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

© The Author(s) 2021.[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.GOF’s lab was supported by Cancer Research UK [C48390/A21153], Worldwide Cancer Research [16-1153], and King’s Health Partners [King’s Medical Research Trust Joint Research Committee studentship to A.V.]. B.F. was supported by a King’s Health Partners studentship to V.S.M. and G.O.F. V.S.M.’s lab was supported by Cancer Research UK [C33043/A12065] and [C33043/A24478] (V.S.M., E.C.M., J.L.O., L.B. and GC), the Royal Society [RG110591] (V.S.M.), The Harry J. Lloyd Charitable Trust (J.L.O. and V.S.M.), the Barts Charity (V.S.M., J.L.O., O.M., I.R.H. and E.C.M.), the Fundacion Alfonso Martin Escudero and Marie Sklodowska-Curie Action [H2020-MSCA-IF-2014-EF-ST] (I.R.H.), and Fundacion Ramon Areces (E.C.M.). F.M. was supported by an MRC Career Development Award (MR/P009417/1). This work was further supported by the Department of Health (DoH) via the National Institute for Health Research (NIHR) Comprehensive Biomedical Research Centre award to King’s Health Partners, and the Wellcome/EPSRC Centre for Medical Engineering [WT203148/Z/16/Z]. Views expressed are those of the authors and not necessarily those of the NHS, NIHR or DoH

The Myosin II cytoskeleton as a new vulnerability in therapy-resistant melanoma

Trabajo presentado en VIB Conference: Tumor Heterogeneity, Plasticity and Therapy, celebrado en modalidad virtual del 05 al 06 de mayo de 2021.MAPK-targeted therapies (MAPKi) and immune checkpoint blockers (ICB) improve survival of subsets of melanoma patients. However, therapy resistance is a persistent problem. Cross-resistance to MAPKi and ICB has been suggested to be driven, in part, by common transcriptomic alterations in pathways controlling invasion and metastasis. We find that adaptation to treatment and acquisition of resistance to MAPKi involve cytoskeletal remodelling and changes in expression levels in the ROCK-Myosin II pathway, which plays a key role in cancer invasion and metastasis. Myosin II activity is decreased shortly after MAPK is blocked. However, resistant cells promptly restore Myosin II activity to increase survival, and this becomes a vulnerability, since survival of MAPKi- and ICB-resistant cells is highly dependent on ROCK-Myosin II. Efficacy of MAPKi and ICB can be improved by combination with ROCK inhibitors, which have a dual action by impairing melanoma cell survival (through induction of lethal reactive oxygen species and unresolved DNA damage) and myeloid- and lymphoiddriven immunosuppression, overcoming cross-resistance. In human tumours, high ROCK-Myosin II activity and their associated transcriptome identify MAPKi-, ICBresistant melanomas, and treatment-naïve melanomas with worse prognosis. Therefore, a subset of MAPKi- and ICB-resistant melanomas is intrinsically more susceptible to ROCK-Myosin II inhibition, suggesting clinical opportunities for combination therapies

The Myosin II cytoskeleton as a new vulnerability in therapy-resistant melanoma

Trabajo presentado en la EACR-AstraZeneca Virtual Conference ‘Drug Tolerant Persister Cells’, celebrada del 07 al 08 de diciembre de 2021.MAPK-targeted therapies (MAPKi) and immune checkpoint blockers (ICB) improve survival of subsets of melanoma patients. However, therapy resistance is a persistent problem. Cross-resistance to MAPKi and ICB may be driven by common transcriptomic alterations in pathways controlling invasion and metastasis. We find that adaptation to treatment and acquisition of resistance to MAPKi involve cytoskeletal remodelling and changes in expression levels in the ROCK-non-muscle Myosin II (NMII) pathway, which is essential for cancer invasion and metastasis. NMII activity is decreased shortly after MAPK is blocked. However, persister cells promptly restore NMII activity to increase survival, and this becomes a vulnerability, since survival of MAPKi- and ICB-resistant cells is highly dependent on ROCK-NMII. Efficacy of MAPKi and ICB can be improved by combination with ROCK inhibitors, which have a dual action by impairing melanoma cell survival (through induction of lethal reactive oxygen species and unresolved DNA damage) and reducing myeloid- and lymphoid-driven immunosuppression, ultimately overcoming cross-resistance. In human tumours, high ROCK-NMII levels identify MAPKi-, ICB-resistant melanomas, and treatment-naïve melanomas with worse prognosis. Therefore, a subset of MAPKi- and ICB-resistant melanomas is more susceptible to ROCK-NMII blockade, suggesting clinical opportunities for combination therapies

The Myosin II cytoskeleton as a new vulnerability in therapy-resistant melanoma

Trabajo presentado en el XIX Congreso de la Sociedad Española de Biología Celular, celebrado en Boadilla del Monte (España) del 26 al 29 de octubre de 2021.MAPK-targeted therapies (MAPKi) and immune checkpoint blockers (ICB) improve survival of subsets of melanoma patients. However, therapy resistance is a persistent problem. Cross-resistance to MAPKi and ICB has been suggested to be driven, in part, by common transcriptomic alterations in pathways controlling invasion and metastasis. We find that adaptation to treatment and acquisition of resistance to MAPKi involve cytoskeletal remodelling and changes in expression levels in the ROCK-Myosin II pathway, which plays a key role in cancer invasion and metastasis. Myosin II activity is decreased shortly after MAPK is blocked. However, resistant cells promptly restore Myosin II activity to increase survival, and this becomes a vulnerability, since survival of MAPKi- and ICB-resistant cells is highly dependent on ROCK-Myosin II. Efficacy of MAPKi and ICB can be improved by combination with ROCK inhibitors, which have a dual action by impairing melanoma cell survival (through induction of lethal reactive oxygen species, unresolved DNA damage and cell cycle arrest) and myeloid- and lymphoid-driven immunosuppression, ultimately overcoming cross-resistance. In human tumours, high ROCK-Myosin II activity and their associated transcriptome identify MAPKi-, ICB-resistant melanomas, and treatment-naïve melanomas with worse prognosis. Therefore, a subset of MAPKi- and ICB-resistant melanomas is intrinsically more susceptible to ROCK-Myosin II inhibition, suggesting clinical opportunities for combination therapies

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.The work was supported by Cancer Research UK (CRUK) C33043/A24478 (to V.S.-M., E.C.-M., O.M., and L.K.); Barts Charity (to V.S.M., R.S., and S.L.G.); The Harry J. Lloyd Charitable Trust (to J.L.O. and V.S.-M); and Comunidad de Madrid (Atraccion de Talento-2019-T1/BMD-13642) (to J.L.O.)

In vitro cell migration, invasion, and adhesion assays: From cell imaging to data analysis

© 2019 Pijuan, Barceló, Moreno, Maiques, Sisó, Marti, Macià and Panosa.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. In vitro assays are excellent approaches to extrapolate to in vivo situations and study live cells behavior. Here we present four in vitro protocols that describe step-by-step cell migration, invasion and adhesion strategies and their corresponding image data quantification. These current protocols are based on two-dimensional wound healing assays (comparing traditional pipette tip-scratch assay vs. culture insert assay), 2D individual cell-tracking experiments by live cell imaging and three-dimensional 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.This work was supported by grants from ISCIII/FEDER Una manera de hacer Europa (PI12/00260, PI15/00711 to RM and PI18/00573 to RM and AM) and CIBERONC. CB holds predoctoral fellowship from UdL-IRBLleida. AM holds postdoctoral fellowship from AECC

The non-muscle Myosin II cytoskeleton as a new vulnerability in therapy-resistant melanoma

Trabajo presentado en el 19th International Congress of the Society For Melanoma Research, celebrado en Edimburgo (Escocia) del 17 al 20 de octubre de 2022.MAPK-targeted therapies (MAPKi) and immune checkpoint blockers (ICB) improve survival of subsets of melanoma patients. However, therapy resistance is a persistent problem. Cross-resistance to MAPKi and ICB may be driven by common transcriptomic alterations in pathways controlling invasion and metastasis. We find that adaptation to treatment and acquisition of resistance to MAPKi involve cytoskeletal remodelling and changes in expression levels in the ROCK-non-muscle Myosin II (NMII) pathway, which is essential for cancer invasion and metastasis. Persister cells overactivate NMII to increase survival, and this becomes a vulnerability, since survival of MAPKiand ICB-resistant cells is highly dependent on ROCK-NMII. Efficacy of MAPKi and ICB can be improved by combination with ROCK inhibitors, which have a dual action by impairing melanoma cell survival (through induction of lethal reactive oxygen species and unresolved DNA damage) and reducing myeloid- and lymphoiddriven immunosuppression, ultimately overcoming cross-resistance. In human tumours, high ROCK-NMII levels identify MAPKi-, ICB-resistant melanomas, and treatment-naïve melanomas with worse prognosis. Therefore, a subset of MAPKi- and ICB-resistant melanomas is more susceptible to ROCK-NMII blockade, suggesting clinical opportunities for combination therapies