The establishment of epithelial architecture is a complex process involving cross-talk between cells and the basement membrane. Basement membrane assembly requires integrin activity but the role of the associated actomyosin cytoskeleton is poorly understood. Here, we identify the actin-bundling protein T-plastin (Pls3) as a regulator of basement membrane assembly and epidermal morphogenesis. In utero depletion of Pls3 transcripts in mouse embryos caused basement membrane and polarity defects in the epidermis but had little effect on cell adhesion and differentiation. Loss-of-function experiments demonstrated that the apicobasal polarity defects were secondary to the disruption of the basement membrane. However, the basement membrane itself was profoundly sensitive to subtle perturbations in the actin cytoskeleton. We further show that Pls3 localized to the cell cortex, where it was essential for the localization and activation of myosin II. Inhibition of myosin II motor activity disrupted basement membrane organization. Our results provide insights into the regulation of cortical actomyosin and its importance for basement membrane assembly and skin morphogenesis.

Previous studies from our lab demonstrated that the crosstalk between brain-metastasizing melanoma cells and microglia, the macrophage-like cells of the central nervous system, fuels progression to metastasis. In the present study, an in-depth investigation of melanoma-microglia interactions elucidated a pro-metastatic molecular mechanism that drives a vicious melanoma-brain-metastasis cycle. We employed RNA-Sequencing, HTG miRNA whole transcriptome assay, and reverse phase protein arrays (RPPA) to analyze the impact of melanoma-microglia interactions on sustainability and progression of four different human brain-metastasizing melanoma cell lines. Microglia cells exposed to melanoma-derived IL-6 exhibited upregulated levels of STAT3 phosphorylation and SOCS3 expression, which, in turn, promoted melanoma cell viability and metastatic potential. IL-6/STAT3 pathway inhibitors diminished the pro-metastatic functions of microglia and reduced melanoma progression. SOCS3 overexpression in microglia cells evoked microglial support in melanoma brain metastasis by increasing melanoma cell migration and proliferation. Different melanomas exhibited heterogeneity in their microglia-activating capacity as well as in their response to microglia-derived signals. In spite of this reality and based on the results of the present study, we concluded that the activation of the IL-6/STAT3/SOCS3 pathway in microglia is a major mechanism by which reciprocal melanoma-microglia signaling engineers the interacting microglia to reinforce the progression of melanoma brain metastasis. This mechanism may operate differently in different melanomas.

RNF5 is implicated in ERAD and in negative regulation of macroautophagy/autophagy. To better understand the function of RNF-5 under ER-stress conditions, we studied the ability of Caenorhabditis elegans rnf-5(tm794) mutant animals to cope with stress in the background of impaired UPR machinery. We demonstrate that downregulation of RNF-5 decreased sensitivity to tunicamycin both in wild type and in an ire-1 mutant. Double-mutant rnf-5;ire-1 animals showed increased starvation resistance and extended lifespan when compared to the ire-1 mutant. This partial rescue of ire-1 required functional autophagy. Downregulation of RNF-5 rescued ER maturation defects and protein secretion of a DAF-28::GFP intestinal reporter in the ire-1 background. Proteomics and functional studies revealed an increase in lysosomal protease levels, in the frequency of intestinal lysosomes, and in lysosomal protease activity in rnf-5(tm794) animals. Together, these data suggest that RNF-5 is a negative regulator of ER stress, and that inactivation of RNF-5 promotes IRE-1-independent elevation of ER capacity.

Mitotic spindle orientation (SO) is a conserved mechanism that governs cell fate and tissue morphogenesis. In the developing epidermis, a balance between self-renewing symmetric divisions and differentiative asymmetric divisions is necessary for normal development. While the cellular machinery that executes SO is well characterized, the extrinsic cues that guide it are poorly understood. Here, we identified the basal cell adhesion molecule (BCAM), a β1 integrin coreceptor, as a novel regulator of epidermal morphogenesis. In utero RNAi-mediated depletion of Bcam in the mouse embryo did not hinder β1 integrin distribution or cell adhesion and polarity. However, Bcam depletion promoted apoptosis, thinning of the epidermis, and symmetric cell division, and the defects were reversed by concomitant overexpression of the apoptosis inhibitor Xiap. Moreover, in mosaic epidermis, depletion of Bcam or Xiap induced symmetric divisions in neighboring wild-type cells. These results identify apoptosis and epidermal architecture as extrinsic cues that guide SO in the developing epidermis.

Reciprocal signaling between melanoma brain metastatic (MBM) cells and microglia reprograms the phenotype of both interaction partners, including upregulation of the transcription factor JunB in microglia. Here, we aimed to elucidate the impact of microglial JunB upregulation on MBM progression. For molecular profiling, we employed RNA-seq and reverse-phase protein array (RPPA). To test microglial JunB functions, we generated microglia variants stably overexpressing JunB (JunB^hi) or with downregulated levels of JunB (JunB^lo). Melanoma-derived factors, namely leukemia inhibitory factor (LIF), controlled JunB upregulation through Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) signaling. The expression levels of JunB in melanoma-associated microglia were heterogeneous. Flow cytometry analysis revealed the existence of basal-level JunB-expressing microglia alongside microglia highly expressing JunB. Proteomic profiling revealed a differential protein expression in JunB^hi and JunB^lo cells, namely the expression of microglia activation markers Iba-1 and CD150, and the immunosuppressive molecules SOCS3 and PD-L1. Functionally, JunB^hi microglia displayed decreased migratory capacity and phagocytic activity. JunB^lo microglia reduced melanoma proliferation and migration, while JunB^hi microglia preserved the ability of melanoma cells to proliferate in three-dimensional co-cultures, that was abrogated by targeting leukemia inhibitory factor receptor (LIFR) in control microglia–melanoma spheroids. Altogether, these data highlight a melanoma-mediated heterogenous effect on microglial JunB expression, dictating the nature of their functional involvement in MBM progression. Targeting microglia highly expressing JunB may potentially be utilized for MBM theranostics.

Background/Objectives: The interaction between brain-metastasizing melanoma cells and surrounding microglia shapes the immune tumor microenvironment and influences tumor progression. Gene expression analysis revealed that sphingosine-1-phosphate receptor 1 (S1PR1), encoding the S1P1 receptor, is upregulated in microglia upon interaction with melanoma cells. Here, we investigated the functions of S1P1 in microglia and its contribution to melanoma–microglia crosstalk. Methods: We examined the effects of S1P1 inhibition on microglia and four brain-metastasizing human melanoma cell lines in monocultures and co-cultures using the selective S1P1 antagonist NIBR0213 and S1PR1 gene knockdown. Results: We found that melanoma-secreted IL-6 upregulated S1PR1 expression in microglia. S1P1 inhibition increased expression of CD32, CD150, and CD163 in microglia; however, CD150 and CD163 upregulation was abolished in the presence of melanoma cells. S1P1 inhibition downregulated immunosuppressive and anti-inflammatory factors in microglia, including CD274, SOCS3, TGFBR1, TGFBR2, and JunB, promoting a pro-inflammatory phenotype. It also reduced viability of both melanoma and microglia cells, inducing apoptosis in melanoma-associated microglia, possibly via downregulation of CH25H, an upstream regulator of SREBPs. In co-cultures, melanoma cells were more sensitive than microglia to NIBR0213-induced growth arrest. In 3D spheroid cultures, NIBR0213 delayed melanoma–microglia aggregation. Combined treatment with the BRAF inhibitor Vemurafenib and NIBR0213 enhanced Vemurafenib efficacy in three of four melanoma lines. Conclusions: S1P1 contributes to the immunosuppressive phenotype of microglia. Inhibiting the S1P/S1P1 axis impairs viability and crosstalk between melanoma cells and tumor-activated microglia, offering a potential therapeutic strategy for melanoma brain metastases.