ZEB1 controls a lineage-specific transcriptional program essential for melanoma cell state transitions.

Cell plasticity sustains intra-tumor heterogeneity and treatment resistance in melanoma. Deciphering the transcriptional mechanisms governing reversible phenotypic transitions between proliferative/differentiated and invasive/stem-like states is required. Expression of the ZEB1 transcription factor is frequently activated in melanoma, where it fosters adaptive resistance to targeted therapies. Here, we performed a genome-wide characterization of ZEB1 transcriptional targets, by combining ChIP-sequencing and RNA-sequencing, upon phenotype switching in melanoma models. We identified and validated ZEB1 binding peaks in the promoter of key lineage-specific genes crucial for melanoma cell identity. Mechanistically, ZEB1 negatively regulates SOX10-MITF dependent proliferative/melanocytic programs and positively regulates AP-1 driven invasive and stem-like programs. Comparative analyses with breast carcinoma cells revealed lineage-specific ZEB1 binding, leading to the design of a more reliable melanoma-specific ZEB1 regulon. We then developed single-cell spatial multiplexed analyses to characterize melanoma cell states intra-tumoral heterogeneity in human melanoma samples. Combined with scRNA-Seq analyses, our findings confirmed increased ZEB1 expression in Neural-Crest-like cells and mesenchymal cells, underscoring its significance in vivo in both populations. Overall, our results define ZEB1 as a major transcriptional regulator of cell states transitions and provide a better understanding of lineage-specific transcriptional programs sustaining intra-tumor heterogeneity in melanoma.

Cancer Cell Biomechanical Properties Accompany Tspan8-Dependent Cutaneous Melanoma Invasion.

The intrinsic biomechanical properties of cancer cells remain poorly understood. To decipher whether cell stiffness modulation could increase melanoma cells' invasive capacity, we performed both in vitro and in vivo experiments exploring cell stiffness by atomic force microscopy (AFM). We correlated stiffness properties with cell morphology adaptation and the molecular mechanisms underlying epithelial-to-mesenchymal (EMT)-like phenotype switching. We found that melanoma cell stiffness reduction was systematically associated with the acquisition of invasive properties in cutaneous melanoma cell lines, human skin reconstructs, and Medaka fish developing spontaneous MAP-kinase-induced melanomas. We observed a systematic correlation of stiffness modulation with cell morphological changes towards mesenchymal characteristic gains. We accordingly found that inducing melanoma EMT switching by overexpressing the ZEB1 transcription factor, a major regulator of melanoma cell plasticity, was sufficient to decrease cell stiffness and transcriptionally induce tetraspanin-8-mediated dermal invasion. Moreover, ZEB1 expression correlated with Tspan8 expression in patient melanoma lesions. Our data suggest that intrinsic cell stiffness could be a highly relevant marker for human cutaneous melanoma development.

CDYL2 Epigenetically Regulates MIR124 to Control NF-κB/STAT3-Dependent Breast Cancer Cell Plasticity.

Epigenetic deregulation of gene transcription is central to cancer cell plasticity and malignant progression but remains poorly understood. We found that the uncharacterized epigenetic factor chromodomain on Y-like 2 (CDYL2) is commonly over-expressed in breast cancer, and that high CDYL2 levels correlate with poor prognosis. Supporting a functional role for CDYL2 in malignancy, it positively regulated breast cancer cell migration, invasion, stem-like phenotypes, and epithelial-to-mesenchymal transition. CDYL2 regulation of these plasticity-associated processes depended on signaling via p65/NF-κB and STAT3. This, in turn, was downstream of CDYL2 regulation of MIR124 gene transcription. CDYL2 co-immunoprecipitated with G9a/EHMT2 and GLP/EHMT1 and regulated the chromatin enrichment of G9a and EZH2 at MIR124 genes. We propose that CDYL2 contributes to poor prognosis in breast cancer by recruiting G9a and EZH2 to epigenetically repress MIR124 genes, thereby promoting NF-κB and STAT3 signaling, as well as downstream cancer cell plasticity and malignant progression.

Tspan8-ß-catenin positive feedback loop promotes melanoma invasion.

Due to its high proclivity to metastasize, and despite the recent development of targeted and immune therapy strategies, melanoma is still the deadliest form of skin cancer. Therefore, understanding the molecular mechanisms underlying melanoma invasion remains crucial. We previously characterized Tspan8 for its ability to prompt melanoma cell detachment from their microenvironment and trigger melanoma cell invasiveness, but the signaling events by which Tspan8 regulates the invasion process still remain unknown. Here, we demonstrated that ß-catenin stabilization is a molecular signal subsequent to the onset of Tspan8 expression, and that, in turn, ß-catenin triggers the direct transcriptional activation of Tspan8 expression, leading to melanoma invasion. Moreover, we showed that ß-catenin activation systematically correlates with a high expression of Tspan8 protein in melanoma lesions from transgenic Nras; bcat* mice, as well as in deep penetrating naevi, a type of human pre-melanoma neoplasm characterized by a combined activation of ß-catenin and MAP kinase signaling. Overall, our data suggest that ß-catenin and Tspan8 are part of a positive feedback loop, which sustains a high Tspan8 expression level, conferring to melanoma cells the invasive properties required for tumor progression and dissemination.

Tetraspanin 8 is a novel regulator of ILK-driven ß1 integrin adhesion and signaling in invasive melanoma cells.

Melanoma is well known for its propensity for lethal metastasis and resistance to most current therapies. Tumor progression and drug resistance depend to a large extent on the interplay between tumor cells and the surrounding matrix. We previously identified Tetraspanin 8 (Tspan8) as a critical mediator of melanoma invasion, whose expression is absent in healthy skin. The present study investigated whether Tspan8 may influence cell-matrix anchorage and regulate downstream molecular pathways leading to an aggressive behavior. Using silencing and ectopic expression strategies, we showed that Tspan8-mediated invasion of melanoma cells resulted from defects in cell-matrix anchorage by interacting with ß1 integrins and by interfering with their clustering, without affecting their surface or global expression levels. These effects were associated with impaired phosphorylation of integrin-linked kinase (ILK) and its downstream target Akt-S473, but not FAK. Specific blockade of Akt or ILK activity strongly affected cell-matrix adhesion. Moreover, expression of a dominant-negative form of ILK reduced ß1 integrin clustering and cell-matrix adhesion. Finally, we observed a tumor-promoting effect of Tspan8 in vivo and a mutually exclusive expression pattern between Tspan8 and phosphorylated ILK in melanoma xenografts and human melanocytic lesions. Altogether, the in vitro, in vivo and in situ data highlight a novel regulatory role for Tspan8 in melanoma progression by modulating cell-matrix interactions through ß1 integrin-ILK axis and establish Tspan8 as a negative regulator of ILK activity. These findings emphasize the importance of targeting Tspan8 as a means of switching from low- to firm-adhesive states, mandatory to prevent tumor dissemination.

MicroRNA-23b-3p regulates human keratinocyte differentiation through repression of TGIF1 and activation of the TGF-ß-SMAD2 signalling pathway.

MicroRNAs (miRNAs) are a class of short non-coding RNAs capable of repressing gene expression at the post-transcriptional level. miRNAs participate in the control of numerous cellular mechanisms, including skin homeostasis and epidermal differentiation. However, few miRNAs involved in these processes have been identified so far in human skin, and the gene networks they control remain largely unknown. Here, we focused on miR-23b-3p, a miRNA that is expressed during the late step of human keratinocyte differentiation. We report that miR-23b-3p silencing modulates epidermal differentiation in human skin reconstructs. The SMAD transcriptional corepressor TGIF1 was identified on bioinformatic analysis as a potential target of miR-23b-3p. Expression analysis and reporter gene assays confirmed direct regulation of TGIF1 expression by miR-23b-3p. Finally, we showed that miR-23-3p was able to activate TGF-ß signalling in human keratinocytes by increasing SMAD2 phosphorylation through TGIF1 repression. Taken together, these data identify miR-23b-3p as a new regulator of human epidermal differentiation in line with TGF-ß signalling.

GATA3 inhibits proliferation and induces expression of both early and late differentiation markers in keratinocytes of the human epidermis.

GATA3 belongs to the GATA transcription factor family and is a crucial regulator of lymphocyte differentiation. More recently, GATA3 was shown to be involved in skin cell lineage determination, in morphogenesis and maintenance of hair follicle keratinocytes as well as in epidermal barrier formation in mouse. In human, the potential role of GATA3 in the regulation of interfollicular epidermal homeostasis was still poorly explored. We thus investigated whether GATA3 could play a role in the regulation of proliferation and/or differentiation processes in human primary keratinocytes. We silenced the expression of GATA3 by small interfering RNA in either proliferating or differentiated human primary keratinocytes and analyzed the effect on cell proliferation and differentiation. We showed that GATA3 inhibition increased cell number, BrdU incorporation and expression of the proliferation markers PCNA and Ki67, demonstrating that GATA3 can inhibit keratinocyte proliferation. Moreover, GATA3 seems to be able to induce keratinocyte differentiation since its silencing leads to a decrease of both early and late differentiation markers such as Keratins 1 and 10, Involucrin and Loricrin. Our results demonstrate that GATA3 transcription factor inhibits proliferation and induces differentiation of primary keratinocytes, which suggest that it may regulate human interfollicular epidermal renewal.