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.
Cell migration is a key procedure involved in many biological processes including embryological development, tissue formation, immune defense or inflammation, and cancer progression. How physical, ...chemical, and molecular aspects can affect cell motility is a challenge to understand migratory cells behavior.
assays are excellent approaches to extrapolate to
situations and study live cells behavior. Here we present four
protocols that describe step-by-step cell migration, invasion and adhesion strategies and their corresponding image data quantification. These current protocols are based on
wound healing assays (comparing traditional pipette tip-scratch assay vs. culture insert assay), 2D individual cell-tracking experiments by live cell imaging and
spreading and transwell assays. All together, they cover different phenotypes and hallmarks of cell motility and adhesion, providing orthogonal information that can be used either individually or collectively in many different experimental setups. These optimized protocols will facilitate physiological and cellular characterization of these processes, which may be used for fast screening of specific therapeutic cancer drugs for migratory function, novel strategies in cancer diagnosis, and for assaying new molecules involved in adhesion and invasion metastatic properties of cancer cells.
Cell migration is crucial for cancer dissemination. We find that AMP-activated protein kinase (AMPK) controls cell migration by acting as an adhesion sensing molecular hub. In 3-dimensional matrices, ...fast-migrating amoeboid cancer cells exert low adhesion/low traction linked to low ATP/AMP, leading to AMPK activation. In turn, AMPK plays a dual role controlling mitochondrial dynamics and cytoskeletal remodelling. High AMPK activity in low adhering migratory cells, induces mitochondrial fission, resulting in lower oxidative phosphorylation and lower mitochondrial ATP. Concurrently, AMPK inactivates Myosin Phosphatase, increasing Myosin II-dependent amoeboid migration. Reducing adhesion or mitochondrial fusion or activating AMPK induces efficient rounded-amoeboid migration. AMPK inhibition suppresses metastatic potential of amoeboid cancer cells in vivo, while a mitochondrial/AMPK-driven switch is observed in regions of human tumours where amoeboid cells are disseminating. We unveil how mitochondrial dynamics control cell migration and suggest that AMPK is a mechano-metabolic sensor linking energetics and the cytoskeleton.
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 β (TGF-β) 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-β 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-β-driven signature. We observe that downstream of TGF-β, 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-β-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-β-induced transcription sustains actomyosin force in melanoma cells and thereby promotes melanoma progression independently of EMT.
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•TGF-β-SMAD promotes amoeboid migration in melanoma•Downstream of TGF-β, the adaptor CITED1 controls actomyosin contractility•Amoeboid features correlate with CITED1 levels in cell lines, xenografts, and patients•TGF-β-SMAD-CITED1 transcriptional network controls melanoma metastatic ability
Cantelli et al. find that, in melanoma, TGF-β-SMAD-CITED1 controls amoeboid behavior through activation of a transcriptional program. As a result, melanoma cells detach from keratinocytes, increase their invasive potential, and efficiently colonize the lung. This is a new function of TGF-β, independent of epithelial-to-mesenchymal transition.
Background The combined use of total-body photography and digital dermatoscopy, named “two-step method of digital follow-up,” allowed the detection of incipient melanoma as a result of dermatoscopic ...or macroscopic changes during follow-up. Objective We sought to assess dermatoscopic features and dynamic changes leading to excision of melanocytic lesions during our 10-year experience of monitoring patients at high risk for melanoma. Methods We analyzed 1152 lesions excised during the surveillance of 618 patients at high risk for melanoma from 1999 to 2008. Results A total of 779 excised lesions had been previously recorded: 728 were removed because of dermatoscopic changes during follow-up and 51 were removed even though no significant change was noted. The remaining 373 excised lesions were new or undetected on previous total-body photography. A total of 98 melanomas were detected, 60 in the monitored lesions, and 38 among the “new” lesions. The most frequent dermatoscopic changes detected were asymmetric enlargement in almost 60% (n = 418), focal changes in structure in 197 (27%) and in pigmentation in 122 (17%), the latter two being more frequently seen in melanomas than in nevi (both P < .001). No significant differences were detected between dermatoscopic or histopathological characteristics of the melanomas in each group, with a considerable proportion of melanomas misclassified as benign in both groups (26.3% and 38.3%, respectively). Limitations The dermatoscopy pattern of stable lesions and the histopathology of lesions not removed were not included in the study. Conclusion The most frequent dermatoscopic features associated with melanoma were focal change in pigmentation or structure. Melanomas detected by dermatoscopic changes were remarkably similar to those detected in total-body photography. Almost 40% of melanomas diagnosed in individuals at high risk corresponded to lesions that were not under dermatoscopic surveillance.
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.
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•Invasive tumor fronts are enriched in rounded-amoeboid cancer cells high in Myosin II•Myosin II activity in these cells regulates an immunomodulatory secretome•The secreted cytokines and chemokines can induce tumor-promoting macrophages•ROCK-Myosin II and IL-1α/NF-κB cross-talk supports this secretory phenotype
Myosin II activation at the tumor edge promotes invasion and also a secretory phenotype that reshapes the local environment and vasculature to support tumor growth.
We solve the Ein–Lazarsfeld–Mustopa conjecture for the blow up of a projective space along a linear subspace. More precisely, let
X
be the blow up of
P
n
at a linear subspace and let
L
be any ample ...line bundle on
X
. We show that the syzygy bundle
M
L
defined as the kernel of the evalution map
H
0
(
X
,
L
)
⊗
O
X
→
L
is
L
-stable. In the last part of this note we focus on the rigidness of
M
L
to study the local shape of the moduli space around the point
M
L
.
Melanoma is a malignant neoplasia that is highly resistant to chemotherapy and radiotherapy and is associated with poor prognosis in advanced stage. Targeting melanoma that harbors the common ...BRAFV600E mutation with kinase inhibitors, such as vemurafenib, reduces tumor burden, but these tumors frequently acquire resistance to these drugs. We previously proposed that T-type calcium channel (TTCC) expression may serve as a biomarker for melanoma progression and prognosis, and we showed that TTCC blockers reduce migration and invasion rates because of autophagy blockade only in BRAFV600E-mutant melanoma cells. Here, we demonstrated that high expression of the TTCC Cav3.1 isoform is related to autophagic status in vemurafenib-resistant BRAFV600E-mutant melanoma cells and human biopsies, and in silico analysis revealed an enrichment of Cav3.1 expression in post-treatment melanomas. We also demonstrated that the TTCC blocker mibefradil induces apoptosis and impairs migration and invasion via inhibition of autophagy in resistant melanoma cells and mouse xenograft models. Moreover, we identified an association between PTEN status and Cav3.1 expression in these cells as a marker of sensitivity to combination therapy in resistant cells. Together, our results suggest that TTCC blockers offer a potential targeted therapy in resistant BRAFV600E-mutant melanoma and a therapeutic strategy to reduce progression toward BRAF inhibitor resistance.
Current strategies for preventing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are limited to nonpharmacologic interventions. Hydroxychloroquine has been proposed as a ...postexposure therapy to prevent coronavirus disease 2019 (Covid-19), but definitive evidence is lacking.
We conducted an open-label, cluster-randomized trial involving asymptomatic contacts of patients with polymerase-chain-reaction (PCR)-confirmed Covid-19 in Catalonia, Spain. We randomly assigned clusters of contacts to the hydroxychloroquine group (which received the drug at a dose of 800 mg once, followed by 400 mg daily for 6 days) or to the usual-care group (which received no specific therapy). The primary outcome was PCR-confirmed, symptomatic Covid-19 within 14 days. The secondary outcome was SARS-CoV-2 infection, defined by symptoms compatible with Covid-19 or a positive PCR test regardless of symptoms. Adverse events were assessed for up to 28 days.
The analysis included 2314 healthy contacts of 672 index case patients with Covid-19 who were identified between March 17 and April 28, 2020. A total of 1116 contacts were randomly assigned to receive hydroxychloroquine and 1198 to receive usual care. Results were similar in the hydroxychloroquine and usual-care groups with respect to the incidence of PCR-confirmed, symptomatic Covid-19 (5.7% and 6.2%, respectively; risk ratio, 0.86 95% confidence interval, 0.52 to 1.42). In addition, hydroxychloroquine was not associated with a lower incidence of SARS-CoV-2 transmission than usual care (18.7% and 17.8%, respectively). The incidence of adverse events was higher in the hydroxychloroquine group than in the usual-care group (56.1% vs. 5.9%), but no treatment-related serious adverse events were reported.
Postexposure therapy with hydroxychloroquine did not prevent SARS-CoV-2 infection or symptomatic Covid-19 in healthy persons exposed to a PCR-positive case patient. (Funded by the crowdfunding campaign YoMeCorono and others; BCN-PEP-CoV2 ClinicalTrials.gov number, NCT04304053.).
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