Krüppel-like factor 4 (KLF4) regulates the miR-183~96~182 cluster under physiologic and pathologic conditions

Cèl·lules mare embrionàries; Melanoma; MicroARNCélulas madre embrionarias; Melanoma; MicroARNEmbryonic stem cells; Melanoma; MicroRNAMicroRNAs (miRNAs) are a class of endogenous non-coding small RNAs that post-transcriptionally control the translation and stability of target mRNAs in a sequence-dependent manner. MiRNAs are essential for key cellular processes including proliferation, differentiation, cell death and metabolism, among others. Consequently, alterations of miRNA expression contribute to developmental defects and a myriad of diseases. The expression of miRNAs can be altered by several mechanisms including gene copy number alterations, aberrant DNA methylation, defects of the miRNA processing machinery or unscheduled expression of transcription factors. In this work, we sought to analyze the regulation of the miR-182 cluster, located at the 7q32 locus, which encodes three different miRNAs that are abundantly expressed in human embryonic stem cells and de-regulated in cancer. We have found that the Krüppel-like factor 4 (KLF4) directly regulates miR-182 cluster expression in human embryonic stem cells (hESCs) and in melanoma tumors, in which the miR-182 cluster is highly expressed and has a pro-metastatic role. Furthermore, higher KLF4 expression was found to be associated with metastatic progression and poor patient outcome. Loss of function experiments revealed that KLF4 is required for melanoma cell maintenance. These findings provide new insights into the regulation of the miR-182 cluster expression and new opportunities for therapeutic intervention in tumors in which the KLF4-miR-182 cluster axis is deregulated.This work was supported by NCI/NIH Grant (5R01CA155234), Instituto de Salud Carlos III (CP11/00052 and RD12/0036/0016) co-financed by the European Regional Development Fund (ERDF), and European Commission’s Framework Programme 7 through the Marie Curie Career Integration Grants

Engineering pH-Sensitive Stable Nanovesicles for Delivery of MicroRNA Therapeutics

Nanovesicles; Neuroblastoma; Pediatric cancerNanovesículas; Neuroblastoma; Cáncer pediátricoNanovesícules; Neuroblastoma; Càncer pediàtricMicroRNAs (miRNAs) are small non-coding endogenous RNAs, which are attracting a growing interest as therapeutic molecules due to their central role in major diseases. However, the transformation of these biomolecules into drugs is limited due to their unstability in the bloodstream, caused by nucleases abundantly present in the blood, and poor capacity to enter cells. The conjugation of miRNAs to nanoparticles (NPs) could be an effective strategy for their clinical delivery. Herein, the engineering of non-liposomal lipid nanovesicles, named quatsomes (QS), for the delivery of miRNAs and other small RNAs into the cytosol of tumor cells, triggering a tumor-suppressive response is reported. The engineered pH-sensitive nanovesicles have controlled structure (unilamellar), size (24 weeks), and are prepared by a green, GMP compliant, and scalable one-step procedure, which are all unavoidable requirements for the arrival to the clinical practice of NP based miRNA therapeutics. Furthermore, QS protect miRNAs from RNAses and when injected intravenously, deliver them into liver, lung, and neuroblastoma xenografts tumors. These stable nanovesicles with tunable pH sensitiveness constitute an attractive platform for the efficient delivery of miRNAs and other small RNAs with therapeutic activity and their exploitation in the clinics.The funding was received by Ministerio de Educación, Cultura y Deporte (Grant no. FPU16/01099), Ministerio de Economía, Industria y Competividad (Grants MAT2016-80820-R, MAT2016-80826-R and SAF2016-75241-R), the Ministry of Science and Innovation (MINECO) of Spain through grant PID2019-105622RB-I00, from Instituto de Salud Carlos III (Grant no. CP16/00006, PI17/00564, PI20/00530, DTS20/00018) (Co-funded by European Regional Development Fund/European Social Fund) “Investing in your future”), from the EuroNanoMed II platform through the NanoVax project, from CIBER-BBN through grant TAG-SMARTLY, Joan Petit Foundation, Asociación Matem Lo Bitxo and Asociación Española Contra el Cáncer (Grant no. LABAE18009SEGU), as well as, Generalitat de Catalunya through the Centres de Recerca de Catalunya (CERCA) programme and grant no. 2017-SGR-918, and from Agency for Management of University and Research Grants (AGAUR) (Grant no 2018LLAV0064 and SIFECAT IU68-010017). Furthermore, ICMAB-CSIC acknowledges support from the MINECO through the Severo Ochoa Programme for Centres of Excellence in R&D (SEV-2015-0496 and CEX2019-000917-S)

Structural disruption of BAF chromatin remodeller impairs neuroblastoma metastasis by reverting an invasiveness epigenomic program

Background Epigenetic programming during development is essential for determining cell lineages, and alterations in this programming contribute to the initiation of embryonal tumour development. In neuroblastoma, neural crest progenitors block their course of natural differentiation into sympathoadrenergic cells, leading to the development of aggressive and metastatic paediatric cancer. Research of the epigenetic regulators responsible for oncogenic epigenomic networks is crucial for developing new epigenetic-based therapies against these tumours. Mammalian switch/sucrose non-fermenting (mSWI/SNF) ATP-dependent chromatin remodelling complexes act genome-wide translating epigenetic signals into open chromatin states. The present study aimed to understand the contribution of mSWI/SNF to the oncogenic epigenomes of neuroblastoma and its potential as a therapeutic target. Methods Functional characterisation of the mSWI/SNF complexes was performed in neuroblastoma cells using proteomic approaches, loss-of-function experiments, transcriptome and chromatin accessibility analyses, and in vitro and in vivo assays. Results Neuroblastoma cells contain three main mSWI/SNF subtypes, but only BRG1-associated factor (BAF) complex disruption through silencing of its key structural subunits, ARID1A and ARID1B, impairs cell proliferation by promoting cell cycle blockade. Genome-wide chromatin remodelling and transcriptomic analyses revealed that BAF disruption results in the epigenetic repression of an extensive invasiveness-related expression program involving integrins, cadherins, and key mesenchymal regulators, thereby reducing adhesion to the extracellular matrix and the subsequent invasion in vitro and drastically inhibiting the initiation and growth of neuroblastoma metastasis in vivo. Conclusions We report a novel ATPase-independent role for the BAF complex in maintaining an epigenomic program that allows neuroblastoma invasiveness and metastasis, urging for the development of new BAF pharmacological structural disruptors for therapeutic exploitation in metastatic neuroblastoma

The oral KIF11 inhibitor 4SC-205 exhibits antitumor activity and potentiates standard and targeted therapies in primary and metastatic neuroblastoma models

Inhibidor de KIF11; Terapias dirigidas; MetástasisInhibidor de KIF11; Teràpies dirigides; MetàstasiKIF11 inhibitor; Targeted therapies; MetastasisIn summary, our study provides a rationale for the future therapeutic integration in clinical trials of 4SC-205, an structurally distinct oral KIF11 inhibitor that shows potent antitumor activity in multiple preclinical neuroblastoma models and sensitizes neuroblastoma cells to standard chemotherapy and specific neuroblastoma-targeted therapies.The financial support for this research was provided by Instituto de Salud Calos III (PI20/00530 to Miguel F. Segura; PI20/01107 to Rosa Noguera; PI17/02248 and CPII18/00027 to Anna Santamaria; PI19/01320 to Alberto Villanueva); Ministerio de Educación, Cultura y Deporte (Grant no. FPU16/01099 to Marc Masanas). This work was also supported by the Asociación NEN (Nico contra el cancer infantil 2017–PVR00157)

Engineering pH-Sensitive Stable Nanovesicles for Delivery of MicroRNA Therapeutics

MicroRNAs (miRNAs) are small non-coding endogenous RNAs, which are attracting a growing interest as therapeutic molecules due to their central role in major diseases. However, the transformation of these biomolecules into drugs is limited due to their unstability in the bloodstream, caused by nucleases abundantly present in the blood, and poor capacity to enter cells. The conjugation of miRNAs to nanoparticles (NPs) could be an effective strategy for their clinical delivery. Herein, the engineering of non-liposomal lipid nanovesicles, named quatsomes (QS), for the delivery of miRNAs and other small RNAs into the cytosol of tumor cells, triggering a tumor-suppressive response is reported. The engineered pH-sensitive nanovesicles have controlled structure (unilamellar), size (24 weeks), and are prepared by a green, GMP compliant, and scalable one-step procedure, which are all unavoidable requirements for the arrival to the clinical practice of NP based miRNA therapeutics. Furthermore, QS protect miRNAs from RNAses and when injected intravenously, deliver them into liver, lung, and neuroblastoma xenografts tumors. These stable nanovesicles with tunable pH sensitiveness constitute an attractive platform for the efficient delivery of miRNAs and other small RNAs with therapeutic activity and their exploitation in the clinics

Mechanisms of inactivation of the tumour suppressor gene RHOA in colorectal cancer

Reduced RHOA signalling has been shown to increase the growth/metastatic potential of colorectal tumours. However, the mechanisms of inactivation of RHOA signalling in colon cancer have not been characterised. A panel of colorectal cancer cell lines and large cohorts of primary tumours were used to investigate the expression and activity of RHOA, as well as the presence of RHOA mutations/deletions and promoter methylation affecting RHOA. Changes in RHOA expression were assessed by western blotting and qPCR after modulation of microRNAs, SMAD4 and c-MYC. We show here that RHOA point mutations and promoter hypermethylation do not significantly contribute to the large variability of RHOA expression observed among colorectal tumours. However, RHOA copy number loss was observed in 16% of colorectal tumours and this was associated with reduced RHOA expression. Moreover, we show that miR-200a/b/429 downregulates RHOA in colorectal cancer cells. In addition, we found that TGF- β /SMAD4 upregulates the RHOA promoter. Conversely, RHOA expression is transcriptionally downregulated by canonical Wnt signalling through the Wnt target gene c-MYC that interferes with the binding of SP1 to the RHOA promoter in colon cancer cells. We demonstrate a complex pattern of inactivation of the tumour suppressor gene RHOA in colon cancer cells through genetic, transcriptional and post-transcriptional mechanisms

Prognostic and therapeutic significance of KIF11 in neuroblastoma

Els neuroblastomes d'alt risc són tumors embrionaris molt agressius que proliferen molt ràpidament. Encara que actualment s'utilitzen teràpies multimodals per tractar els pacients, més del 50% recau, i la seva supervivència al cap de 5 anys és menor del 50%. Per tant, és necessari trobar noves teràpies més efectives que incrementin les probabilitats de supervivència d'aquests pacients. KIF11 és una proteïna motora que és indispensable per la formació del fus acromàtic i per la progressió de la cèl·lula a les diferents fases de la mitosis. KIF11 també ha estat descrita com a diana terapèutica en varis tipus de tumors. En aquesta tesis hem investigat els efectes de l'inhibidor oral de KIF11 4SC-205 en neuroblastoma d'alt risc utilitzant models preclínics de tumors primaris i de metàstasis. El valor pronòstic de KIF11 es va investigar analitzant el nivell d'expressió de ARNm en bases de dades públiques i per immunohistoquímica en microarrays de teixits. A partir d'aquestes dades, vam observar que l'alta expressió de KIF11 està relacionada a pitjor supervivència i a alteracions genètiques associades a tumors agressius, com l'amplificació del gen MCYN, la pèrdua de heterozigositat del 1p36 o el guany del 17q23. A continuació vam investigar la inhibició gènica i farmacològica de KIF11 en línies cel·lulars de neuroblastoma. Vam veure que l'inhibidor 4SC-205 recapitula els efectes fenotípics del silenciament gènic, tant in vitro com in vivo, induint arrest en mitosis durant el cicle cel·lular i la subseqüent mort per apoptosis. In vivo, vam observar que el 4SC-205 inhibeix el creixement del tumor primari en models xenografts subcutanis de ratolí, en models PDOX (sigles de l'anglès Patient-Derived Orthotopic Xenograft) i models de metàstasis. Finalment, vam veure que el 4SC-205 potencia l'efecte de la quimioteràpia estàndard (cisplatí, doxorubicina i topotecan) i teràpies dirigides com la inhibició d'ALK i de la via de RAS/MAPK. Aquests resultats suggereixen que la inhibició de KIF11 utilitzant el 4SC-205 tot sol o en combinació amb quimioteràpia convencional i/o teràpies dirigides podria ser una nova estratègia terapèutica per tractar els pacients amb neuroblastoma d'alt risc.Los neuroblastomas de alto riesgo son tumores embrionarios muy agresivos que proliferan muy rápidamente. A pesar del uso de terapias multimodales, aproximadamente un 50% de los pacientes presenta recaídas, y su supervivencia a los 5 años es inferior al 50%. Por lo tanto, es necesario encontrar nuevas terapias más eficaces que permitan aumentar la probabilidad de supervivencia de estos pacientes. KIF11 es una proteína motora que es indispensable para la formación del huso acromático y para la progresión de la célula a las distintas fases de la mitosis. KIF11 ha sido descrita como diana terapéutica en varios tipos de tumores del adulto. En esta tesis hemos investigado los efectos del inhibidor oral de KIF11 4SC-205 en neuroblastoma de alto riesgo usando modelos preclínicos de tumores primarios y de metástasis. El valor pronóstico de KIF11 se investigó analizando los niveles de expresión de ARNm en bases de datos públicos y por inmunohistoquímica en microarrays de tejidos. Usando estos datos, observamos que la expresión de KIF11 correlaciona con una peor supervivencia y con alteraciones génicas asociadas a tumores agresivos, como la amplificación del gen MYCN, la pérdida de heterocigosidad del 1p36 o la ganancia del 17q23. A continuación investigamos la inhibición génica y farmacológica de KIF11 en líneas celulares de neuroblastoma. Vimos que el inhibidor 4SC-205 recapitula los efectos fenotípicos del silenciamento génico, tanto in vitro como in vivo, induciendo arresto en mitosis durante el ciclo celular y la subsecuente muerte por apoptosis. In vivo, observamos que el 4SC-205 inhibe el crecimiento del tumor primario en modelos de xenotrasplates subcutáneos de ratón, en modelos PDOX (siglas del inglés Patient-Derived Orthotopic Xenograft) y modelos de metástasis. Finalmente, vimos que el 4SC-205 potencia los efectos de la quimioterapia estándar (cisplatino, doxorubicina y topotecan) y terapias dirigidas como la inhibición de ALK y de la vía de RAS/MAPK. Estos resultados sugieren que la inhibición de KIF11 utilizando el 4SC-205 sólo o en combinación con quimioterapia convencional y/o terapias dirigidas podría ser una nueva estrategia terapéutica para tratar pacientes con neuroblastoma de alto riesgo.High-risk neuroblastomas are highly proliferative embryonal tumors with extremely aggressive behavior. Despite multimodal therapies, more than 50% of patients relapse, and their 5-year overall survival is still below than 50%. Therefore, it is necessary to find more effective therapies to increase the survival chances of these patients. KIF11 is a motor protein that is essential for bipolar spindle formation and mitotic progression in human cells and is a therapeutic target in multiple tumor types. In this thesis, we investigated the effects of the oral KIF11 inhibitor, 4SC-205, in high-risk neuroblastoma using preclinical primary and metastatic models. KIF11 prognostic value was analyzed by mining publicly available mRNA expression datasets and immunohistochemistry in neuroblastoma tissue microarrays. We found that high KIF11 expression correlated with poor overall survival and genetic variables associated with aggressive neuroblastomas, such as MYCN amplification, -1p36, and +17q23. We next investigated the genetic and pharmacological inhibition of KIF11 in neuroblastoma cell lines. We demonstrated that 4SC-205 recapitulated KIF11 silencing phenotypic effects in vitro and in vivo, inducing cell cycle arrest during mitosis and the subsequent induction of apoptosis. In vivo, 4SC-205 inhibited primary tumor growth in subcutaneous and orthotopic patient-derived xenografts and reduced the metastatic burden. Finally, 4SC-205 potentiated the therapeutic effect of standard chemotherapies (cisplatin, doxorubicin, and topotecan) and targeted therapies such as ALK and RAS/MAPK inhibitors. These findings suggest that KIF11 inhibition using 4SC-205 alone or in combination with conventional and/or targeted therapeutics is a potential new therapeutic strategy to treat high-risk neuroblastomas

FAIM Is Regulated by MiR-206, MiR-1-3p and MiR-133b

Apoptosis plays an important role during development, control of tissue homeostasis and in pathological contexts. Apoptosis is executed mainly through the intrinsic pathway or the death receptor pathway, i.e., extrinsic pathway. These processes are tightly controlled by positive and negative regulators that dictate pro- or anti-apoptotic death receptor signaling. One of these regulators is the Fas Apoptotic Inhibitory Molecule (FAIM). This death receptor antagonist has two main isoforms, FAIM-S (short) which is the ubiquitously expressed, and a longer isoform, FAIM-L (long), which is mainly expressed in the nervous system. Despite its role as a death receptor antagonist, FAIM also participates in cell death-independent processes such as nerve growth factor-induced neuritogenesis or synaptic transmission. Moreover, FAIM isoforms have been implicated in blocking the formation of protein aggregates under stress conditions or de-regulated in certain pathologies such as Alzheimer's and Parkinson's diseases. Despite the role of FAIM in physiological and pathological processes, little is known about the molecular mechanisms involved in the regulation of its expression. Here, we seek to investigate the post-transcriptional regulation of FAIM isoforms by microRNAs (miRNAs). We found that miR-206, miR-1-3p, and miR-133b are direct regulators of FAIM expression. These findings provide new insights into the regulation of FAIM and may provide new opportunities for therapeutic intervention in diseases in which the expression of FAIM is altered

Neuronal Differentiation-Related Epigenetic Regulator ZRF1 Has Independent Prognostic Value in Neuroblastoma but Is Functionally Dispensable In Vitro

Neuroblastoma is a pediatric tumor of the peripheral nervous system that accounts for up to ~15% of all cancer-related deaths in children. Recently, it has become evident that epigenetic deregulation is a relevant event in pediatric tumors such as high-risk neuroblastomas, and a determinant for processes, such as cell differentiation blockade and sustained proliferation, which promote tumor progression and resistance to current therapies. Thus, a better understanding of epigenetic factors implicated in the aggressive behavior of neuroblastoma cells is crucial for the development of better treatments. In this study, we characterized the role of ZRF1, an epigenetic activator recruited to genes involved in the maintenance of the identity of neural progenitors. We combined analysis of patient sample expression datasets with loss- and gain-of-function studies on neuroblastoma cell lines. Functional analyses revealed that ZRF1 is functionally dispensable for those cellular functions related to cell differentiation, proliferation, migration, and invasion, and does not affect the cellular response to chemotherapeutic agents. However, we found that high levels of ZRF1 mRNA expression are associated to shorter overall survival of neuroblastoma patients, even when those patients with the most common molecular alterations used as prognostic factors are removed from the analyses, thereby suggesting that ZRF1 expression could be used as an independent prognostic factor in neuroblastoma