CD73 controls Myosin II-driven invasion, metastasis, and immunosuppression in amoeboid pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) has a very poor prognosis because of its high propensity to metastasize and its immunosuppressive microenvironment. Using a panel of pancreatic cancer cell lines, three-dimensional (3D) invasion systems, microarray gene signatures, microfluidic devices, mouse models, and intravital imaging, we demonstrate that ROCK-Myosin II activity in PDAC cells supports a transcriptional program conferring amoeboid invasive and immunosuppressive traits and in vivo metastatic abilities. Moreover, we find that immune checkpoint CD73 is highly expressed in amoeboid PDAC cells and drives their invasive, metastatic, and immunomodulatory traits. Mechanistically, CD73 activates RhoA-ROCK-Myosin II downstream of PI3K. Tissue microarrays of human PDAC biopsies combined with bioinformatic analysis reveal that rounded-amoeboid invasive cells with high CD73-ROCK-Myosin II activity and their immunosuppressive microenvironment confer poor prognosis to patients. We propose targeting amoeboid PDAC cells as a therapeutic strategy.

Cytoskeletal Remodelling as an Achilles' Heel for Therapy Resistance in Melanoma.

Melanoma is an aggressive skin cancer with a poor prognosis when diagnosed late. MAPK-targeted therapies and immune checkpoint blockers benefit a subset of melanoma patients; however, acquired therapy resistance inevitably arises within a year. In addition, some patients display intrinsic (primary) resistance and never respond to therapy. There is mounting evidence that resistant cells adapt to therapy through the rewiring of cytoskeleton regulators, leading to a profound remodelling of the actomyosin cytoskeleton. Importantly, this renders therapy-resistant cells highly dependent on cytoskeletal signalling pathways for sustaining their survival under drug pressure, which becomes a vulnerability that can be exploited therapeutically. Here, we discuss the current knowledge on cytoskeletal pathways involved in mainly targeted therapy resistance and future avenues, as well as potential clinical interventions.

Rho GTPase signaling in cancer progression and dissemination.

Rho GTPases are a family of small G proteins that regulate a wide array of cellular processes related to their key roles controlling the cytoskeleton. Cancer is a multistep disease caused by the accumulation of genetic mutations and epigenetic alterations, from the initial stages of cancer development when cells in normal tissues undergo transformation, to the acquisition of invasive and metastatic traits, responsible for a large number of cancer related deaths. In this review, we discuss the role of Rho GTPase signaling in cancer in every step of disease progression. Rho GTPases contribute to tumor initiation and progression, by regulating proliferation and apoptosis, but also metabolism, senescence, and cancer cell stemness. Rho GTPases play a major role in cell migration and in the metastatic process. They are also involved in interactions with the tumor microenvironment and regulate inflammation, contributing to cancer progression. After years of intensive research, we highlight the importance of relevant models in the Rho GTPase field, and we reflect on the therapeutic opportunities arising for cancer patients.

A preclinical pipeline to evaluate migrastatics as therapeutic agents in metastatic melanoma.

BACKGROUND: Metastasis is a hallmark of cancer and responsible for most cancer deaths. Migrastatics were defined as drugs interfering with all modes of cancer cell invasion and thus cancers' ability to metastasise. First anti-metastatic treatments have recently been approved. METHODS: We used bioinformatic analyses of publicly available melanoma databases. Experimentally, we performed in vitro target validation (including 2.5D cell morphology analysis and mass spectrometric analysis of RhoA binding partners), developed a new traceable spontaneously metastasising murine melanoma model for in vivo validation, and employed histology (haematoxylin/eosin and phospho-myosin II staining) to confirm drug action in harvested tumour tissues. RESULTS: Unbiased and targeted bioinformatic analyses identified the Rho kinase (ROCK)-myosin II pathway and its various components as potentially relevant targets in melanoma. In vitro validation demonstrated redundancy of several RhoGEFs upstream of RhoA and confirmed ROCK as a druggable target downstream of RhoA. The anti-metastatic effects of two ROCK inhibitors were demonstrated through in vivo melanoma metastasis tracking and inhibitor effects also confirmed ex vivo by digital pathology. CONCLUSIONS: We proposed a migrastatic drug development pipeline. As part of the pipeline, we provide a new traceable spontaneous melanoma metastasis model for in vivo quantification of metastasis and anti-metastatic effects by non-invasive imaging.

WNT11-FZD7-DAAM1 signalling supports tumour initiating abilities and melanoma amoeboid invasion.

Melanoma is a highly aggressive tumour that can metastasize very early in disease progression. Notably, melanoma can disseminate using amoeboid invasive strategies. We show here that high Myosin II activity, high levels of ki-67 and high tumour-initiating abilities are characteristic of invasive amoeboid melanoma cells. Mechanistically, we find that WNT11-FZD7-DAAM1 activates Rho-ROCK1/2-Myosin II and plays a crucial role in regulating tumour-initiating potential, local invasion and distant metastasis formation. Importantly, amoeboid melanoma cells express both proliferative and invasive gene signatures. As such, invasive fronts of human and mouse melanomas are enriched in amoeboid cells that are also ki-67 positive. This pattern is further enhanced in metastatic lesions. We propose eradication of amoeboid melanoma cells after surgical removal as a therapeutic strategy.

CDC42EP5/BORG3 modulates SEPT9 to promote actomyosin function, migration, and invasion.

Fast amoeboid migration is critical for developmental processes and can be hijacked by cancer cells to enhance metastatic dissemination. This migratory behavior is tightly controlled by high levels of actomyosin contractility, but how it is coupled to other cytoskeletal components is poorly understood. Septins are increasingly recognized as novel cytoskeletal components, but details on their regulation and contribution to migration are lacking. Here, we show that the septin regulator Cdc42EP5 is consistently required for amoeboid melanoma cells to invade and migrate into collagen-rich matrices and locally invade and disseminate in vivo. Cdc42EP5 associates with actin structures, leading to increased actomyosin contractility and amoeboid migration. Cdc42EP5 affects these functions through SEPT9-dependent F-actin cross-linking, which enables the generation of F-actin bundles required for the sustained stabilization of highly contractile actomyosin structures. This study provides evidence that Cdc42EP5 is a regulator of cancer cell motility that coordinates actin and septin networks and describes a unique role for SEPT9 in melanoma invasion and metastasis.

What does not kill you makes you stronger: surviving anti-cancer therapies by cytoskeletal remodeling and Myosin II reactivation.

Myosin II and its regulator Rho-associated coiled-coil containing protein kinase (ROCK) are essential for cell invasion and metastatic dissemination. Our recent findings show that this molecular machinery is also involved in drug resistance in melanoma by playing a dual role: protection of tumor cells from reactive oxygen species (ROS) and DNA damage (intrinsic), and co-option of myeloid and lymphoid populations to establish immunosuppression (extrinsic).

Myosin II Reactivation and Cytoskeletal Remodeling as a Hallmark and a Vulnerability in Melanoma Therapy Resistance.

Despite substantial clinical benefit of targeted and immune checkpoint blockade-based therapies in melanoma, resistance inevitably develops. We show cytoskeletal remodeling and changes in expression and activity of ROCK-myosin II pathway during acquisition of resistance to MAPK inhibitors. MAPK regulates myosin II activity, but after initial therapy response, drug-resistant clones restore myosin II activity to increase survival. High ROCK-myosin II activity correlates with aggressiveness, identifying targeted therapy- and immunotherapy-resistant melanomas. Survival of resistant cells is myosin II dependent, regardless of the therapy. ROCK-myosin II ablation specifically kills resistant cells via intrinsic lethal reactive oxygen species and unresolved DNA damage and limits extrinsic myeloid and lymphoid immunosuppression. Efficacy of targeted therapies and immunotherapies can be improved by combination with ROCK inhibitors.

Regional Activation of Myosin II in Cancer Cells Drives Tumor Progression via a Secretory Cross-Talk with the Immune Microenvironment.

ROCK-Myosin II drives fast rounded-amoeboid migration in cancer cells during metastatic dissemination. Analysis of human melanoma biopsies revealed that amoeboid melanoma cells with high Myosin II activity are predominant in the invasive fronts of primary tumors in proximity to CD206+CD163+ tumor-associated macrophages and vessels. Proteomic analysis shows that ROCK-Myosin II activity in amoeboid cancer cells controls an immunomodulatory secretome, enabling the recruitment of monocytes and their differentiation into tumor-promoting macrophages. Both amoeboid cancer cells and their associated macrophages support an abnormal vasculature, which ultimately facilitates tumor progression. Mechanistically, amoeboid cancer cells perpetuate their behavior via ROCK-Myosin II-driven IL-1α secretion and NF-κB activation. Using an array of tumor models, we show that high Myosin II activity in tumor cells reprograms the innate immune microenvironment to support tumor growth. We describe an unexpected role for Myosin II dynamics in cancer cells controlling myeloid function via secreted factors.

T-type calcium channels drive migration/invasion in BRAFV600E melanoma cells through Snail1.

Melanoma is a malignant tumor derived from melanocytes. Once disseminated, it is usually highly resistant to chemotherapy and is associated with poor prognosis. We have recently reported that T-type calcium channels (TTCCs) are overexpressed in melanoma cells and play an important role in melanoma progression. Importantly, TTCC pharmacological blockers reduce proliferation and deregulate autophagy leading to apoptosis. Here, we analyze the role of autophagy during migration/invasion of melanoma cells. TTCC Cav3.1 and LC3-II proteins are highly expressed in BRAFV600E compared with NRAS mutant melanomas, both in cell lines and biopsies. Chloroquine, pharmacological blockade, or gene silencing of TTCCs inhibit the autophagic flux and impair the migration and invasion capabilities, specifically in BRAFV600E melanoma cells. Snail1 plays an important role in motility and invasion of melanoma cells. We show that Snail1 is strongly expressed in BRAFV600E melanoma cells and patient biopsies, and its expression decreases when autophagy is blocked. These results demonstrate a role of Snail1 during BRAFV600E melanoma progression and strongly suggest that targeting macroautophagy and, particularly TTCCs, might be a good therapeutic strategy to inhibit metastasis of the most common melanoma type (BRAFV600E).

Actomyosin contractility and collective migration: may the force be with you.

Collective cell migration is essential during physiological processes such as development or wound healing and in pathological conditions such as cancer dissemination. Cells migrating within multicellular tissues experiment different forces which play an intricate role during tissue formation, development and maintenance. How cells are able to respond to these forces depends largely on how they interact with the extracellular matrix. In this review, we focus on mechanics and mechanotransduction in collective migration. In particular, we discuss current knowledge on how cells integrate mechanical signals during collective migration and we highlight actomyosin contractility as a central hub coordinating mechanosensing and mechanotransduction responses.

Modes of invasion during tumour dissemination.

Cancer cell migration and invasion underlie metastatic dissemination, one of the major problems in cancer. Tumour cells exhibit a striking variety of invasion strategies. Importantly, cancer cells can switch between invasion modes in order to cope with challenging environments. This ability to switch migratory modes or plasticity highlights the challenges behind antimetastasis therapy design. In this Review, we present current knowledge on different tumour invasion strategies, the determinants controlling plasticity and arising therapeutic opportunities. We propose that targeting master regulators controlling plasticity is needed to hinder tumour dissemination and metastasis.

Reactivation of p53 by a Cytoskeletal Sensor to Control the Balance Between DNA Damage and Tumor Dissemination.

BACKGROUND: Abnormal cell migration and invasion underlie metastasis, and actomyosin contractility is a key regulator of tumor invasion. The links between cancer migratory behavior and DNA damage are poorly understood. METHODS: Using 3D collagen systems to recapitulate melanoma extracellular matrix, we analyzed the relationship between the actomyosin cytoskeleton of migrating cells and DNA damage. We used multiple melanoma cell lines and microarray analysis to study changes in gene expression and in vivo intravital imaging (n = 7 mice per condition) to understand how DNA damage impacts invasive behavior. We used Protein Tissue Microarrays (n = 164 melanomas) and patient databases (n = 354 melanoma samples) to investigate the associations between markers of DNA damage and actomyosin cytoskeletal features. Data were analyzed with Student's and multiple t tests, Mann-Whitney's test, one-way analysis of variance, and Pearson correlation. All statistical tests were two-sided. RESULTS: Melanoma cells with low levels of Rho-ROCK-driven actomyosin are subjected to oxidative stress-dependent DNA damage and ATM-mediated p53 protein stabilization. This results in a specific transcriptional signature enriched in DNA damage/oxidative stress responsive genes, including Tumor Protein p53 Inducible Protein 3 (TP53I3 or PIG3). PIG3, which functions in DNA damage repair, uses an unexpected catalytic mechanism to suppress Rho-ROCK activity and impair tumor invasion in vivo. This regulation was suppressed by antioxidants. Furthermore, PIG3 levels decreased while ROCK1/2 levels increased in human metastatic melanomas (ROCK1 vs PIG3; r = -0.2261, P < .0001; ROCK2 vs PIG3: r = -0.1381, P = .0093). CONCLUSIONS: The results suggest using Rho-kinase inhibitors to reactivate the p53-PIG3 axis as a novel therapeutic strategy; we suggest that the use of antioxidants in melanoma should be very carefully evaluated.

TGF-ß-Induced Transcription Sustains Amoeboid Melanoma Migration and Dissemination.

Cell migration underlies metastatic dissemination of cancer cells, and fast "amoeboid" migration in the invasive fronts of tumors is controlled by high levels of actomyosin contractility. How amoeboid migration is regulated by extracellular signals and sustained over time by transcriptional changes is not fully understood. Transforming growth factor ß (TGF-ß) is well known to promote epithelial-to-mesenchymal transition (EMT) and contribute to metastasis, but melanocytes are neural crest derivatives that have undergone EMT during embryonic development. Surprisingly, we find that in melanoma, TGF-ß promotes amoeboid features such as cell rounding, membrane blebbing, high levels of contractility, and increased invasion. Using genome-wide transcriptomics, we find that amoeboid melanoma cells are enriched in a TGF-ß-driven signature. We observe that downstream of TGF-ß, SMAD2 and its adaptor CITED1 control amoeboid behavior by regulating the expression of key genes that activate contractile forces. Moreover, CITED1 is highly upregulated during melanoma progression, and its high expression is associated with poor prognosis. CITED1 is coupled to a contractile-rounded, amoeboid phenotype in a panel of 16 melanoma cell lines, in mouse melanoma xenografts, and in 47 human melanoma patients. Its expression is also enriched in the invasive fronts of lesions. Functionally, we show how the TGF-ß-SMAD2-CITED1 axis promotes different steps associated with progression: melanoma detachment from keratinocytes, 2D and 3D migration, attachment to endothelial cells, and in vivo lung metastatic initial colonization and outgrowth. We propose a novel mechanism by which TGF-ß-induced transcription sustains actomyosin force in melanoma cells and thereby promotes melanoma progression independently of EMT.

Nuclear DICKKOPF-1 as a biomarker of chemoresistance and poor clinical outcome in colorectal cancer.

Sporadic colorectal cancer (CRC) insurgence and progression depend on the activation of Wnt/ß-catenin signaling. Dickkopf (DKK)-1 is an extracellular inhibitor of Wnt/ß-catenin signaling that also has undefined ß-catenin-independent actions. Here we report for the first time that a proportion of DKK-1 locates within the nucleus of healthy small intestine and colon mucosa, and of CRC cells at specific chromatin sites of active transcription. Moreover, we show that DKK-1 regulates several cancer-related genes including the cancer stem cell marker aldehyde dehydrogenase 1A1 (ALDH1A1) and Ral-binding protein 1-associated Eps domain-containing 2 (REPS2), which are involved in detoxification of chemotherapeutic agents. Nuclear DKK-1 expression is lost along CRC progression; however, it remains high in a subset (15%) of CRC patients (n = 699) and associates with decreased progression-free survival (PFS) after chemotherapy administration and overall survival (OS) [adjusted HR, 1.65; 95% confidence interval (CI), 1.23-2.21; P = 0.002)]. Overexpression of ALDH1A1 and REPS2 associates with nuclear DKK-1 expression in tumors and correlates with decreased OS (P = 0.001 and 0.014) and PFS. In summary, our findings demonstrate a novel location of DKK-1 within the cell nucleus and support a role of nuclear DKK-1 as a predictive biomarker of chemoresistance in colorectal cancer.

Rho GTPases modulate malignant transformation of tumor cells.

Rho GTPases are involved in the acquisition of all the hallmarks of cancer, which comprise 6 biological capabilities acquired during the development of human tumors. The hallmarks include proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, and activating invasion and metastasis programs, as defined by Hanahan and Weinberg. (1) Controlling these hallmarks are genome instability and inflammation. Emerging hallmarks are reprogramming of energy metabolism and evading immune destruction. To give a different view to the readers, we will not be focusing on invasion, metastasis, or cytoskeletal remodeling, but we will review here how Rho GTPases contribute to other hallmarks of cancer with a special emphasis on malignant transformation.

Diverse matrix metalloproteinase functions regulate cancer amoeboid migration.

Rounded-amoeboid cancer cells use actomyosin contractility driven by Rho-ROCK and JAK-STAT3 to migrate efficiently. It has been suggested that rounded-amoeboid cancer cells do not require matrix metalloproteinases (MMPs) to invade. Here we compare MMP levels in rounded-amoeboid and elongated-mesenchymal melanoma cells. Surprisingly, we find that rounded-amoeboid melanoma cells secrete higher levels of several MMPs, including collagenase MMP-13 and gelatinase MMP-9. As a result, rounded-amoeboid melanoma cells degrade collagen I more efficiently than elongated-mesenchymal cells. Furthermore, using a non-catalytic mechanism, MMP-9 promotes rounded-amoeboid 3D migration through regulation of actomyosin contractility via CD44 receptor. MMP-9 is upregulated in a panel of rounded-amoeboid compared with elongated-mesenchymal melanoma cell lines and its levels are controlled by ROCK-JAK-STAT3 signalling. MMP-9 expression increases during melanoma progression and it is particularly prominent in the invasive fronts of lesions, correlating with cell roundness. Therefore, rounded-amoeboid cells use both catalytic and non-catalytic activities of MMPs for invasion.

Cellular plasticity confers migratory and invasive advantages to a population of glioblastoma-initiating cells that infiltrate peritumoral tissue.

Glioblastoma (GBM) is associated with infiltration of peritumoral (PT) parenchyma by isolated tumor cells that leads to tumor regrowth. Recently, GBM stem-like or initiating cells (GICs) have been identified in the PT area, but whether these GICs have enhanced migratory and invasive capabilities compared with GICs from the tumor mass (TM) is presently unknown. We isolated GICs from the infiltrated PT tissue and the TM of three patients and found that PT cells have an advantage over TM cells in two-dimensional and three-dimensional migration and invasion assays. Interestingly, PT cells display a high plasticity in protrusion formation and cell shape and their migration is insensitive to substrate stiffness, which represent advantages to infiltrate microenvironments of different rigidity. Furthermore, mouse and chicken embryo xenografts revealed that only PT cells showed a dispersed distribution pattern, closely associated to blood vessels. Consistent with cellular plasticity, simultaneous Rac and RhoA activation are required for the enhanced invasive capacity of PT cells. Moreover, Rho GTPase signaling modulators αVß3 and p27 play key roles in GIC invasiveness. Of note, p27 is upregulated in TM cells and inhibits RhoA activity. Gene silencing of p27 increased the invasive capacity of TM GICs. Additionally, ß3 integrin is upregulated in PT cells. Blockade of dimeric integrin αVß3, a Rac activator, reduced the invasive capacity of PT GICs in vitro and abrogated the spreading of PT cells into chicken embryos. Thus, our results describe the invasive features acquired by a unique subpopulation of GICs that infiltrate neighboring tissue.

(V600E)BRAF promotes invasiveness of thyroid cancer cells by decreasing E-cadherin expression through a Snail-dependent mechanism.

BRAF is a main oncogene in human thyroid cancer. Here, we show that BRAF depletion by siRNA or inhibition of its activity by treatment with BRAF inhibitor PLX4720 decreases migration and invasion in thyroid cancer cells expressing oncogenic (V600E)BRAF through a MEK/ERK-dependent mechanism, since treatment with the MEK inhibitor U0126 exerts the same effect. Moreover, over-expression of (V600E)BRAF increases migration and invasion of wild-type BRAF thyroid cells. Using the same strategies, we demonstrate that these effects are mediated by upregulation of the transcriptional repressor Snail with a concomitant decrease of its target E-cadherin, both hallmarks of EMT. These results reveal a novel (V600E)BRAF-induced mechanism in thyroid tumours progression and provides a rationale for using the PLX4720 inhibitor to target (V600E)BRAF signalling to effectively control progression of thyroid cancer.

Emerging molecular targets in melanoma invasion and metastasis.

Metastatic cutaneous melanoma accounts for the majority of skin cancer deaths due to its aggressiveness and high resistance to current therapies. To efficiently metastasize, invasive melanoma cells need to change their cytoskeletal organization and alter contacts with the extracellular matrix and the surrounding stromal cells. Melanoma cells can use different migratory strategies depending on varying environments to exit the primary tumour mass and invade surrounding and later distant tissues. In this review, we have focused on tumour cell plasticity or the interconvertibility that melanoma cells have as one of the factors that contribute to melanoma metastasis. This has been an area of very intense research in the last 5 yr yielding a vast number of findings. We have therefore reviewed all the possible clinical opportunities that this new knowledge offers to both stratify and treat cutaneous malignant melanoma patients.