Connecting the dots: Melanoma cell of origin,tumor cell plasticity,trans-differentiation,and drug resistance

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.     

3D chromatin architecture and epigenetic regulation in cancer stem cells

Dedifferentiation of cell identity to a progenitor-like or stem cell-like state with increased cellular plasticity is frequently observed in cancer formation.During this process,a subpopulation of cells in tumours acquires a stem cell-like state partially resembling to naturally occurring pluripotent stem cells that are temporarily present during early embryogenesis.Such characteris-tics allow these cancer stem cells (CSCs) to give rise to the whole tumour with its entire cellular heterogeneity and thereby support metastases formation while being resistant to current cancer therapeutics.Cancer devel-opment and progression are demarcated by transcrip-tional dysregulation.In this article,we explore the epigenetic mechanisms shaping gene expression dur-ing tumorigenesis and cancer stem cell formation,with an emphasis on 3D chromatin architecture.Comparing the pluripotant stem cell state and epigenetic repro-gramming to dedifferentiation in cellular transformation provides intriguing insight to chromatin dynamics.We suggest that the 3D chromatin architecture could be used as a target for re-sensitizing cancer stem cells to therapeutics.     

Potential for Cripto-1 in defining stem cell-like characteristics in human malignant melanoma

The diagnosis of melanoma is becoming ever more frequent. Although surgical excision of early lesions is associated with relatively significant high cure rates, treatment modalities are largely unsuccessful for advanced disease. Characteristics such as cellular heterogeneity and plasticity, expression of certain molecules such as the multidrug resistance protein-1 (MDR1) or the aberrant expression of embryonic signaling molecules and morphogens like Nodal, important for self renewal and pluripotency, suggest that a stem cell-like population may reside in aggressive melanomas. This perspective focuses on preliminary findings obtained in our laboratory which indicate that the expression of the Nodal co-receptor, Cripto-1, in a subset of malignant melanoma cells may be exploited to identify possible melanoma stem cells (MSC). In fact, the use of anti-Cripto-1 antibodies to cell sort Cripto-1-positive cells in the metastatic melanoma cell line C8161 has identified a slow growing, sphere forming subpopulation that expresses increased levels of Oct4, Nanog and MDR1. If current in vivo studies confirm the self renewal and tumorigenic characteristics of these cells, the expression of Cripto-1 may represent a useful marker to identify cancer stem cells in melanoma, and possibly other aggressive tumors as well.     

Cancer stem cell plasticity and tumor hierarchy

The origins of the complex process of intratumoral heterogeneity have been highly debated and different cellular mechanisms have been hypothesized to account for the diversity within a tumor. The clonal evolution and cancer stem cell(CSC) models have been proposed as drivers of this heterogeneity. However, the concept of cancer stem cell plasticity and bidirectional conversion between stem and non-stem cells has added additional complexity to these highly studied paradigms and may help explain the tumor heterogeneity observed in solid tumors. The process of cancer stem cell plasticity in which cancer cel s harbor the dynamic ability of shifting from a non-CSC state to a CSC state and vice versa may be modulated by specific microenvironmental signals and cellular interactions arising in the tumor niche. In addition to promoting CSC plasticity, these interactions may contribute to the cellular transformation of tumor cells and affect response to chemotherapeutic and radiation treatments by providing CSCs protection from these agents. Herein, we review the literature in support of this dynamic CSC state, discuss the effectors of plasticity, and examine their role in the development and treatment of cancer.     

Understanding cancer stem cell heterogeneity and plasticity

Heterogeneity is an omnipresent feature of mammalian cells in vitro and in vivo. It has been recently realized that even mouse and human embryonic stem cells under the best culture conditions are heterogeneous containing pluripotent as well as partially committed cells. Somatic stem cells in adult organs are also heterogeneous, containing many subpopulations of self-renewing cells with distinct regenerative capacity. The differentiated progeny of adult stem cells also retain significant developmental plasticity that can be induced by a wide variety of experimental approaches. Like normal stem cells, recent data suggest that cancer stem cells (CSCs) similarly display significant phenotypic and functional heterogeneity, and that the CSC progeny can manifest diverse plasticity. Here, I discuss CSC heterogeneity and plasticity in the context of tumor development and progression, and by comparing with normal stem cell development. Appreciation of cancer cell plasticity entails a revision to the earlier concept that only the tumorigenic subset in the tumor needs to be targeted. By understanding the interrelationship between CSCs and their differentiated progeny, we can hope to develop better therapeutic regimens that can prevent the emergence of tumor cell variants that are able to found a new tumor and distant metastases.     

The Nerve Growth Factor Receptor CD271 Is Crucial to Maintain Tumorigenicity and Stem-Like Properties of Melanoma Cells

Background

Large-scale genomic analyses of patient cohorts have revealed extensive heterogeneity between individual tumors, contributing to treatment failure and drug resistance. In malignant melanoma, heterogeneity is thought to arise as a consequence of the differentiation of melanoma-initiating cells that are defined by cell-surface markers like CD271 or CD133.

Results

Here we confirmed that the nerve growth factor receptor (CD271) is a crucial determinant of tumorigenicity, stem-like properties, heterogeneity and plasticity in melanoma cells. Stable shRNA mediated knock-down of CD271 in patient-derived melanoma cells abrogated their tumor-initiating and colony-forming capacity. A genome-wide expression profiling and gene-set enrichment analysis revealed novel connections of CD271 with melanoma-associated genes like CD133 and points to a neural crest stem cell (NCSC) signature lost upon CD271 knock-down. In a meta-analysis we have determined a shared set of 271 differentially regulated genes, linking CD271 to SOX10, a marker that specifies the neural crest. To dissect the connection of CD271 and CD133 we have analyzed 10 patient-derived melanoma-cell strains for cell-surface expression of both markers compared to established cell lines MeWo and A375. We found CD271⁺ cells in the majority of cell strains analyzed as well as in a set of 16 different patient-derived melanoma metastases. Strikingly, only 2/12 cell strains harbored a CD133⁺ sub-set that in addition comprised a fraction of cells of a CD271⁺/CD133⁺ phenotype. Those cells were found in the label-retaining fraction and in vitro deduced from CD271⁺ but not CD271 knock-down cells.

Conclusions

Our present study provides a deeper insight into the regulation of melanoma cell properties and points CD271 out as a regulator of several melanoma-associated genes. Further, our data strongly suggest that CD271 is a crucial determinant of stem-like properties of melanoma cells like colony-formation and tumorigenicity.     

DDX3X Induces Primary EGFR-TKI Resistance Based on Intratumor Heterogeneity in Lung Cancer Cells Harboring EGFR-Activating Mutations

The specific mechanisms how lung cancer cells harboring epidermal growth factor receptor (EGFR) activating mutations can survive treatment with EGFR-tyrosine kinase inhibitors (TKIs) until they eventually acquire treatment-resistance genetic mutations are unclear. The phenotypic diversity of cancer cells caused by genetic or epigenetic alterations (intratumor heterogeneity) confers treatment failure and may foster tumor evolution through Darwinian selection. Recently, we found DDX3X as the protein that was preferentially expressed in murine melanoma with cancer stem cell (CSC)-like phenotypes by proteome analysis. In this study, we transfected PC9, human lung cancer cells harboring EGFR exon19 deletion, with cDNA encoding DDX3X and found that DDX3X, an ATP-dependent RNA helicase, induced CSC-like phenotypes and the epithelial-mesenchymal transition (EMT) accompanied with loss of sensitivity to EGFR-TKI. DDX3X expression was associated with upregulation of Sox2 and increase of cancer cells exhibiting CSC-like phenotypes, such as anchorage-independent proliferation, strong expression of CD44, and aldehyde dehydrogenase (ALDH). The EMT with switching from E-cadherin to N-cadherin was also facilitated by DDX3X. Either ligand-independent or ligand-induced EGFR phosphorylation was inhibited in lung cancer cells that strongly expressed DDX3X. Lack of EGFR signal addiction resulted in resistance to EGFR-TKI. Moreover, we found a small nonadherent subpopulation that strongly expressed DDX3X accompanied by the same stem cell-like properties and the EMT in parental PC9 cells. The unique subpopulation lacked EGFR signaling and was highly resistant to EGFR-TKI. In conclusion, our data indicate that DDX3X may play a critical role for inducing phenotypic diversity, and that treatment targeting DDX3X may overcome primary resistance to EGFR-TKI resulting from intratumor heterogeneity.     

Novel Functions of CD147 in the Mitochondria Exacerbates Melanoma Metastasis

Melanoma is an aggressive form of skin cancer characterized by rapid invasion and metastasis. CD147 is known to be functioning in cell invasion. In this study, we showed that CD147 was translocated from the cell membrane to the mitochondria in advanced melanoma. Melanoma patients with CD147 localized to the mitochondria confer a worse prognosis. The mitochondrial CD147 levels are correlated with the invasion potential of various melanoma cell lines as well as mitochondrial energy metabolism. Depletion of CD147 decreased the activity of mitochondrial complex V. STRING analysis for protein-protein interaction networks (PPIN) in CD147-depleted melanoma cells showed that mitochondrial proteins HSP60 and ATP5B, a subunit of mitochondrial complex V, were node proteins. HSP60 upregulation was correlated with a worse prognosis of melanoma patients. Co-immunoprecipitation (Co-IP) assay indicates that CD147 interacts with HSP60. These data suggested that mitochondrial CD147 may prompt HSP60 to activate ATP5B, thereby promoting the mitochondrial aerobic oxidation and the invasive abilities of melanoma cells. Correlation analysis of the data acquired from patients was helpful to draw a 5-year survival curve for patients who screened positive and negative for mitochondrial CD147. This study unravels the function of CD147 in tumor invasion and highlights it as a potential tumor therapeutic target.     

Stacking the DEK: From chromatin topology to cancer stem cells

Stem cells are essential for development and tissue maintenance and display molecular markers and functions distinct from those of differentiated cell types in a given tissue. Malignant cells that exhibit stem cell-like activities have been detected in many types of cancers and have been implicated in cancer recurrence and drug resistance. Normal stem cells and cancer stem cells have striking commonalities, including shared cell surface markers and signal transduction pathways responsible for regulating quiescence vs. proliferation, self-renewal, pluripotency and differentiation. As the search continues for markers that distinguish between stem cells, progenitor cells and cancer stem cells, growing evidence suggests that a unique chromatin-associated protein called DEK may confer stem cell-like qualities. Here, we briefly describe current knowledge regarding stem and progenitor cells. We then focus on new findings that implicate DEK as a regulator of stem and progenitor cell qualities, potentially through its unusual functions in the regulation of local or global chromatin organization.     

Phenotyping of Human Melanoma Cells Reveals a Unique Composition of Receptor Targets and a Subpopulation Co-Expressing ErbB4, EPO-R and NGF-R

Malignant melanoma is a life-threatening skin cancer increasingly diagnosed in the western world. In advanced disease the prognosis is grave. Growth and metastasis formation in melanomas are regulated by a network of cytokines, cytokine-receptors, and adhesion molecules. However, little is known about surface antigens and target expression profiles in human melanomas. We examined the cell surface antigen profile of human skin melanoma cells by multicolor flow cytometry, and compared their phenotype with 4 melanoma cell lines (A375, 607B, Mel-Juso, SK-Mel28). Melanoma cells were defined as CD45−/CD31− cells co-expressing one or more melanoma-related antigens (CD63, CD146, CD166). In most patients, melanoma cells exhibited ErbB3/Her3, CD44/Pgp-1, ICAM-1/CD54 and IGF-1-R/CD221, but did not express CD20, ErbB2/Her2, KIT/CD117, AC133/CD133 or MDR-1/CD243. Melanoma cell lines were found to display a similar phenotype. In most patients, a distinct subpopulation of melanoma cells (4–40%) expressed the erythropoietin receptor (EPO-R) and ErbB4 together with PD-1 and NGF-R/CD271. Both the EPO-R+ and EPO-R− subpopulations produced melanoma lesions in NOD/SCID IL-2Rgamma^null (NSG) mice in first and secondary recipients. Normal skin melanocytes did not express ErbB4 or EPO-R, but expressed a functional KIT receptor (CD117) as well as NGF-R, ErbB3/Her3, IGF-1-R and CD44. In conclusion, melanoma cells display a unique composition of surface target antigens and cytokine receptors. Malignant transformation of melanomas is accompanied by loss of KIT and acquisition of EPO-R and ErbB4, both of which are co-expressed with NGF-R and PD-1 in distinct subfractions of melanoma cells. However, expression of EPO-R/ErbB4/PD-1 is not indicative of a selective melanoma-initiating potential.     

Melanoma chemotherapy leads to the selection of ABCB5-expressing cells

Metastatic melanoma is the most aggressive skin cancer. Recently, phenotypically distinct subpopulations of tumor cells were identified. Among them, ABCB5-expressing cells were proposed to display an enhanced tumorigenicity with stem cell-like properties. In addition, ABCB5(+) cells are thought to participate to chemoresistance through a potential efflux function of ABCB5. Nevertheless, the fate of these cells upon drugs that are used in melanoma chemotherapy remains to be clarified. Here we explored the effect of anti-melanoma treatments on the ABCB5-expressing cells. Using a melanoma xenograft model (WM266-4), we observed in vivo that ABCB5-expressing cells are enriched after a temozolomide treatment that induces a significant tumor regression. These results were further confirmed in a preliminary study conducted on clinical samples from patients that received dacarbazine. In vitro, we showed that ABCB5-expressing cells selectively survive when exposed to dacarbazine, the reference treatment of metastatic melanoma, but also to vemurafenib, a new inhibitor of the mutated kinase V600E BRAF and other various chemotherapeutic drugs. Our results show that anti-melanoma chemotherapy might participate to the chemoresistance acquisition by selecting tumor cell subpopulations expressing ABCB5. This is of particular importance in understanding the relapses observed after anti-melanoma treatments and reinforces the interest of ABCB5 and ABCB5-expressing cells as potential therapeutic targets in melanoma.     

The Human Melanoma Side Population Displays Molecular and Functional Characteristics of Enriched Chemoresistance and Tumorigenesis

Melanoma remains the most lethal skin cancer, mainly because of high resistance to therapy. Side population (SP) cells are found in many types of cancer and are usually enriched in therapy-resistant as well as tumorigenic cells. Here, we identified a Hoechst dye-effluxing SP in a large series of human melanoma samples representing different progression phases. The SP size did not change with disease stage but was correlated with the prognostic “Breslow’s depth” in the primary (cutaneous) tumors. When injected into immunodeficient mice, the SP generated larger tumors than the bulk “main population” (MP) melanoma cells in two consecutive generations, and showed tumorigenic capacity at lower cell numbers than the MP. In addition, the SP reconstituted the heterogeneous composition of the human A375 melanoma cell line, and its clonogenic activity was 2.5-fold higher than that of the MP. Gene-expression analysis revealed upregulated expression in the melanoma SP (versus the MP) of genes associated with chemoresistance and anti-apoptosis. Consistent with these molecular characteristics, the SP increased in proportion when A375 cells were exposed to the melanoma standard chemotherapeutic agent dacarbazine, and to the aggravating condition of hypoxia. In addition, the SP showed enhanced expression of genes related to cell invasion and migration, as well as to putative (melanoma) cancer stem cells (CSC) including ABCB1 and JARID1B. ABCB1 immunoreactivity was detected in a number of tumor cells in human melanomas, and in particular in clusters at the invasive front of the primary tumors. Together, our findings support that the human melanoma SP is enriched in tumorigenic and chemoresistant capacity, considered key characteristics of CSC. The melanoma SP may therefore represent an interesting therapeutic target.