Molecular hallmarks of gliomas

Gliomas are a heterogeneous group of neoplasias that account for the majority of primary tumors of the central nervous system, of which glioblastoma multiforme is by far the most common and malignant subtype. These are particularly dramatic diseases, as they rank first among all human tumor types for the tumor‐related average years of life lost, and for which curative therapies are not yet available. Their etiology remains mostly undetermined: so far, only exposure to high‐dose therapeutic radiation has been firmly established as a risk factor, but other plausible causes include genetic syndromes, familial aggregation, and genetic polymorphisms. Equally mysterious are truly clinically‐relevant prognostic factors of glioma patients; patient age at diagnosis and clinical performance status are classic features associated with patient outcome, but recent evidences suggest that tumor’s molecular traits are also major determinants of prognosis. The outcome of glioblastoma patients is remarkably variable and unpredictable. Regardless, all patients are equally treated with a standardized therapeutic approach. It is widely acknowledged that a set of molecular (genetic and epigenetic) markers predictive of patient outcome, and/or tumor response to specific therapies, will be the basis of a molecular stratification of subgroups of glioblastoma and may prove crucial in rationalizing treatment decisions. However, well-established and clinically relevant biomarkers of prognosis of glioblastoma patients are still lacking. The methylation status of the promoter region of MGMT is currently the most promising, but has not reached clinical applicability. In this context, one of the most important research fields in neuro-oncology today is the identification of molecular determinants of survival and therapy response in glioma patients. This is very relevant because if patients with poor prognosis could be identified at the time of surgery, they would be followed more closely and would be promptly directed to potentially effective experimental therapies, rather than suffering the effects of ineffective and expensive treatments. Identifying new molecular markers and understanding their functional mechanisms in these aggressive therapy-insensitive gliomas may also be the first step in designing novel therapies. The general aim of this chapter is to review some of the most relevant genetic and epigenetic hallmarks of glial tumors, with a particular emphasis on those molecular alterations that have been suggested to affect the prognosis of glioma patients. Naturally, a special focus will be given to glioblastoma, one of the highest devastating human tumors. The chapter is organized in individual subsections, which are more interrelated than autonomous, starting with a brief summary of the classification, epidemiology, and treatment of gliomas. Subsequently, the different types of molecular alterations are defined and discussed in the context of brain gliomas, as well as the interaction between these different features. The molecular alterations are specific to tumor subtype and grade, and may be genetic, including deletion, gain, amplification, mutation, and translocation, or epigenetic, such as DNA CpG island hypermethylation, gene-specific and global genome-wide hypomethylation, and aberrant post-translational histone modifications. Together, these molecular aberrations result in altered gene expression profiles, including oncogene activation, tumor suppressor gene inactivation, loss of imprinting, and chromosomal instability, which ultimately culminate in uncontrolled cell division, deregulation of programmed cell death mechanisms, and limitless replication potential, favoring tumor expansion, angiogenesis, and infiltration into surrounding normal brain tissue. Although epigenetic alterations are usually studied independently of genetic alterations, there is interaction on specific genes, signaling pathways and within chromosomal domains. For example, we recently found that a chromosomal domain of transcriptional aberrant activation encompassing the HOXA genes is present in a subgroup of glioblastoma, and demonstrated that a PI3K‐associated epigenetic mechanism reversibly regulates this domain via histone modifications. In addition, we showed that reactivation of HOXA9 expression, one of the genes within this domain, is a novel, independent, and negative prognostic factor in glioblastoma patients, highlighting the clinical relevance of concurrent genetic and epigenetic events in brain tumors. A particular emphasis will be given to some of the currently most promising biomarkers of glioma prognosis (e.g., MGMT promoter methylation, IDH genes mutations, HOXA genes activation), and some of the challenges for future studies in the field of glioma biomarkers will be discussed

Subversion of Ras Small GTPases in Cutaneous Melanoma Aggressiveness

The rising incidence and mortality rate associated with the metastatic ability of cutaneous melanoma represent a major public health concern. Cutaneous melanoma is one of the most invasive human cancers, but the molecular mechanisms are poorly understood. Moreover, currently available therapies are not efficient in avoiding melanoma lethality. In this context, new biomarkers of prognosis, metastasis, and response to therapy are necessary to better predict the disease outcome. Additionally, the knowledge about the molecular alterations and dysregulated pathways involved in melanoma metastasis may provide new therapeutic targets. Members of the Ras superfamily of small GTPases regulate various essential cellular activities, from signaling to membrane traffic and cytoskeleton dynamics. Therefore, it is not surprising that they are differentially expressed, and their functions subverted in several types of cancer, including melanoma. Indeed, Ras small GTPases were found to regulate melanoma progression and invasion. Hence, a better understanding of the mechanisms regulated by Ras small GTPases that are involved in melanoma tumorigenesis and progression may provide new therapeutic strategies to block these processes. Here, we review the current knowledge on the role of Ras small GTPases in melanoma aggressiveness and the molecular mechanisms involved. Furthermore, we summarize the known involvement of these proteins in melanoma metastasis and how these players influence the response to therapy.publishersversionpublishe

Take advantage of glutamine anaplerosis, the kernel of the metabolic rewiring in malignant gliomas

Glutamine is a non-essential amino acid that plays a key role in the metabolism of proliferating cells including neoplastic cells. In the central nervous system (CNS), glutamine metabolism is particularly relevant, because the glutamine-glutamate cycle is a way of controlling the production of glutamate-derived neurotransmitters by tightly regulating the bioavailability of the amino acids in a neuron-astrocyte metabolic symbiosis-dependent manner. Glutamine-related metabolic adjustments have been reported in several CNS malignancies including malignant gliomas that are considered ‘glutamine addicted’. In these tumors, glutamine becomes an essential amino acid preferentially used in energy and biomass production including glutathione (GSH) generation, which is crucial in oxidative stress control. Therefore, in this review, we will highlight the metabolic remodeling that gliomas undergo, focusing on glutamine metabolism. We will address some therapeutic regimens including novel research attempts to target glutamine metabolism and a brief update of diagnosis strategies that take advantage of this altered profile. A better understanding of malignant glioma cell metabolism will help in the identification of new molecular targets and the design of new therapies.publishersversionpublishe

Unraveling the relevance of arl gtpases in cutaneous melanoma prognosis through integrated bioinformatics analysis

Funding Information: Funding: This research was funded by iNOVA4Health—UIDB/04462/2020, a program financially supported by the Fundação para a Ciência e Tecnologia/Ministério da Educação e Ciência. M.P. was funded by the Liga Portuguesa Contra o Cancro ‒ Núcleo Regional do Sul (LPCC‐NRS). D.B. was funded by the FCT Investigator Program (IF/00501/2014/CP1252/CT0001). Publisher Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland.Cutaneous melanoma (CM) is the deadliest skin cancer, whose molecular pathways underlying its malignancy remain unclear. Therefore, new information to guide evidence‐based clinical decisions is required. Adenosine diphosphate (ADP)‐ribosylation factor‐like (ARL) proteins are membrane trafficking regulators whose biological relevance in CM is undetermined. Here, we investigated ARL expression and its impact on CM prognosis and immune microenvironment through integrated bioinformatics analysis. Our study found that all 22 ARLs are differentially expressed in CM. Specifically, ARL1 and ARL11 are upregulated and ARL15 is downregulated regardless of mutational frequency or copy number variations. According to TCGA data, ARL1 and ARL15 represent independent prognostic factors in CM as well as ARL11 based on GEPIA and OncoLnc. To investigate the mechanisms by which ARL1 and ARL11 increase patient survival while ARL15 reduces it, we evaluated their correlation with the immune microenvironment. CD4+ T cells and neutrophil infiltrates are significantly increased by ARL1 expression. Furthermore, ARL11 expression was correlated with 17 out of 21 immune infiltrates, including CD8+ T cells and M2 macrophages, described as having anti‐tumoral activity. Likewise, ARL11 is interconnected with ZAP70, ADAM17, and P2RX7, which are implicated in immune cell activation. Collectively, this study provides the first evidence that ARL1, ARL11, and ARL15 may influence CM progression, prognosis, and immune microenvironment remodeling.publishersversionpublishe

SPRY4 as a Potential Mediator of the Anti-Tumoral Role of Macrophages in Anaplastic Thyroid Cancer Cells

Funding Information: This work was funded by MERCK in collaboration with Grupo de Estudos da Tiroide (GET) from Sociedade Portuguesa de Endocrinologia Diabetes e Metabolismo (SPEDM) (MERCK/GET/SPEDM/2017), by Fundação para a Ciência e Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior (FCT/MCTES, Portugal) through national funds to iNOVA4Health (UIDB/04462/2020 and UIDP/04462/2020) and the Associated Laboratory LS4FUTURE (LA/P/0087/2020), by Associação de Endocrinologia Oncológica (AEO/2017), and by Instituto Português de Oncologia de Lisboa Francisco Gentil (IPOLFG/2017). Marta Pojo was granted by Liga Portuguesa Contra o Cancro—Núcleo Regional do Sul (LPCC-NRS/2017). Carolina Pires was granted with a Ph.D. scholarship by FCT—2020.07120.BD. Ricardo Rodrigues was granted with a Ph.D. scholarship by iNOVA4Health Research Unit—UIDP/04462/2020; UI/BD/154256/2022. Publisher Copyright: © 2023 by the authors.Anaplastic thyroid carcinoma (ATC) is the most lethal subtype of thyroid cancer, with high invasive and metastatic potential, not responding to conventional treatments. Its aggressiveness may be influenced by macrophages, which are abundant cells in the tumor microenvironment. To investigate the role of macrophages in ATC aggressiveness, indirect co-cultures were established between ATC cell lines and THP-1-derived macrophages. Macrophages significantly increased both the migration and invasion of T235 cells (p < 0.01; p < 0.01), contrasting with a decrease in C3948 (p < 0.001; p < 0.05), with mild effects in T238 migration (p < 0.01) and C643 invasion (p < 0.05). Flow cytometry showed upregulation of CD80 (pro-inflammatory, anti-tumoral) and downregulation of CD163 (anti-inflammatory, pro-tumoral) in macrophages from co-culture with T235 (p < 0.05) and C3948 (p < 0.05), respectively. Accordingly, we found an upregulation of secreted pro-inflammatory mediators (e.g., GM-CSF, IL-1α; p < 0.05) in C3948–macrophage co-cultures. Proteomic analysis showed the upregulation of SPRY4, an inhibitor of the MAPK pathway, in C3948 cells from co-culture. SPRY4 silencing promoted cancer cell invasion, reverting the reduced invasion of C3948 caused by macrophages. Our findings support that macrophages play a role in ATC cell aggressiveness. SPRY4 is a possible modulator of macrophage–ATC cell communication, with a tumor suppressor role relevant for therapeutic purposes.publishersversionpublishe

WNT6 is a novel oncogenic prognostic biomarker in human glioblastoma

Glioblastoma (GBM) is a universally fatal brain cancer, for which novel therapies targeting specific underlying oncogenic events are urgently needed. While the WNT pathway has been shown to be frequently activated in GBM, constituting a potential therapeutic target, the relevance of WNT6, an activator of this pathway, remains unknown. Methods: WNT6 protein and mRNA levels were evaluated in GBM. WNT6 levels were silenced or overexpressed in GBM cells to assess functional effects in vitro and in vivo. Phospho-kinase arrays and TCF/LEF reporter assays were used to identify WNT6-signaling pathways, and significant associations with stem cell features and cancer-related pathways were validated in patients. Survival analyses were performed with Cox regression and Log-rank tests. Meta-analyses were used to calculate the estimated pooled effect. Results: We show that WNT6 is significantly overexpressed in GBMs, as compared to lower-grade gliomas and normal brain, at mRNA and protein levels. Functionally, WNT6 increases typical oncogenic activities in GBM cells, including viability, proliferation, glioma stem cell capacity, invasion, migration, and resistance to temozolomide chemotherapy. Concordantly, in in vivo orthotopic GBM mice models, using both overexpressing and silencing models, WNT6 expression was associated with shorter overall survival, and increased features of tumor aggressiveness. Mechanistically, WNT6 contributes to activate typical oncogenic pathways, including Src and STAT, which intertwined with the WNT pathway may be critical effectors of WNT6-associated aggressiveness in GBM. Clinically, we establish WNT6 as an independent prognostic biomarker of shorter survival in GBM patients from several independent cohorts. Conclusion: Our findings establish WNT6 as a novel oncogene in GBM, opening opportunities to develop more rational therapies to treat this highly aggressive tumor.FCT - Foundation for Science and Technology (PTDC/SAU-GMG/113795/2009 and IF/00601/2012 to B.M.C.; SFRH/BD/92786/2013 to C.S.G.; SFRH/BD/88121/2012 to J.V.C.; SFRH/BD/81042/2011 to M.P.; SFRH/BD/93443/2013 to S.Q.) and Fundação Calouste Gulbenkian (B.M.C.), by FEDER funds through the Operational Programme Competitiveness Factors - COMPETE and National Funds through FCT under the project POCI-01-0145-FEDER-007038; by the project NORTE-01-0145-FEDER-000013 and NORTE-01-0246-FEDER-000012, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF); and by the project NORTE-01-0145-FEDER-000023, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER)info:eu-repo/semantics/publishedVersio

HOXA9 promotes glioblastoma initiation, aggressiveness and resistance to therapy

Glioblastoma is the most common and malignant subtype of glioma, exhibiting remarkable resistance to treatment. Here we investigated the oncogenic potential of HOXA9 in gliomagenesis, the molecular and cellular mechanisms by which HOXA9 may render glioblastoma more aggressive, and how HOXA9 affects response to chemotherapy and prognosis. Expression microarrays were used to identify HOXA9 target genes. Stable glioblastoma cell lines with ectopic HOXA9 overexpression or shRNA-­mediated knockdown of HOXA9 were established to evaluate the roles of HOXA9 in cell viability, death, invasion, and response to temozolomide. Subcutaneous and orthotopic intracranial xenograft models of glioblastoma were established to evaluate the oncogenic potential of HOXA9 in vivo, and its role in response to temozolomide and overall survival. Transcriptomic analyses identified novel HOXA9-­target genes that have key roles in critical cancer processes, including cell proliferation, adhesion, DNA metabolism and repair, and stem cell maintenance. Functional assays with a variety of glioblastoma cells revealed that HOXA9 promotes cell viability, stemness, and invasion; conversely, HOXA9 displayed anti-­apoptotic functions. Additionally, ectopic expression of HOXA9 promoted the malignant transformation of human immortalized astrocytes in an intracranial orthotopic mouse model of glioblastoma, and caused tumor-­associated death. HOXA9 also mediated resistance to temozolomide treatment both in vitro and in vivo. Mechanistically, BCL2 was identified as a novel HOXA9 target that may be therapeutically targeted. Indeed, the pharmacological inhibition of BCL2 with ABT-­737 specifically reverted temozolomide resistance in HOXA9-­positive cells. These data establish HOXA9 as a critical driver of glioma initiation, aggressiveness and resistance to therapy

Therapeutic targeting of PD-1/PD-L1 blockade by novel small-molecule inhibitors recruits cytotoxic T cells into solid tumor microenvironment

© Author(s) (or their employer(s)) 2022.This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See http://creativecommons.org/licenses/by-nc/4.0/.Background: Inhibiting programmed cell death protein 1 (PD-1) or PD-ligand 1 (PD-L1) has shown exciting clinical outcomes in diverse human cancers. So far, only monoclonal antibodies are approved as PD-1/PD-L1 inhibitors. While significant clinical outcomes are observed on patients who respond to these therapeutics, a large proportion of the patients do not benefit from the currently available immune checkpoint inhibitors, which strongly emphasize the importance of developing new immunotherapeutic agents. Methods: In this study, we followed a transdisciplinary approach to discover novel small molecules that can modulate PD-1/PD-L1 interaction. To that end, we employed in silico analyses combined with in vitro, ex vivo, and in vivo experimental studies to assess the ability of novel compounds to modulate PD-1/PD-L1 interaction and enhance T-cell function. Results: Accordingly, in this study we report the identification of novel small molecules, which like anti-PD-L1/PD-1 antibodies, can stimulate human adaptive immune responses. Unlike these biological compounds, our newly-identified small molecules enabled an extensive infiltration of T lymphocytes into three-dimensional solid tumor models, and the recruitment of cytotoxic T lymphocytes to the tumor microenvironment in vivo, unveiling a unique potential to transform cancer immunotherapy. Conclusions: We identified a new promising family of small-molecule candidates that regulate the PD-L1/PD-1 signaling pathway, promoting an extensive infiltration of effector CD8 T cells to the tumor microenvironment.C and RCA are supported by the Fundação para a Ciência e a Tecnologia, Ministério da Ciência, Tecnologia e Ensino Superior (FCT-MCTES) (PhD grants PD/BD/128238/2016 (RCA) and SFRH/BD/131969/2017 (BC)). The authors thank the funding received from the European Structural & Investment Funds through the COMPETE Programme and from National Funds through FCT under the Programme grant LISBOA-01-0145-FEDER016405 - SAICTPAC/0019/2015 (HF and RCG). HFF and RCA received additional support from FCT-MCTES (UIDB/04138/2020, PTDC/BTM-SAL/4350/2021 and UTAPEXPL/NPN/0041/2021; EXPL/MED-QUI/1316/2021, respectively). The MultiNano@MBM project was supported by The Israeli Ministry of Health, and FCTMCTES, under the frame of EuroNanoMed-II (ENMed/0051/2016; HF and RS-F). HF and RS-F thank the generous financial support from ‘La Caixa’ Foundation under the framework of the Healthcare Research call 2019 (NanoPanther; LCF/PR/HR19/52160021), as well as CaixaImpulse (Co-Vax; LCF/TR/CD20/52700005). MP thanks the financial support from Liga Portuguesa Contra o Cancro – Nucleo Regional do Sul and ‘iNOVA4Health – UIDB/04462/2020’, a program financially supported by Fundação para a Ciência e Tecnologia/Ministério da Educação e Ciência. RS-F thanks the following funding agencies for their generous support: the European Research Council (ERC) Advanced Grant Agreement No. (835227)–3DBrainStrom, ERC PoC Grant Agreement no. 862580 – 3DCanPredict, The Israel Science Foundation (Grant No. 1969/18), The Melanoma Research Alliance (MRA Established Investigator Award n°615808), the Israel Cancer Research Fund (ICRF) Professorship award (n° PROF-18-682), and the Morris Kahn Foundation.info:eu-repo/semantics/publishedVersio