and metastasizing derivatives. This review will present the specific biomarkers of MSCs, including CD133, ATP binding cassette subfamily B member 5, CD271, CD20 and aldehyde dehydrogenase, which can regulate the transduction of tumor-related signals. These signal molecules can reversely act on tumor cells and regulate tumor angiogenesis, leading to the occurrence of melanoma metastasis. Targeting these specific biomarkers could inhibit the progression of melanoma and may help the development of novel therapeutic strategies for melanoma.

Requirement of splicing factor hnRNP A2B1 for tumorigenesis of melanoma stem cells

BackgroundCancer stem cells play essential roles in tumorigenesis, thus forming an important target for tumor therapy. The hnRNP family proteins are important splicing factors that have been found to be associated with tumor progression. However, the influence of hnRNPs on cancer stem cells has not been extensively explored.MethodsQuantitative real-time PCR and Western blot were used to examine gene expressions. RNA immunoprecipitation assays were conducted to identify the RNAs interacted with hnRNP A2B1. The in vivo assays were performed in nude mice.ResultsIn this study, the results showed that out of 19 evaluated hnRNPs, hnRNP A2B1 was significantly upregulated in melanoma stem cells compared with non-stem cells, suggesting an important role of hnRNP A2B1 in cancer stem cells. Silencing of hnRNP A2B1 triggered cell cycle arrest in G2 phase, leading to apoptosis of melanoma stem cells. The results also revealed that hnRNP A2B1 could bind to the precursor mRNAs of pro-apoptosis genes (DAPK1, SYT7, and RNF128) and anti-apoptosis genes (EIF3H, TPPP3, and DOCK2) to regulate the splicing of these 6 genes, thus promoting the expressions of anti-apoptosis genes and suppressing the expressions of pro-apoptosis genes. The in vivo data indicated that hnRNP A2B1 was required for tumorigenesis by affecting the splicing of TPPP3, DOCK2, EIF3H, RNF128, DAPK1, and SYT7, thus suppressing apoptosis of melanoma stem cells.ConclusionOur findings showed the requirement of hnRNP A2B1 for tumorigenesis, thus presenting novel molecular insights into the role of hnRNPs in cancer stem cells.

CD133 Antigen as a Potential Marker of Melanoma Stem Cells: In Vitro and In Vivo Studies.

Melanoma is the most dangerous type of skin cancer. Cancer stem cells (CSCs) are suspected to be responsible for the cancer recurrence and in the consequence for cancer therapy failure. CD133 is a potential marker for detection of melanoma CSCs. Experiments were performed on the B16-F10 mouse melanoma cell line. CD133+ cells were isolated using an immunomagnetic cell sorting technique. After isolation proliferative and clonogenic potential of CD133+, CD133- and CD133+/- were evaluated. The potential of CD133+ and CD133- cells for tumor induction was conducted on C57BL/6J mouse model. Three different cell quantities (100, 1000, 10000) were tested. Tumor morphology, number of mitoses, and tumor necrosis area were analyzed. Average 0.12% CD133+ cells were isolated. Compared to CD133- and unsorted CD133+/- cells, CD133+ cells were characterized by the higher proliferative and clonogenic potential. These properties were not confirmed in vivo, as both CD133+ and CD133- cells induced tumor growth in mouse model. No statistical differences in mitosis number and tumor necrosis area were observed. Simultaneous detection of CD133 antigen with other markers is necessary for accurate identification of these melanoma cancer stem cells.

Cancer Stem Cells and the Slow Cycling Phenotype: How to Cut the Gordian Knot Driving Resistance to Therapy in Melanoma.

Simple SummaryCancer stem cells play a central role in the development of cancer and are poorly sensitive to standard chemotherapy and radiotherapy. Furthermore, they are also responsible for the onset of drug resistance. This also occurs in malignant melanoma, the deadliest form of skin cancer. Hence, cancer stem cells eradication is one of the main challenges for medical oncology. Here, we conducted a bioinformatics approach aimed to identify the main circuits and proteins underpinning cancer stem cell fitness in melanoma. Several lessons emerged from our work and may help to conceptualize future therapeutic approaches to prolong the efficacy of current therapies.Cancer stem cells (CSCs) have historically been defined as slow cycling elements that are able to differentiate into mature cells but without dedifferentiation in the opposite direction. Thanks to advances in genomic and non-genomic technologies, the CSC theory has more recently been reconsidered in a dynamic manner according to a “phenotype switching” plastic model. Transcriptional reprogramming rewires this plasticity and enables heterogeneous tumors to influence cancer progression and to adapt themselves to drug exposure by selecting a subpopulation of slow cycling cells, similar in nature to the originally defined CSCs. This model has been conceptualized for malignant melanoma tailored to explain resistance to target therapies. Here, we conducted a bioinformatics analysis of available data directed to the identification of the molecular pathways sustaining slow cycling melanoma stem cells. Using this approach, we identified a signature of 25 genes that were assigned to four major clusters, namely (1) kinases and metabolic changes, (2) melanoma-associated proteins, (3) Hippo pathway and (4) slow cycling/CSCs factors. Furthermore, we show how a protein−protein interaction network may be the main driver of these melanoma cell subpopulations. Finally, mining The Cancer Genome Atlas (TCGA) data we evaluated the expression levels of this signature in the four melanoma mutational subtypes. The concomitant alteration of these genes correlates with the worst overall survival (OS) for melanoma patients harboring BRAF-mutations. All together these results underscore the potentiality to target this signature to selectively kill CSCs and to achieve disease control in melanoma.

LncRNA LHFPL3-AS1 contributes to tumorigenesis of melanoma stem cells via the miR-181a-5p/BCL2 pathway

Long noncoding RNAs (lncRNAs) are recognized as a new area for cancer therapy. B-cell lymphoma-2 (Bcl-2)-mediated suppression of apoptosis is an important molecular hallmark of cancer. However, the influence of lncRNA on the regulation of oncogenic Bcl-2 in cancer stem cells has not been explored. In this study, our findings revealed that the lncRNA LHFPL3-AS1-long, generated from the polypyrimidine tract binding protein 1 (PTBP1)-mediated splicing of the LHFPL3-AS1 precursor, upregulated BCL2 protein to contribute to tumorigenesis of melanoma stem cells. The in vitro and in vivo results showed that LHFPL3-AS1-long directly interacted with miR-181a-5p to inhibit the mRNA degradation of Bcl-2 (the target of miR-181), thus suppressing apoptosis of melanoma stem cells. The splicing factor PTBP1 regulated the alternative splicing of LHFPL3-AS1 transcript by preferentially binding to the motifs located in exon3 of LHFPL3-AS1 precursor, leading to the biogenesis of LHFPL3-AS1-long in melanoma stem cells. In patients with melanoma, the expressions of PTBP1 and LHFPL3-AS1 were significantly upregulated compared with the healthy donors. Therefore, our study revealed a mechanistic crosstalk among an onco-splicing factor, lncRNA and tumorigenesis of melanoma stem cells, enabling PTBP1 and LHFPL3-AS1 to serve as the attractive therapeutic targets for melanoma.

Melanoma stem cell maintenance and chemo-resistance are mediated by CD133 signal to PI3K-dependent pathways.

Melanoma stem cells (MSCs) are characterized by their unique cell surface proteins and aberrant signaling pathways. These stemness properties are either in a causal or consequential relationship to melanoma progression, treatment resistance and recurrence. The functional analysis of CD133+ and CD133- cells in vitro and in vivo revealed that melanoma progression and treatment resistance are the consequences of CD133 signal to PI3K pathway. CD133 signal to PI3K pathway drives two downstream pathways, the PI3K/Akt/MDM2 and the PI3K/Akt/MKP-1 pathways. Activation of PI3K/Akt/MDM2 pathway results in the destabilization of p53 protein, while the activation of PI3K/Akt/MKP-1 pathway results in the inhibition of mitogen-activated protein kinases (MAPKs) JNK and p38. Activation of both pathways leads to the inhibition of fotemustine-induced apoptosis. Thus, the disruption of CD133 signal to PI3K pathway is essential to overcome Melanoma resistance to fotemustine. The pre-clinical verification of in vitro data using xenograft mouse model of MSCs confirmed the clinical relevance of CD133 signal as a therapeutic target for melanoma treatment. In conclusion, our study provides an insight into the mechanisms regulating MSCs growth and chemo-resistance and suggested a clinically relevant approach for melanoma treatment.

Cancer stem cells, epigenetics, tumor microenvironment and future therapeutics in cutaneous malignant melanoma: a review.

This review provides an overview of the current understanding of the ontogeny and biology of melanoma stem cells in cutaneous malignant melanoma. This article also summarizes and evaluates the current knowledge of the underlying epigenetic mechanisms, the regulation of melanoma progress by the tumor microenvironment as well as the therapeutic implications and applications of these novel insights, in the setting of personalized medicine. Unraveling the complex ecosystem of cutaneous malignant melanoma and the interplay between its components, aims to provide novel insights into the establishment of efficient therapeutic strategies.

Integrated proteogenomic deep sequencing and analytics accurately identify non-canonical peptides in tumor immunopeptidomes

Efforts to precisely identify tumor human leukocyte antigen (HLA) bound peptides capable of mediating T cell-based tumor rejection still face important challenges. Recent studies suggest that non-canonical tumor-specific HLA peptides derived from annotated non-coding regions could elicit anti-tumor immune responses. However, sensitive and accurate mass spectrometry (MS)-based proteogenomics approaches are required to robustly identify these non-canonical peptides. We present an MS-based analytical approach that characterizes the non-canonical tumor HLA peptide repertoire, by incorporating whole exome sequencing, bulk and single-cell transcriptomics, ribosome profiling, and two MS/MS search tools in combination. This approach results in the accurate identification of hundreds of shared and tumor-specific non-canonical HLA peptides, including an immunogenic peptide derived from an open reading frame downstream of the melanoma stem cell marker gene ABCB5. These findings hold great promise for the discovery of previously unknown tumor antigens for cancer immunotherapy.

B4GALNT1 induces angiogenesis, anchorage independence growth and motility, and promotes tumorigenesis in melanoma by induction of ganglioside GM2/GD2

β-1,4-N-Acetyl-Galactosaminyltransferase 1 (B4GALNT1) encodes the key enzyme B4GALNT1 to generate gangliosides GM2/GD2. GM2/GD2 gangliosides are surface glycolipids mainly found on brain neurons as well as peripheral nerves and skin melanocytes and are reported to exacerbate the malignant potential of melanomas. In order to elucidate the mechanism, we performed functional analyses of B4GALNT1-overexpressing cells. We analyzed ganglioside pattern on four melanoma and two neuroblastoma cell lines by high performance liquid chromatography (HPLC). We overexpressed B4GALNT1 in GM2/GD2-negative human melanoma cell line (SH4) and confirmed production of GM2/GD2 by HPLC. They showed higher anchorage independence growth (AIG) in colony formation assay, and exhibited augmented motility. In vitro, cell proliferation was not affected by GM2/GD2 expression. In vivo, GM2/GD2-positive SH4 clones showed significantly higher tumorigenesis in NOD/Scid/IL2Rγ-null mice, and immunostaining of mouse CD31 revealed that GM2/GD2 induced remarkable angiogenesis. No differences were seen in melanoma stem cell and Epithelial-Mesenchymal Transition markers between GM2/GD2-positive and -negative SH4 cells. We therefore concluded that B4GALNT1, and consequently GM2/GD2, enhanced tumorigenesis via induction of angiogenesis, AIG, and cell motility. RNA-Seq suggested periostin as a potential key factor for angiogenesis and AIG. These findings may lead to development of novel therapy for refractory melanoma.

Dual-procedural separation of CTCs in cutaneous melanoma provides useful information for both molecular diagnosis and prognosis.

Background:Circulating tumor cells (CTCs) have recently emerged as a new dynamic soluble marker for several malignancies including cutaneous melanoma (CM) and are suitable for prognostic evaluations and treatment monitoring. However, to date many limitations still hamper the wide-scale application of CTCs in CM setting, including the lack of standardized methods as well as both low levels and heterogeneity of these cells.Methods:We developed a protocol for CTC detection in CM based on immune-magnetic sorting to deplete CD45-, CD31- or CD34-positive cells, followed by dielectrophoretic DEPArray separation according to cell morphology and immunophenotype. To this end, we explored the expression of melanoma stem cell antigens (CD271, ABCB5, and RANK) and the epithelial-to-mesenchymal transition markers (N-Cad, -CD44, and -MCAM/CD146) on CTCs from 17 stage IV CM patients, and investigated their BRAF mutational status by droplet digital PCR.Results:The number of CTCs isolated from CM patients ranged from 2 to 91 cells (38 ± 6.4) with respect to healthy donors (p < 0.0002). To confirm the melanoma origin of isolated cells, we observed an 80% agreement between their BRAFV600 mutational status and matched primary tumors. The characterization of the immune phenotype of isolated cells revealed high interindividual and intraindividual heterogeneity that was found to correlate with the clinical outcome.Conclusions:The dual-step protocol of immune-magnetic sorting and subsequent dielectrophoretic DEPArray separation, turned out to be a suitable method to isolate viable CTCs from stage IV melanoma patients and enabled quantitative and qualitative analyses on these cells, which may deserve prospective evaluation for potential use in the clinical practice.

Aldehyde dehydrogenase-positive melanoma stem cells in tumorigenesis, drug resistance and anti-neoplastic immunotherapy.

Cancer stem cells (CSCs), a rare subset of cancer cells, are well known for their self-renewing capacity. CSCs play a critical role in therapeutic failure and are responsible for poor prognosis in leukemia and various solid tumors. However, it is still unclear how CSCs initiate carcinogenesis and evade the immune response. In humans, the melanoma initiating cells (MICs) are recognized as the CSCs in melanomas, and were verified to possess CSC potentials. The enzymatic system, aldehyde dehydrogenase (ALDH) is considered to be a specific marker for CSCs in several tumors. The expression of ALDH in MICs may be closely correlated with phenotypic heterogeneity, melanoma-genesis, metastasis, and drug resistance. The ALDH+ CSCs/MICs not only serve as an indicator for therapeutic efficacy, but have also become a target for the treat of melanoma. In this review, we initially introduce the multiple capacities of MICs in melanoma. Then, we summarize in vivo and in vitro studies that illustrate the relationship between ALDH and MICs. Furthermore, understanding of chemotherapy resistance in melanoma relies on ALDH+ MICs. Finally, we review studies that focus on melanoma immunotherapies, rendering ALDH a potential marker to evaluate the efficacy of anti-neoplastic therapies or an adjuvant anti-melanoma target.

ABCB5 is activated by MITF and β-catenin and is associated with melanoma differentiation.

Defining markers of different phenotypic states in melanoma is important for understanding disease progression, determining the response to therapy, and defining the molecular mechanisms underpinning phenotype-switching driven by the changing intratumor microenvironment. The ABCB5 transporter is implicated in drug-resistance and has been identified as a marker of melanoma-initiating cells. Indeed ongoing studies are using ABCB5 to define stem cell populations. However, we show here that the ABCB5 is a direct target for the microphthalmia-associated transcription factor MITF and its expression can be induced by β-catenin, a key activator and co-factor for MITF. Consequently, ABCB5 mRNA expression is primarily associated with melanoma cells exhibiting differentiation markers. The results suggest first that ABCB5 is unlikely to represent a marker of de-differentiated melanoma stem cells, and second that ABCB5 may contribute to the non-genetic drug-resistance associated with highly differentiated melanoma cells. To reconcile the apparently conflicting observations in the field, we propose a model in which ABCB5 may mark a slow-cycling differentiated population of melanoma cells.

Mitochondrial Damage Mediated by miR-1 Overexpression in Cancer Stem Cells

It is well known that cells rely on mitochondrial respiration for survival. However, the effect of microRNAs (miRNAs) on mitochondria of cells has not been extensively explored. Our results indicated that the overexpression of a miRNA (miR-1) could destroy mitochondria of cancer stem cells. miR-1 was downregulated in melanoma stem cells (MSCs) and breast cancer stem cells (BCSCs) compared with cancer non-stem cells. However, the upregulation of miR-1 in cancer non-stem cells did not induce mitochondrial damage. miR-1 overexpression caused mitochondrial damage of cancer stem cells by directly targeting the 3′ UTRs of MINOS1 (mitochondrial inner membrane organizing system 1) and GPD2 (glycerol-3-phosphate dehydrogenase 2) genes and interacting with LRPPRC (leucine-rich pentatricopeptide-repeat containing) protein, a protein localized in mitochondria. MINOS1, GPD2, and LRPPRC in mitochondria were required for mitochondrial inner membrane. The results of in vitro and in vivo assays demonstrated that miR-1 overexpression induced mitophagy of cancer stem cells. Therefore, our study contributed novel insights into the mechanism of miRNA-mediated regulation of mitochondria morphology of cancer stem cells.

Requirement of the transcription factor YB-1 for maintaining the stemness of cancer stem cells and reverting differentiated cancer cells into cancer stem cells

BackgroundCancer stem cells always express high levels of stemness-associated transcription factors to maintain their features. However, the regulatory mechanism of the stemness of cancer stem cells mediated by transcription factors has not been extensively explored.MethodsThe YB-1 gene in cancer stem cells was knocked out by the CRISPR/Cas9 system. The YB-1 knockout cancer stem cells were transfected with a vector expressing YB-1 to rescue YB-1, and then the cell proliferation, cell cycle, apoptosis, and stemness, as well as tumorigenesis in nude mice, were assessed to examine the effect of YB-1 in cancer stem cells. The target genes of YB-1 were confirmed by CHIP-seq. The totipotency or pluripotency of differentiated cancer stem cells were detected by tumorsphere formation assay and quantitative real-time PCR.ResultsThe deletion of YB-1 gene inhibited the proliferation of breast cancer stem cells and melanoma stem cells, leading to cell cycle arrest and apoptosis, and induced irreversible differentiation of cancer stem cells. The tumorigenicity ability of YB-1-deleted cancer stem cells was significantly reduced in vitro and in vivo. The results of ChIP-seq showed that YB-1 maintained the stemness of cancer stem cells by promoting the expressions of stemness-associated genes (FZD-1, p21, GLP-1, GINS1, and Notch2). Furthermore, simultaneous expressions of YB-1 and the other four (SOX2, POU3F2, OCT-4, and OLIG1) or five (SOX2, SALL2, OCT-4, POU3F2, and Bmi-1) transcription factors in YB-1 knockout cancer stem cells restored the stemness of YB-1 knockout cancer stem cells.ConclusionsOur study indicated that YB-1 was required for maintaining the stemness of cancer stem cells and reverting the differentiated tumor cells into cancer stem cells.