In lung cancer, the Epidermal Growth Factor Receptor (EGFR) is one of the main targets for clinical management of this disease. The effectiveness of therapies toward this receptor has already been linked to the expression of integrin receptor subunit β1 in NSCLC A549 cells. In this work we demonstrate that azurin, an anticancer therapeutic protein originated from bacterial cells, controls the levels of integrin β1 and its appropriate membrane localization, impairing the intracellular signaling cascades downstream these receptors and the invasiveness of cells. We show evidences that azurin when combined with gefitinib and erlotinib, tyrosine kinase inhibitors which targets specifically the EGFR, enhances the sensitivity of these lung cancer cells to these molecules. The broad effect of azurin at the cell surface level was examined by Atomic Force Microscopy. The Young 's module (E) shows that the stiffness of A549 lung cancer cells decreased with exposure to azurin and also gefitinib, suggesting that the alterations in the membrane properties may be the basis of the broad anticancer activity of this protein. Overall, these results show that azurin may be relevant as an adjuvant to improve the effects of other anticancer agents already in clinical use, to which patients often develop resistance hampering its full therapeutic response.

Exosomes are nano-sized vesicles of endolysosomal origin, released by several cytotypes in physiological and pathological conditions. Tumor derived exosomes, interacting with other cells of the tumor microenvironment, modulate tumor progression, angiogenic switch, metastasis, and immune escape. Recently, extracellular vesicles were proposed as excellent biomarkers for disease monitoring and prognosis in cancer patients. Non-small cell lung cancer (NSCLC) has a poor 5-year survival rate due to the delay in the detection of the disease. The majority of patients are diagnosed in an advanced disease stage. Exosomes might be promising beneficial tools as biomarker candidates in the scenario of NSCLC, since they contain both, proteins and miRNAs. The clinical case reported in this manuscript is a proof of concept revealing that NSCLC exosomes and sorted miRNAs might constitute, in a near future, novel biomarkers. This review summarizes the role of exosomes in NSCLC, focusing on the importance of exosomal microRNAs in lung cancer diagnosis and prognosis.

Introdução – Uma das abordagens terapêuticas no carcinoma do pulmão de não pequenas células (CPNPC) é a quimioterapia e radioterapia (RT) concomitante. O planeamento de RT pode ser realizado com base em diferentes técnicas de imagem, existindo vários fatores que podem influenciar a qualidade das mesmas, nomeadamente o movimento respiratório. Objetivos – Este estudo tem como objetivos: 1) avaliar a importância da 4DCT e da 4DPET/CT para a correção de movimento e definição do volume alvo no planeamento de RT para CPNPC; e 2) comparar a delimitação de volumes entre as técnicas PET/CT, 4DCT e 4DPET/CT, identificando as suas vantagens e limitações. Métodos – Revisão da literatura, recorrendo-se à metodologia PRISMA para a seleção de artigos nas bases de dados PubMed e Science Direct e outras fontes (b-on e Google Scholar). Foram considerados artigos publicados entre janeiro de 2014 e janeiro de 2020. Foram comparados volumes delimitados através de técnicas 3D e 4D, avaliando o impacto do movimento respiratório na aquisição de imagens e posterior planeamento de RT. Resultados – Obtiveram-se 230 artigos, mas apenas cinco foram elegíveis para estudo. As técnicas 4DCT e 4DPET/CT apresentaram melhores resultados, diminuindo os artefactos de movimento. Os volumes delimitados pelas várias técnicas em estudo foram semelhantes, apesar de se registarem pequenas variações quando a técnica 4D não é aplicada. Conclusão – Atualmente a 4DPET/CT apresenta melhores resultados na delimitação de volumes alvo para o planeamento de RT do que a técnica 4DCT.

The critical role of angiogenesis in tumor development makes its inhibition a valuable new approach in therapy, rapidly making anti-angiogenesis a major focus in research. While the VEGF/VEGFR pathway is the main target of the approved anti-angiogenic molecules in NSCLC treatment, the results obtained are still modest, especially due to resistance mechanisms. Accumulating scientific data show that vessel co-option is an alternative mechanism to angiogenesis during tumor development in well-vascularized organs such as the lungs, where tumor cells highjack the existing vasculature to obtain its blood supply in a non-angiogenic fashion. This can explain the low/lack of response to current anti-angiogenic strategies. The same principle applies to lung metastases of other primary tumors. The exact mechanisms of vessel co-option need to be further elucidated, but it is known that the co-opted vessels regress by the action of Angiopoietin-2 (Ang-2), a vessel destabilizing cytokine expressed by the endothelial cells of the pre-existing mature vessels. In the absence of VEGF, vessel regression leads to tumor cell loss and hypoxia, with a subsequent switch to a neoangiogenic phenotype by the remaining tumor cells. Unravelling the vessel co-option mechanisms and involved players may be fruitful for numerous reasons, and the particularities of this form of vascularization should be carefully considered when planning anti-angiogenic interventions or designing clinical trials for this purpose. In view of the current knowledge, rationale for therapeutic approaches of dual inhibition of Ang-2 and VEGF are swiftly gaining strength and may serve as a launchpad to more successful NSCLC anti-vascular treatments.

The formation of distant metastasis resulting from vascular dissemination is one of the leading causes of mortality in non-small cell lung cancer (NSCLC). This metastatic dissemination initiates with the adhesion of circulating cancer cells to the endothelium. The minimal requirement for the binding of leukocytes to endothelial E-selectins and subsequent transmigration is the epitope of the fucosylated glycan, sialyl Lewis x (sLex), attached to specific cell surface glycoproteins. sLex and its isomer sialyl Lewis a (sLea) have been described in NSCLC, but their functional role in cancer cell adhesion to endothelium is still poorly understood. In this study, it was hypothesised that, similarly to leukocytes, sLe glycans play a role in NSCLC cell adhesion to E-selectins. To assess this, paired tumour and normal lung tissue samples from 18 NSCLC patients were analyzed. Immunoblotting and immunohistochemistry assays demonstrated that tumour tissues exhibited significantly stronger reactivity with anti‑sLex/sLea antibody and E-selectin chimera than normal tissues (2.2- and 1.8-fold higher, respectively), as well as a higher immunoreactive score. High sLex/sLea expression was associated with bone metastasis. The overall α1,3-fucosyltransferase (FUT) activity was increased in tumour tissues, along with the mRNA levels of FUT3, FUT6 and FUT7, whereas FUT4 mRNA expression was decreased. The expression of E-selectin ligands exhibited a weak but significant correlation with the FUT3/FUT4 and FUT7/FUT4 ratios. Additionally, carcinoembryonic antigen (CEA) was identified in only 8 of the 18 tumour tissues; CEA-positive tissues exhibited significantly increased sLex/sLea expression. Tumour tissue areas expressing CEA also expressed sLex/sLea and showed reactivity to E-selectin. Blot rolling assays further demonstrated that CEA immunoprecipitates exhibited sustained adhesive interactions with E-selectin-expressing cells, suggesting CEA acts as a functional protein scaffold for E-selectin ligands in NSCLC. In conclusion, this work provides the first demonstration that sLex/sLea are increased in primary NSCLC due to increased α1,3-FUT activity. sLex/sLea is carried by CEA and confers the ability for NSCLC cells to bind E-selectins, and is potentially associated with bone metastasis. This study contributes to identifying potential future diagnostic/prognostic biomarkers and therapeutic targets for lung cancer.

Introduction – One of the therapeutic approaches in non-small cell lung carcinoma (NSCLC) is concomitant chemotherapy and radiotherapy (RT). The planning of RT can be performed based on different imaging techniques, and several factors may influence their quality, including respiratory motion. Objectives – This study aims to: 1) assess the importance of 4DCT and 4DPET/CT for motion correction and target volume definition in planning RT for NSCLC; and 2) compare volume delimitation between PET/CT, 4DCT, and 4DPET/CT techniques, identifying their advantages and limitations. Methods – Literature review, using the PRISMA methodology for the selection of articles in PubMed and Science Direct databases and other sources (b-on and Google Scholar). Articles published between January 2014 and January 2020 were considered. Delineated volumes using 3D and 4D techniques were compared, assessing the impact of respiratory motion on image acquisition and subsequent RT planning. Results – 230 articles were obtained, but only five were eligible for the study. The 4DCT and 4DPET/CT techniques showed better results, decreasing motion artifacts. The volumes delimited by the various techniques under study were similar, although small variations were recorded when the 4D technique is not applied. Conclusion – Currently 4DPET/CT shows better results in the delimitation of target volumes for RT planning than the 4DCT technique.

NSCLC is marked by low survival and resistance to platinum-based chemotherapy. The XPG endonuclease has emerged as a promising biomarker for predicting the prognosis of cisplatin-treated patients and its downregulation having been reported to increase cisplatin efficacy. This study presents an integrated strategy for identifying small molecule inhibitors of XPG to improve cisplatin therapy in NSCLC. A structure-based virtual screening approach was adopted, including a structural and physicochemical analysis of the protein, and a library of small molecules with reported inhibitory activities was retrieved. This analysis identified Lys84 as a crucial residue for XPG activity by targeting its interaction with DNA. After molecular docking and virtual screening calculations, 61 small molecules were selected as potential XPG inhibitors, acquired from the ChemBridge database and then validated in H1299 cells, a NSCLC cell line exhibiting the highest ERCC5 expression. The MTS assay was performed as a first screening approach to determine whether these potential inhibitors could enhance cisplatin-induced cytotoxicity. Overall, among the eight compounds identified as the most promising, three of them revealed to significantly increase the impact of cisplatin. The inherent cytotoxicity of these compounds was further investigated in a non-tumoral lung cell line (BEAS-2B cells), which resulted in the identification of two non-cytotoxic candidates to be used in combination with cisplatin in order to improve its efficacy in NSCLC therapy.

Modifications in DNA repair pathways are recognized as prognostic markers and potential therapeutic targets in various cancers, including non-small cell lung cancer (NSCLC). Overexpression of ERCC1 correlates with poorer prognosis and response to platinum-based chemotherapy. As a result, there is a pressing need to discover new inhibitors of the ERCC1–XPF complex that can potentiate the efficacy of cisplatin in NSCLC. In this study, we developed a structure-based virtual screening strategy targeting the inhibition of ERCC1 and XPF interaction. Analysis of crystal structures and a library of small molecules known to act against the complex highlighted the pivotal role of Phe293 (ERCC1) in maintaining complex stability. This residue was chosen as the primary binding site for virtual screening. Using an optimized docking protocol, we screened compounds from various databases, ultimately identifying more than one hundred potential inhibitors. Their capability to amplify cisplatin-induced cytotoxicity was assessed in NSCLC H1299 cells, which exhibited the highest ERCC1 expression of all the cell lines tested. Of these, 22 compounds emerged as promising enhancers of cisplatin efficacy. Our results underscore the value of pinpointing crucial molecular characteristics in the pursuit of novel modulators of the ERCC1–XPF interaction, which could be combined with cisplatin to treat NSCLC more effectively.