LKB1-SIK2 loss drives uveal melanoma proliferation and hypersensitivity to SLC8A1 and ROS inhibition.

Metastatic uveal melanomas are highly resistant to all existing treatments. To address this critical issue, we performed a kinome-wide CRISPR-Cas9 knockout screen, which revealed the LKB1-SIK2 module in restraining uveal melanoma tumorigenesis. Functionally, LKB1 loss enhances proliferation and survival through SIK2 inhibition and upregulation of the sodium/calcium (Na+ /Ca2+ ) exchanger SLC8A1. This signaling cascade promotes increased levels of intracellular calcium and mitochondrial reactive oxygen species, two hallmarks of cancer. We further demonstrate that combination of an SLC8A1 inhibitor and a mitochondria-targeted antioxidant promotes enhanced cell death efficacy in LKB1- and SIK2-negative uveal melanoma cells compared to control cells. Our study also identified an LKB1-loss gene signature for the survival prognostic of patients with uveal melanoma that may be also predictive of response to the therapy combination. Our data thus identify not only metabolic vulnerabilities but also new prognostic markers, thereby providing a therapeutic strategy for particular subtypes of metastatic uveal melanoma.

Cholesterol efflux pathways hinder KRAS-driven lung tumor progenitor cell expansion.

Cholesterol efflux pathways could be exploited in tumor biology to unravel cancer vulnerabilities. A mouse model of lung-tumor-bearing KRASG12D mutation with specific disruption of cholesterol efflux pathways in epithelial progenitor cells promoted tumor growth. Defective cholesterol efflux in epithelial progenitor cells governed their transcriptional landscape to support their expansion and create a pro-tolerogenic tumor microenvironment (TME). Overexpression of the apolipoprotein A-I, to raise HDL levels, protected these mice from tumor development and dire pathologic consequences. Mechanistically, HDL blunted a positive feedback loop between growth factor signaling pathways and cholesterol efflux pathways that cancer cells hijack to expand. Cholesterol removal therapy with cyclodextrin reduced tumor burden in progressing tumor by suppressing the proliferation and expansion of epithelial progenitor cells of tumor origin. Local and systemic perturbations of cholesterol efflux pathways were confirmed in human lung adenocarcinoma (LUAD). Our results position cholesterol removal therapy as a putative metabolic target in lung cancer progenitor cells.

Secretion of IL1 by Dedifferentiated Melanoma Cells Inhibits JAK1-STAT3-Driven Actomyosin Contractility of Lymph Node Fibroblastic Reticular Cells.

Fibroblastic reticular cells (FRC) are immunologically specialized myofibroblasts that control the elasticity of the lymph node, in part through their contractile properties. Swelling of tumor-draining lymph nodes is a hallmark of lymphophilic cancers such as cutaneous melanoma. Melanoma displays high intratumoral heterogeneity with the coexistence of melanoma cells with variable differentiation phenotypes from melanocytic to dedifferentiated states. Factors secreted by melanoma cells promote premetastatic lymph node reprograming and tumor spreading. Elucidating the impact of the melanoma secretome on FRC could help identify approaches to prevent metastasis. Here we show that melanocytic and dedifferentiated melanoma cells differentially impact the FRC contractile phenotype. Factors secreted by dedifferentiated cells, but not by melanocytic cells, strongly inhibited actomyosin-dependent contractile forces of FRC by decreasing the activity of the RHOA-RHO-kinase (ROCK) pathway and the mechano-responsive transcriptional coactivator Yes1 associated transcriptional regulator (YAP). Transcriptional profiling and biochemical analyses indicated that actomyosin cytoskeleton relaxation in FRC is driven by inhibition of the JAK1-STAT3 pathway. This FRC relaxation was associated with increased FRC proliferation and activation and with elevated tumor invasion in vitro. The secretome of dedifferentiated melanoma cells also modulated the biomechanical properties of distant lymph node in premetastatic mouse models. Finally, IL1 produced by dedifferentiated cells was involved in the inhibition of FRC contractility. These data highlight the role of the JAK1-STAT3 and YAP pathways in spontaneous contractility of resting FRC. They also suggest that dedifferentiated melanoma cells specifically target FRC biomechanical properties to favor tumor spreading in the premetastatic lymph node niche. Targeting this remote communication could be an effective strategy to prevent metastatic spread of the disease. SIGNIFICANCE: Communication between dedifferentiated melanoma cells and lymph node fibroblasts reprograms the biomechanical properties of the premetastatic lymph node niche to promote tumor invasion. See related commentary by Lund, p. 1692.

Blockade of the pro-fibrotic reaction mediated by the miR-143/-145 cluster enhances the responses to targeted therapy in melanoma.

Lineage dedifferentiation toward a mesenchymal-like state displaying myofibroblast and fibrotic features is a common mechanism of adaptive and acquired resistance to targeted therapy in melanoma. Here, we show that the anti-fibrotic drug nintedanib is active to normalize the fibrous ECM network, enhance the efficacy of MAPK-targeted therapy, and delay tumor relapse in a preclinical model of melanoma. Acquisition of this resistant phenotype and its reversion by nintedanib pointed to miR-143/-145 pro-fibrotic cluster as a driver of this mesenchymal-like phenotype. Upregulation of the miR-143/-145 cluster under BRAFi/MAPKi therapy was observed in melanoma cells in vitro and in vivo and was associated with an invasive/undifferentiated profile. The 2 mature miRNAs generated from this cluster, miR-143-3p and miR-145-5p, collaborated to mediate transition toward a drug-resistant undifferentiated mesenchymal-like state by targeting Fascin actin-bundling protein 1 (FSCN1), modulating the dynamic crosstalk between the actin cytoskeleton and the ECM through the regulation of focal adhesion dynamics and mechanotransduction pathways. Our study brings insights into a novel miRNA-mediated regulatory network that contributes to non-genetic adaptive drug resistance and provides proof of principle that preventing MAPKi-induced pro-fibrotic stromal response is a viable therapeutic opportunity for patients on targeted therapy.

Metastasis-suppressor NME1 controls the invasive switch of breast cancer by regulating MT1-MMP surface clearance.

Membrane Type 1 Matrix Metalloprotease (MT1-MMP) contributes to the invasive progression of breast cancers by degrading extracellular matrix tissues. Nucleoside diphosphate kinase, NME1/NM23-H1, has been identified as a metastasis suppressor; however, its contribution to local invasion in breast cancer is not known. Here, we report that NME1 is up-regulated in ductal carcinoma in situ (DCIS) as compared to normal breast epithelial tissues. NME1 levels drop in microinvasive and invasive components of breast tumor cells relative to synchronous DCIS foci. We find a strong anti-correlation between NME1 and plasma membrane MT1-MMP levels in the invasive components of breast tumors, particularly in aggressive histological grade III and triple-negative breast cancers. Knockout of NME1 accelerates the invasive transition of breast tumors in the intraductal xenograft model. At the mechanistic level, we find that MT1-MMP, NME1 and dynamin-2, a GTPase known to require GTP production by NME1 for its membrane fission activity in the endocytic pathway, interact in clathrin-coated vesicles at the plasma membrane. Loss of NME1 function increases MT1-MMP surface levels by inhibiting endocytic clearance. As a consequence, the ECM degradation and invasive potentials of breast cancer cells are enhanced. This study identifies the down-modulation of NME1 as a potent driver of the in situ-to invasive transition during breast cancer progression.

UBTD1 regulates ceramide balance and endolysosomal positioning to coordinate EGFR signaling.

To adapt in an ever-changing environment, cells must integrate physical and chemical signals and translate them into biological meaningful information through complex signaling pathways. By combining lipidomic and proteomic approaches with functional analysis, we have shown that ubiquitin domain-containing protein 1 (UBTD1) plays a crucial role in both the epidermal growth factor receptor (EGFR) self-phosphorylation and its lysosomal degradation. On the one hand, by modulating the cellular level of ceramides through N-acylsphingosine amidohydrolase 1 (ASAH1) ubiquitination, UBTD1 controls the ligand-independent phosphorylation of EGFR. On the other hand, UBTD1, via the ubiquitination of Sequestosome 1 (SQSTM1/p62) by RNF26 and endolysosome positioning, participates in the lysosomal degradation of EGFR. The coordination of these two ubiquitin-dependent processes contributes to the control of the duration of the EGFR signal. Moreover, we showed that UBTD1 depletion exacerbates EGFR signaling and induces cell proliferation emphasizing a hitherto unknown function of UBTD1 in EGFR-driven human cell proliferation.

Single-cell RNA sequencing reveals intratumoral heterogeneity in primary uveal melanomas and identifies HES6 as a driver of the metastatic disease.

Intratumor heterogeneity has been recognized in numerous cancers as a major source of metastatic dissemination. In uveal melanomas, the existence and identity of specific subpopulations, their biological function and their contribution to metastasis remain unknown. Here, in multiscale analyses using single-cell RNA sequencing of six different primary uveal melanomas, we uncover an intratumoral heterogeneity at the genomic and transcriptomic level. We identify distinct transcriptional cell states and diverse tumor-associated populations in a subset of the samples. We also decipher a gene regulatory network underlying an invasive and poor prognosis state driven in part by the transcription factor HES6. HES6 heterogenous expression has been validated by RNAscope assays within primary human uveal melanomas, which further unveils the existence of these cells conveying a dismal prognosis in tumors diagnosed with a favorable outcome using bulk analyses. Depletion of HES6 impairs proliferation, migration and metastatic dissemination in vitro and in vivo using the chick chorioallantoic membrane assay, demonstrating the essential role of HES6 in uveal melanomas. Thus, single-cell analysis offers an unprecedented view of primary uveal melanoma heterogeneity, identifies bona fide biomarkers for metastatic cells in the primary tumor, and reveals targetable modules driving growth and metastasis formation. Significantly, our findings demonstrate that HES6 is a valid target to stop uveal melanoma progression.

Evidences of a Direct Relationship between Cellular Fuel Supply and Ciliogenesis Regulated by Hypoxic VDAC1-ΔC.

Metabolic flexibility is the ability of a cell to adapt its metabolism to changes in its surrounding environment. Such adaptability, combined with apoptosis resistance provides cancer cells with a survival advantage. Mitochondrial voltage-dependent anion channel 1 (VDAC1) has been defined as a metabolic checkpoint at the crossroad of these two processes. Here, we show that the hypoxia-induced cleaved form of VDAC1 (VDAC1-ΔC) is implicated in both the up-regulation of glycolysis and the mitochondrial respiration. We demonstrate that VDAC1-ΔC, due to the loss of the putative phosphorylation site at serine 215, concomitantly with the loss of interaction with tubulin and microtubules, reprograms the cell to utilize more metabolites, favoring cell growth in hypoxic microenvironment. We further found that VDAC1-ΔC represses ciliogenesis and thus participates in ciliopathy, a group of genetic disorders involving dysfunctional primary cilium. Cancer, although not representing a ciliopathy, is tightly linked to cilia. Moreover, we highlight, for the first time, a direct relationship between the cilium and cancer cell metabolism. Our study provides the first new comprehensive molecular-level model centered on VDAC1-ΔC integrating metabolic flexibility, ciliogenesis, and enhanced survival in a hypoxic microenvironment.

Coronin 1C promotes triple-negative breast cancer invasiveness through regulation of MT1-MMP traffic and invadopodia function.

Membrane type 1-matrix metalloproteinase (MT1-MMP), a membrane-tethered protease, is key for matrix breakdown during cancer invasion and metastasis. Assembly of branched actin networks by the Arp2/3 complex is required for MT1-MMP traffic and formation of matrix-degradative invadopodia. Contrasting with the well-established role of actin filament branching factor cortactin in invadopodia function during cancer cell invasion, the contribution of coronin-family debranching factors to invadopodia-based matrix remodeling is not known. Here, we investigated the contribution of coronin 1C to the invasive potential of breast cancer cells. We report that expression of coronin 1C is elevated in invasive human breast cancers, correlates positively with MT1-MMP expression in relation with increased metastatic risk and is a new independent prognostic factor in breast cancer. We provide evidence that, akin to cortactin, coronin 1C is required for invadopodia formation and matrix degradation by breast cancer cells lines and for 3D collagen invasion by multicellular spheroids. Using intravital imaging of orthotopic human breast tumor xenografts, we find that coronin 1C accumulates in structures forming in association with collagen fibrils in the tumor microenvironment. Moreover, we establish the role of coronin 1C in the regulation of positioning and trafficking of MT1-MMP-positive endolysosomes. These results identify coronin 1C as a novel player of the multi-faceted mechanism responsible for invadopodia formation, MT1-MMP surface exposure and invasiveness in breast cancer cells.

Impaired PRC2 activity promotes transcriptional instability and favors breast tumorigenesis.

Alterations of chromatin modifiers are frequent in cancer, but their functional consequences often remain unclear. Focusing on the Polycomb protein EZH2 that deposits the H3K27me3 (trimethylation of Lys27 of histone H3) mark, we showed that its high expression in solid tumors is a consequence, not a cause, of tumorigenesis. In mouse and human models, EZH2 is dispensable for prostate cancer development and restrains breast tumorigenesis. High EZH2 expression in tumors results from a tight coupling to proliferation to ensure H3K27me3 homeostasis. However, this process malfunctions in breast cancer. Low EZH2 expression relative to proliferation and mutations in Polycomb genes actually indicate poor prognosis and occur in metastases. We show that while altered EZH2 activity consistently modulates a subset of its target genes, it promotes a wider transcriptional instability. Importantly, transcriptional changes that are consequences of EZH2 loss are predominantly irreversible. Our study provides an unexpected understanding of EZH2's contribution to solid tumors with important therapeutic implications.

ARF6-JIP3/4 regulate endosomal tubules for MT1-MMP exocytosis in cancer invasion.

Invasion of cancer cells into collagen-rich extracellular matrix requires membrane-tethered membrane type 1-matrix metalloproteinase (MT1-MMP) as the key protease for collagen breakdown. Understanding how MT1-MMP is delivered to the surface of tumor cells is essential for cancer cell biology. In this study, we identify ARF6 together with c-Jun NH2-terminal kinase-interacting protein 3 and 4 (JIP3 and JIP4) effectors as critical regulators of this process. Silencing ARF6 or JIP3/JIP4 in breast tumor cells results in MT1-MMP endosome mispositioning and reduces MT1-MMP exocytosis and tumor cell invasion. JIPs are recruited by Wiskott-Aldrich syndrome protein and scar homologue (WASH) on MT1-MMP endosomes on which they recruit dynein-dynactin and kinesin-1. The interaction of plasma membrane ARF6 with endosomal JIPs coordinates dynactin-dynein and kinesin-1 activity in a tug-of-war mechanism, leading to MT1-MMP endosome tubulation and exocytosis. In addition, we find that ARF6, MT1-MMP, and kinesin-1 are up-regulated in high-grade triple-negative breast cancers. These data identify a critical ARF6-JIP-MT1-MMP-dynein-dynactin-kinesin-1 axis promoting an invasive phenotype of breast cancer cells.

Control of MT1-MMP transport by atypical PKC during breast-cancer progression.

Dissemination of carcinoma cells requires the pericellular degradation of the extracellular matrix, which is mediated by membrane type 1-matrix metalloproteinase (MT1-MMP). In this article, we report a co-up-regulation and colocalization of MT1-MMP and atypical protein kinase C iota (aPKCι) in hormone receptor-negative breast tumors in association with a higher risk of metastasis. Silencing of aPKC in invasive breast-tumor cell lines impaired the delivery of MT1-MMP from late endocytic storage compartments to the surface and inhibited matrix degradation and invasion. We provide evidence that aPKCι, in association with MT1-MMP-containing endosomes, phosphorylates cortactin, which is present in F-actin-rich puncta on MT1-MMP-positive endosomes and regulates cortactin association with the membrane scission protein dynamin-2. Thus, cell line-based observations and clinical data reveal the concerted activity of aPKC, cortactin, and dynamin-2, which control the trafficking of MT1-MMP from late endosome to the plasma membrane and play an important role in the invasive potential of breast-cancer cells.

Endosomal WASH and exocyst complexes control exocytosis of MT1-MMP at invadopodia.

Remodeling of the extracellular matrix by carcinoma cells during metastatic dissemination requires formation of actin-based protrusions of the plasma membrane called invadopodia, where the trans-membrane type 1 matrix metalloproteinase (MT1-MMP) accumulates. Here, we describe an interaction between the exocyst complex and the endosomal Arp2/3 activator Wiskott-Aldrich syndrome protein and Scar homolog (WASH) on MT1-MMP­containing late endosomes in invasive breast carcinoma cells. We found that WASH and exocyst are required for matrix degradation by an exocytic mechanism that involves tubular connections between MT1-MMP­positive late endosomes and the plasma membrane in contact with the matrix. This ensures focal delivery of MT1-MMP and supports pericellular matrix degradation and tumor cell invasion into different pathologically relevant matrix environments. Our data suggest a general mechanism used by tumor cells to breach the basement membrane and for invasive migration through fibrous collagen-enriched tissues surrounding the tumor.

HDAC6 is required for invadopodia activity and invasion by breast tumor cells.

Invasion across tissue boundaries by metastatic tumor cells depends on the proteolytic degradation of the extracellular matrix, initiated by the formation of invadopodia, actin-driven membrane protrusions with matrix-degradative activity. Yet, mechanisms underlying invadopodia formation remain largely unknown. In this report, we examined the role of the histone deacetylase HDAC6 in invadopodia formation and invasion by breast cancer cells. Using small interfering RNA silencing of protein expression in highly invasive MDA-MB-231 breast adenocarcinoma cells, we show that HDAC6 is required for two-dimensional matrix proteolysis. In addition, we demonstrate that HDAC6 acts as a tubulin and cortactin deacetylase. We also report that the inhibition of HDAC6 by siRNA or treatment with HDAC inhibitor TSA results in a decreased invasion capacity of a three-dimensional type I collagen matrix by MDA-MB-231 cells. These data identify HDAC6 as a critical component of the invasive apparatus of tumor cells, in both two- and three-dimensional matrices.

Ankyrin G restricts ion channel diffusion at the axonal initial segment before the establishment of the diffusion barrier.

In mammalian neurons, the precise accumulation of sodium channels at the axonal initial segment (AIS) ensures action potential initiation. This accumulation precedes the immobilization of membrane proteins and lipids by a diffusion barrier at the AIS. Using single-particle tracking, we measured the mobility of a chimeric ion channel bearing the ankyrin-binding motif of the Nav1.2 sodium channel. We found that ankyrin G (ankG) limits membrane diffusion of ion channels when coexpressed in neuroblastoma cells. Site-directed mutants with decreased affinity for ankG exhibit increased diffusion speeds. In immature hippocampal neurons, we demonstrated that ion channel immobilization by ankG is regulated by protein kinase CK2 and occurs as soon as ankG accumulates at the AIS of elongating axons. Once the diffusion barrier is formed, ankG is still required to stabilize ion channels. In conclusion, our findings indicate that specific binding to ankG constitutes the initial step for Nav channel immobilization at the AIS membrane and precedes the establishment of the diffusion barrier.