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Abstract Melanoma progression depends on melanoma stem cells (MSCs), which are distinguished by the distinct dysregulated genes. As the key factors in the dysregulation of genes, long non-coding RNAs (lncRNAs) take great effects on MSCs. However, the underlying mechanism of lncRNAs in MSCs has not been extensively characterized. To address the roles of lncRNAs in MSCs, LINC00698 was characterized in this study. The results revealed that LINC00698 was upregulated in MSCs, showing its important role in MSCs. The further data indicated that the LINC00698 silencing triggered cell cycle arrest in the G0/G1 phase and apoptosis of MSCs. LINC00698 could directly interact with miR-3132 to upregulate the expression of TCF7, which was required for sustaining the stemness and the tumorigenic potency of MSCs. At the same time, LINC00698 could bind to the hnRNPM protein to enhance the protein stability, thus suppressing apoptosis and promoting the stemness of MSCs. Furthermore, the in vivo data demonstrated that LINC00698 was essential for tumorigenesis of MSCs via the LINC00698-miR-3132-TCF7/hnRNPM axis. Therefore, our findings contributed novel insights into the underlying mechanism of melanoma progression.

Melanoma progression depends on melanoma stem cells (MSCs), which are distinguished by the distinct dysregulated genes. As the key factors in the dysregulation of genes, long non-coding RNAs (lncRNAs) take great effects on MSCs. However, the underlying mechanism of lncRNAs in MSCs has not been extensively characterized. To address the roles of lncRNAs in MSCs, LINC00698 was characterized in this study. The results revealed that LINC00698 was upregulated in MSCs, showing its important role in MSCs. The further data indicated that the LINC00698 silencing triggered cell cycle arrest in the G0/G1 phase and apoptosis of MSCs. LINC00698 could directly interact with miR-3132 to upregulate the expression of TCF7, which was required for sustaining the stemness and the tumorigenic potency of MSCs. At the same time, LINC00698 could bind to the hnRNPM protein to enhance the protein stability, thus suppressing apoptosis and promoting the stemness of MSCs. Furthermore, the in vivo data demonstrated that LINC00698 was essential for tumorigenesis of MSCs via the LINC00698-miR-3132-TCF7/hnRNPM axis. Therefore, our findings contributed novel insights into the underlying mechanism of melanoma progression. [ABSTRACT FROM AUTHOR]

CD133, a cancer stem cell (CSC) marker in tumors, including melanoma, is associated with tumor recurrence, chemoresistance, and metastasis. Patient-derived melanoma cell lines were transduced with a Tet-on vector expressing CD133, generating doxycycline (Dox)-inducible cell lines. Cells were exposed to Dox for 24 h to induce CD133 expression, followed by RNA-seq and bioinformatic analyses, revealing genes and pathways that are significantly up- or downregulated by CD133. The most significantly upregulated gene after CD133 was amphiregulin (AREG), validated by qRT-PCR and immunoblot analyses. Induced CD133 expression significantly increased cell growth, percentage of cells in S-phase, BrdU incorporation into nascent DNA, and PCNA levels, indicating that CD133 stimulates cell proliferation. CD133 induction also activated EGFR and the MAPK pathway. Potential mechanisms highlighting the role(s) of CD133 and AREG in melanoma CSC were further delineated using AREG/EGFR inhibitors or siRNA knockdown of AREG mRNA. Treatment with the EGFR inhibitor gefitinib blocked CD133-induced cell growth increase and MAPK pathway activation. Importantly, siRNA knockdown of AREG reversed the stimulatory effects of CD133 on cell growth, indicating that AREG mediates the effects of CD133 on cell proliferation, thus serving as an attractive target for novel combinatorial therapeutics in melanoma and cancers with overexpression of both CD133 and AREG. [ABSTRACT FROM AUTHOR]

Abstract Background Mucosal Melanomas (MM) are highly aggressive neoplasms arising from mucosal melanocytes. Current treatments offer a limited survival benefit for patients with advanced MM; moreover, the lack of pre-clinical cellular systems has significantly limited the understanding of their immunobiology. Methods Five novel cell lines were obtained from patient-derived biopsies of MM arising in the sino-nasal mucosa and designated as SN-MM1-5. The morphology, ultrastructure and melanocytic identity of SN-MM cell lines were validated by transmission electron microscopy and immunohistochemistry. Moreover, in vivo tumorigenicity of SN-MM1-5 was tested by subcutaneous injection in NOD/SCID mice. Molecular characterization of SN-MM cell lines was performed by a mass-spectrometry proteomic approach, and their sensitivity to PI3K chemical inhibitor LY294002 was validated by Akt activation, measured by pAkt(Ser473) and pAkt(Thr308) in immunoblots, and MTS assay. Results This study reports the validation and functional characterization of five newly generated SN-MM cell lines. Compared to the normal counterpart, the proteomic profile of SN-MM is consistent with transformed melanocytes showing a heterogeneous degree of melanocytic differentiation and activation of cancer-related pathways. All SN-MM cell lines resulted tumorigenic in vivo and display recurrent structural variants according to aCGH analysis. Of relevance, the microscopic analysis of the corresponding xenotransplants allowed the identification of clusters of MITF-/CDH1-/CDH2 + /ZEB1 + /CD271 + cells, supporting the existence of melanoma-initiating cells also in MM, as confirmed in clinical samples. In vitro, SN-MM cell lines were sensitive to cisplatin, but not to temozolomide. Moreover, the proteomic analysis of SN-MM cell lines revealed that RICTOR, a subunit of mTORC2 complex, is the most significantly activated upstream regulator, suggesting a relevant role for the PI3K-Akt-mTOR pathway in these neoplasms. Consistently, phosphorylation of NDRG1 and Akt activation was observed in SN-MM, the latter being constitutive and sustained by PTEN loss in SN-MM2 and SN-MM3. The cell viability impairment induced by LY294002 confirmed a functional role for the PI3K-Akt-mTOR pathway in SN-MM cell lines. Conclusions Overall, these novel and unique cellular systems represent relevant experimental tools for a better understanding of the biology of these neoplasms and, as an extension, to MM from other sites.

Background: Mucosal Melanomas (MM) are highly aggressive neoplasms arising from mucosal melanocytes. Current treatments offer a limited survival benefit for patients with advanced MM; moreover, the lack of pre-clinical cellular systems has significantly limited the understanding of their immunobiology. Methods: Five novel cell lines were obtained from patient-derived biopsies of MM arising in the sino-nasal mucosa and designated as SN-MM1-5. The morphology, ultrastructure and melanocytic identity of SN-MM cell lines were validated by transmission electron microscopy and immunohistochemistry. Moreover, in vivo tumorigenicity of SN-MM1-5 was tested by subcutaneous injection in NOD/SCID mice. Molecular characterization of SN-MM cell lines was performed by a mass-spectrometry proteomic approach, and their sensitivity to PI3K chemical inhibitor LY294002 was validated by Akt activation, measured by pAkt(Ser473) and pAkt(Thr308) in immunoblots, and MTS assay. Results: This study reports the validation and functional characterization of five newly generated SN-MM cell lines. Compared to the normal counterpart, the proteomic profile of SN-MM is consistent with transformed melanocytes showing a heterogeneous degree of melanocytic differentiation and activation of cancer-related pathways. All SN-MM cell lines resulted tumorigenic in vivo and display recurrent structural variants according to aCGH analysis. Of relevance, the microscopic analysis of the corresponding xenotransplants allowed the identification of clusters of MITF-/CDH1-/CDH2 + /ZEB1 + /CD271 + cells, supporting the existence of melanoma-initiating cells also in MM, as confirmed in clinical samples. In vitro, SN-MM cell lines were sensitive to cisplatin, but not to temozolomide. Moreover, the proteomic analysis of SN-MM cell lines revealed that RICTOR, a subunit of mTORC2 complex, is the most significantly activated upstream regulator, suggesting a relevant role for the PI3K-Akt-mTOR pathway in these neoplasms. Consistently, phosphorylation of NDRG1 and Akt activation was observed in SN-MM, the latter being constitutive and sustained by PTEN loss in SN-MM2 and SN-MM3. The cell viability impairment induced by LY294002 confirmed a functional role for the PI3K-Akt-mTOR pathway in SN-MM cell lines. Conclusions: Overall, these novel and unique cellular systems represent relevant experimental tools for a better understanding of the biology of these neoplasms and, as an extension, to MM from other sites. [ABSTRACT FROM AUTHOR]

Simple Summary: Transforming growth factor-beta (TGFβ) mediates various biological processes including cell growth, cell death, cellular differentiation and stemness, among others. TGFβ also regulates tumor formation and metastasis in a context-dependent manner. This research aims to investigate and define the role of the TGFβ cell signaling pathway in melanoma, which is a deadly form of skin cancer. Using relevant melanoma cancer cell lines and preclinical models of melanoma, we show that TGFβ acts as a potent tumor suppressor and negative regulator of cancer stemness and metastasis in melanoma. These findings will be instrumental for the future development of targeted therapy in melanoma. The secreted protein transforming growth factor-beta (TGFβ) plays essential roles, ranging from cell growth regulation and cell differentiation in both normal and cancer cells. In melanoma, TGFβ acts as a potent tumor suppressor in melanoma by blocking cell cycle progression and inducing apoptosis. In the present study, we found TGFβ to regulate cancer stemness in melanoma through the Smad signaling pathway. We discovered that TGFβ/Smad signaling inhibits melanosphere formation in multiple melanoma cell lines and reduces expression of the CD133+ cancer stem cell subpopulation in a Smad3-dependent manner. Using preclinical models of melanoma, we further showed that preventing Smad3/4 signaling, by means of CRISPR knockouts, promoted both tumorigenesis and lung metastasis in vivo. Collectively, our results define new functions for the TGFβ/Smad signaling axis in melanoma stem-cell maintenance and open avenues for new therapeutic approaches to this disease. [ABSTRACT FROM AUTHOR]

Melanoma, a lethal malignancy that arises from melanocytes, exhibits a multiplicity of clinico‐pathologically distinct subtypes in sun‐exposed and non‐sun‐exposed areas. Melanocytes are derived from multipotent neural crest cells and are present in diverse anatomical locations, including skin, eyes, and various mucosal membranes. Tissue‐resident melanocyte stem cells and melanocyte precursors contribute to melanocyte renewal. Elegant studies using mouse genetic models have shown that melanoma can arise from either melanocyte stem cells or differentiated pigment‐producing melanocytes depending on a combination of tissue and anatomical site of origin and activation of oncogenic mutations (or overexpression) and/or the repression in expression or inactivating mutations in tumor suppressors. This variation raises the possibility that different subtypes of human melanomas (even subsets within each subtype) may also be a manifestation of malignancies of distinct cells of origin. Melanoma is known to exhibit phenotypic plasticity and trans‐differentiation (defined as a tendency to differentiate into cell lineages other than the original lineage from which the tumor arose) along vascular and neural lineages. Additionally, stem cell‐like properties such as pseudo‐epithelial‐to‐mesenchymal (EMT‐like) transition and expression of stem cell‐related genes have also been associated with the development of melanoma drug resistance. Recent studies that employed reprogramming melanoma cells to induced pluripotent stem cells have uncovered potential relationships between melanoma plasticity, trans‐differentiation, and drug resistance and implications for cell or origin of human cutaneous melanoma. This review provides a comprehensive summary of the current state of knowledge on melanoma cell of origin and the relationship between tumor cell plasticity and drug resistance. [ABSTRACT FROM AUTHOR]

CD133, a cancer stem cell (CSC) marker in tumors, including melanoma, is associated with tumor recurrence, chemoresistance, and metastasis. Patient-derived melanoma cell lines were transduced with a Tet-on vector expressing CD133, generating doxycycline (Dox)-inducible cell lines. Cells were exposed to Dox for 24 h to induce CD133 expression, followed by RNA-seq and bioinformatic analyses, revealing genes and pathways that are significantly up- or downregulated by CD133. The most significantly upregulated gene after CD133 was amphiregulin (AREG), validated by qRT-PCR and immunoblot analyses. Induced CD133 expression significantly increased cell growth, percentage of cells in S-phase, BrdU incorporation into nascent DNA, and PCNA levels, indicating that CD133 stimulates cell proliferation. CD133 induction also activated EGFR and the MAPK pathway. Potential mechanisms highlighting the role(s) of CD133 and AREG in melanoma CSC were further delineated using AREG/EGFR inhibitors or siRNA knockdown of AREG mRNA. Treatment with the EGFR inhibitor gefitinib blocked CD133-induced cell growth increase and MAPK pathway activation. Importantly, siRNA knockdown of AREG reversed the stimulatory effects of CD133 on cell growth, indicating that AREG mediates the effects of CD133 on cell proliferation, thus serving as an attractive target for novel combinatorial therapeutics in melanoma and cancers with overexpression of both CD133 and AREG.

Abstract Background Metastatic melanoma has a high mortality rate and poor survival. This is associated with efficient metastatic colonization, but the underlying mechanisms remain elusive. Communication between cancer stem cells (CSCs) and cancer cells plays an important role in metastatic dissemination. Whether cancer stem cells can alter the metastatic properties of non-CSC cells; and whether exosomal crosstalk can mediate such interaction, have not been demonstrated in melanoma prior to this report. Results The results revealed that exosomes secreted by highly metastatic melanoma CSCs (OL-SCs) promoted the invasiveness of the low metastatic melanoma cells (OL) and accelerated metastatic progression. miR-1268a was up-regulated in cells and exosomes of OL-SCs. Moreover, OL-SCs-derived exosomal miR-1268a, upon taking up by OL cells, promoted the metastatic colonization ability of OL cells in vitro and in vivo. In addition, the pro-metastatic activity of exosomal miR-1268a is achieved through inhibition of autophagy. Conclusion Our study demonstrates that OL cells can acquire the “metastatic ability” from OL-SCs cells. OL-SCs cells achieves this goal by utilizing its exosomes to deliver functional miRNAs, such as miR-1268a, to the targeted OL cells which in turn augments metastatic colonization by inactivating the autophagy pathway in OL 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.

Background: Metastatic melanoma has a high mortality rate and poor survival. This is associated with efficient metastatic colonization, but the underlying mechanisms remain elusive. Communication between cancer stem cells (CSCs) and cancer cells plays an important role in metastatic dissemination. Whether cancer stem cells can alter the metastatic properties of non-CSC cells; and whether exosomal crosstalk can mediate such interaction, have not been demonstrated in melanoma prior to this report. Results: The results revealed that exosomes secreted by highly metastatic melanoma CSCs (OL-SCs) promoted the invasiveness of the low metastatic melanoma cells (OL) and accelerated metastatic progression. miR-1268a was up-regulated in cells and exosomes of OL-SCs. Moreover, OL-SCs-derived exosomal miR-1268a, upon taking up by OL cells, promoted the metastatic colonization ability of OL cells in vitro and in vivo. In addition, the pro-metastatic activity of exosomal miR-1268a is achieved through inhibition of autophagy. Conclusion: Our study demonstrates that OL cells can acquire the "metastatic ability" from OL-SCs cells. OL-SCs cells achieves this goal by utilizing its exosomes to deliver functional miRNAs, such as miR-1268a, to the targeted OL cells which in turn augments metastatic colonization by inactivating the autophagy pathway in OL cells. [ABSTRACT FROM AUTHOR]

We previously described a novel in vitro culture technique for dedifferentiated human adult skin melanocytes. Melanocytes cultured in a defined, cholera toxin and PMA free medium became bipolar, unpigmented, and highly proliferative. Furthermore, TRP‐1 and c‐Kit expression disappeared and EGFR receptor and nestin expression were induced in the cells. Here, we further characterized the phenotype of these dedifferentiated cells and by comparing them to mature pigmented melanocytes we detected crucial steps in their phenotype change. Our data suggest that normal adult melanocytes easily dedifferentiate into pluripotent stem cells given the right environment. This dedifferentiation process described here for normal melanocyte is very similar to what has been described for melanoma cells, indicating that phenotype switching driven by environmental factors is a general characteristic of melanocytes that can occur independent of malignant transformation. [ABSTRACT FROM AUTHOR]

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.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Allogeneic whole cell gene modified therapeutic melanoma vaccine (AGI-101H) comprising of two melanoma cell lines transduced with cDNA encoding fusion protein composed of IL-6 linked with the soluble IL-6 receptor (sIL-6R), referred to as H6 was developed. H6 served as a molecular adjuvant, however, it has altered vaccine cells phenotype towards melanoma stem cells (MSC)-like with high activity of aldehyde dehydrogenase isoenzyme (ALDH1A1). AGI-101H was applied in advanced melanoma patients with non-resected and resected disease. In the adjuvant setting, it was combined with surgery in case of recurring metastases, which were surgically removed and vaccination continued. A significant fraction of AGI-101H treated melanoma patients is still alive (11–19 years). Out of 106 living patients, 39 were HLA-A2 positive and were the subject of the study. Immunization of melanoma patients resulted in the generation of cytotoxic CD8+ T cells specific for ALDH1A1, which were detected in circulation by HLA-A0201 MHC dextramers loaded with ALDH1A188-96(LLYKLADLI) peptide. Phenotypically they were central memory CD8+ T cells. Re-stimulation with ALDH1A188-96 ex vivo resulted in IFN-γ secretion and cells degranulation. Following each vaccine dose administration, the number of ALDH1A1-CD8+ T cells increased in circulation and returned to the previous level until next dose injection (one month). ALDH1A1-CD8+ T cells were also found, however in the lower number than in vaccinated patients, in the circulation of untreated melanoma with stage IV but were not found in stage II or III and healthy donors. Specific anti-ALDH1 antibodies were present in treated patients. Long-term survival suggests immuno-targeting of MSC in treated patients.

Melanoma is one of the most aggressive and extremely resistant to conventional therapies neoplasms. Recently, cellular resistance was linked to the cancer stem cell phenotype, still controversial and not well-defined. In this study, we used a Rhodamine 123 (Rh123) exclusion assay to functionally identify stem-like cells in metastatic human melanomas and melanoma cell lines. We demonstrate that a small subset of Rh123-low-retention (Rh123low) cells is enriched for stem cell-like activities, including the ability to self-renew and produce nonstem Rh123low progeny and to form melanospheres, recapitulating the phenotypic profile of the parental tumor. Rh123low cells are relatively quiescent and chemoresistant. At the molecular level, we show that melanoma Rh123low cells overexpress HIF1α, pluripotency factor OCT4, and the ABCB5 marker of melanoma stem cells and downregulate the expression of Cyclin Dl and CDK4. Interestingly, a short treatment with LY294002, an inhibitor of the PI3K/AKT pathway, specifically reverts a subset of Rh123high cells to the Rh123low phenotype, whereas treatment with inhibitors of mammalian target of rapamycin, phosphatase and tensin homolog or mitogen-activated protein kinase signaling does not. This phenotypic switching was associated with reduced levels of the HIF1α transcript and an increase in the level of phosphorylated nuclear FOXO3a preferentially in Rh123low cells. Moreover, the Rh123low cells became less quiescent and displayed a significant increase in their melanosphere-forming ability. All the above indicates that the Rh123low melanoma stem cell pool is composed of cycling and quiescent cells and that the PI3K/AKT signaling while maintaining the quiescence of RH123low GO cells promotes the exit of cycling cells from the stem cell compartment. [ABSTRACT FROM AUTHOR]

Abstract: Cutaneous melanoma is a significant health problem worldwide. Available treatments can induce objective tumor regression in a small percent of patients, but these responses are not always associated with improved long-term survival. The resistance of melanoma to therapy and its predestined recurrence are related to the genetic heterogeneity and genomic instability of the tumor. For many years these genetic alterations were thought to be linked to the accumulation of random mutations in functionally differentiated cells which transform them into malignant cells that have lost their ability to differentiate and have acquired drug resistance. In the last few years it has been largely demonstrated that melanoma as other solid tumors contains a subpopulation of cells (CSCs) considered the source of the primary tumor mass, of new tumor nodules and responsible for drug resistance and cancer recurrence. In this review, we provide an overview of findings and advances in CSCs research that are relevant to the initiation, natural history, and the response to treatment of malignant melanoma. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

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., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

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. [ABSTRACT FROM AUTHOR]

Sphere assays are widely used in vitro techniques to enrich and evaluate the stem-like cell behavior of both normal and cancer cells. Utilizing three-dimensional in vitro sphere culture conditions provide a better representation of tumor growth in vivo than the more common monolayer cultures. We describe how to perform primary and secondary sphere assays, used for the enrichment and self-renewability studies of melanoma/melanocyte stem-like cells. Spheres are generated by growing melanoma cells at low density in nonadherent conditions with stem cell media. We provide protocols for preparing inexpensive and versatile polyHEMA-coated plates, setting up primary and secondary sphere assays in almost any tissue culture format and quantification methods using standard inverted microscopy. Our protocol is easily adaptable to laboratories with basic cell culture capabilities, without the need for expensive fluidic instruments.

Allogeneic whole cell gene modified therapeutic melanoma vaccine (AGI-101H) comprising of two melanoma cell lines transduced with cDNA encoding fusion protein composed of IL-6 linked with the soluble IL-6 receptor (sIL-6R), referred to as H6 was developed. H6 served as a molecular adjuvant, however, it has altered vaccine cells phenotype towards melanoma stem cells (MSC)-like with high activity of aldehyde dehydrogenase isoenzyme (ALDH1A1). AGI-101H was applied in advanced melanoma patients with non-resected and resected disease. In the adjuvant setting, it was combined with surgery in case of recurring metastases, which were surgically removed and vaccination continued. A significant fraction of AGI-101H treated melanoma patients is still alive (11-19 years). Out of 106 living patients, 39 were HLA-A2 positive and were the subject of the study. Immunization of melanoma patients resulted in the generation of cytotoxic CD8+ T cells specific for ALDH1A1, which were detected in circulation by HLA-A0201 MHC dextramers loaded with ALDH1A188-96(LLYKLADLI) peptide. Phenotypically they were central memory CD8+ T cells. Re-stimulation with ALDH1A188-96 ex vivo resulted in IFN-γ secretion and cells degranulation. Following each vaccine dose administration, the number of ALDH1A1-CD8+ T cells increased in circulation and returned to the previous level until next dose injection (one month). ALDH1A1-CD8+ T cells were also found, however in the lower number than in vaccinated patients, in the circulation of untreated melanoma with stage IV but were not found in stage II or III and healthy donors. Specific anti-ALDH1 antibodies were present in treated patients. Long-term survival suggests immuno-targeting of MSC in treated patients. [ABSTRACT FROM AUTHOR]

MCL-1 (BCL-2 family anti-apoptotic protein) is responsible for melanoma's resistance to therapy. Cancer initiating cells also contribute to resistance and relapse from treatments. Here we examined the effects of the MCL-1 inhibitor SC-2001 in killing non melanoma-initiating-cells (bulk of melanoma), and melanoma-initiating-cells (MICs). By itself, SC-2001 significantly kills melanoma cells under monolayer conditions in vitro and in a conventional mouse xenograft model. However, even at high doses (10μM), SC-2001 does not effectively eliminate MICs. In contrast, the combination of SC-2001 with ABT-737 (a BCL-2/BCL-XL/BCL-W inhibitor) significantly decreases ALDH+ cells, disrupts primary spheres, and inhibits the self-renewability of MICs. These results were observed in multiple melanomas, including short term cultures of relapsed tumors from current treatments, independent of the mutation status of BRAF or NRAS. Using a low-cell-number mouse xenograft model, we examined the effects of these treatments on the tumor initiating ability of MIC-enriched cultures. The combination therapy reduces tumor formation significantly compared to either drug alone. Mechanistic studies using shRNA and the CRISPR-Cas9 technology demonstrated that the upregulation of pro-apoptotic proteins NOXA and BIM contribute to the combination-induced cell death. These results indicate that the MCL-1 inhibitor SC-2001 combined with ABT-737 is a promising treatment strategy for targeting melanoma.

Major limitations of current melanoma treatments are for instances of relapse and the lack of therapeutic options for BRAF wild-type patients who do not respond to immunotherapy. Many studies therefore focus on killing resistant subpopulations, such as Melanoma Initiating Cells (MICs) to prevent relapse. Here we examined whether combining a GSI (γ-Secretase Inhibitor) with ABT-737 (a small molecule BCL-2/BCL-XL/BCL-W inhibitor) can kill both the non-MICs (bulk of melanoma) and MICs. To address the limitations of melanoma therapies, we included multiple tumor samples of patients relapsed from current treatments, with a diverse genetic background (with or without the common BRAF, NRAS or NF1 mutations) in these studies. Excitingly, the combination treatment reduced cell viability and induced apoptosis of the non-MICs; disrupted primary spheres, decreased the ALDH+ cells, and inhibited the self-renewability of the MICs in multiple melanoma cell lines and relapsed patient samples. Using a low-cell-number mouse xenograft model, we demonstrated that the combination significantly reduced the tumor initiating ability of MIC-enriched cultures from relapsed patient samples. Mechanistic studies also indicate that cell death is NOXA-dependent. In summary, this combination may be a promising strategy to address treatment relapse and for triple wild-type patients who do not respond to immunotherapy.

Circulating melanoma cells (CMCs) represent critical mediators of metastatic melanoma progression. However, isolation and characterization of CMCs has been challenging due to the low frequency of these cells and the paucity of melanoma-specific cell surface markers. Herein, we describe a method for the isolation of CMCs that employs two independent markers, displays high sensitivity for CMC enrichment, and can be readily adapted to include additional molecular melanoma markers of interest. CMCs isolated by this method are enriched for ABCB5-positive melanoma stem cells, are tumorigenic in xenotransplantation assays, and can be used for phenotypical, genetic, and functional investigations of CMC biology.

Tumors are incredibly diverse and contain many different subpopulations of cells. The cancer stem cell (CSC) subpopulation is responsible for many aspects of tumorigenesis and has been shown to play an important role in melanoma development, progression, drug resistance and metastasis. However, it is becoming clear that tumor cell populations are dynamic and can be influenced by many factors, such as signals from the tumor microenvironment and somatic evolution. This review will present the current understanding of CSCs and the challenges of identifying and characterizing this dynamic cell population. The known characteristics and functions of melanoma stem cells, and the potential for therapeutic targeting of these cells in melanoma, will be discussed.

History of cancer immunotherapy lasts for more than 120 years. In 1891 William B. Coley injected bacteria into inoperable cancer (bone sarcoma) and observed tumor shrinkage. He is recognized as the "'"Father of Immunotherapy"'". Cancer immunotherapy is based on the ability of the immune system to recognize cancer cells and to affect their growth and expansion. Beside the fact that, tumor cells are genetically distinct from their normal counterparts, and should be recognized and eliminated by immune system, the tumor associated antigens (TAAs) are often poorly immunogenic due to immunoediting. This process allows tumor to evolve during continuous interactions with the host immune system, and eventually escape from immune surveillance. Furthermore, tumor microenvironment consists of immunosuppressive cells that release immunosuppressive factors including IL-6, IL-10, IDO, TGFβ or VEGF. Interactions between cancer and stroma cells create network of immunosuppressive pathways, while activation of immune defense is inhibited. A key to successful immunotherapy is to overcome the local immunosuppression within tumor microenvironment and activate mechanisms that lead to tumor eradication. There are two clinical approaches of immunotherapy: active and passive. Active immunotherapy involves stimulation of immune response to tumor associated antigens (TAAs), either non-specifically via immunomodulating agents or specifically employing cancer vaccines. This review presents the progress and breakthroughs in design, development and clinical application of selected cell-based tumor vaccines achieved due to the generation and development of gene transfer technologies., (Copyright © 2013 Elsevier B.V. All rights reserved.)