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Metabolic cues in cancer diagnosis and therapeutics

Publication . Marques, Vanda; Rodrigues, Cecília Maria Pereira; Afonso, Marta Bento; Saraiva, Lucília Helena Ataíde

The worldwide social and economic impact of cancer keeps increasing and proper disease management is a critical step to reducing the cancer burden on society, where appropriate diagnosis, staging, and treatment are key factors. Concomitantly, the increasing prevalence of obesity brings forward its importance as a risk factor for cancer development but also allows to explore associated metabolites and signaling pathways as tools for cancer research. As such, the studies presented in this thesis aim to tackle two pillars in cancer research: early diagnosis and therapy resistance, by exploring metabolism-related hormones as disease biomarkers and their impact on resistance to chemoradiotherapy, as well as unraveling novel molecules targeting cancer stem cells as major culprits in refractory cancer. In our first study, we focused on nonalcoholic fatty liver disease (NAFLD) as a predisposing risk factor for the development of hepatocellular carcinoma. Our goal was to evaluate serum levels of metabolism hormones as non-invasive biomarkers aiming to produce diagnostic options alternative to liver biopsy. Using two independent cohorts of biopsy-proven obese NAFLD patients and healthy-liver controls, we measured adiponectin, leptin, and insulin-like growth factor 1 (IGF-1) serum levels. Leptin serum levels were able to discriminate NAFLD with obesity posing as a potential confounding factor. Adiponectin serum levels combined with specific serum lipids composed a biomarker panel with good assay performance for the stratification of nonalcoholic steatohepatitis versus nonalcoholic fatty liver, however, it can be influenced by the presence of patatin-like phosphate domain-containing protein 3 (PNPLA3) genetic polymorphisms. In turn, IGF-1 levels combined with INR and ferritin were able to distinguish advanced liver fibrosis outranking other fibrosis evaluation scores used in the clinical practice. Next, we followed to evaluate how adiponectin and leptin modulated cell stemness and influenced resistance to chemoradiotherapy in rectal cancer patients, and ultimately providing novel biomarker candidates to identify patients not responding to neoadjuvant therapy. By evaluating serum and biopsy tissue from a cohort of locally advanced rectal cancer, we observed that leptin and adiponectin serum levels were not able to identify non-responder patients to chemoradiotherapy. Yet, high leptin and low adiponectin levels were associated with worsened clinical outcomes and unfavorable patient survival. At a molecular level, leptin serum profile was associated with increased expression of Yamanaka factors mRNA, particularly of octamer-binding transcription factor 4 (OCT4) and Kruppel-like factor 4 (KLF4), as well as signal transducer and activator of transcription 3 (STAT3) activation in the biopsy tissues, and all three parameters tended to increase in non-responders. Bringing together STAT3 activation, OCT4, and KLF4 mRNA expression with carcinoembryonic antigen levels – the only serological parameter that distinguished responders from non-responders – we were able to establish a high-performance biomarker panel to distinguish rectal cancer patient responders versus non-responders to chemoradiotherapy. In our third study, we focused on cancer stem cells as a cause of cancer relapse and resistance to therapy. Taking advantage of in vitro tumorsphere forming assays, we identified a curcumin analog that was able to reduce cellular viability of several colorectal cancer cell lines, by modulating stemness traits and inducing apoptosis. Importantly, this compound significantly reduced tumor burden in an in vivo xenograft mouse model following the same mechanisms of action. Moreover, in silico analysis identified nuclear factor kappa B (NF-ĸB) and STAT3 as putative molecular targets that were later confirmed both in vitro and in vivo. In conclusion, our studies highlight how hormones associated with obesity may pose as biomarkers in chronic disease diagnosis and valuable tools in cancer prognosis. Moreover, our results support the influence of obesity on cancer development and clinical outcome, and how its associated signaling pathways provide valuable targets for an innovative approach developed to treat cancer. Finally, we discovered a novel molecule that by targeting those signaling pathways could be a promising drug candidate for therapy insensitive tumors.

2022-09Doctoral thesis

Embargoed access

Exploring the phytochemicals of Acacia melanoxylon R. Br.

Publication . Alves, Diana; Duarte, Sidónio; Arsénio, Pedro; Gonçalves, Joana; Rodrigues, Cecíla M.P.; Ana Lourenço, Lourenço; Patrícia Máximo

Invasive species are currently a world menace to the environment, although the study of their chemistry may provide a means for their future beneficial use. From a study of Portuguese Acacia melanoxylon R. Br. five known compounds were isolated: lupeol, 3 -Z-coumaroyl lupeol, 3 -E-coumaroyl lupeol (dioslupecin A), kolavic acid 15-methyl ester and vomifoliol (blumenol A). Their structures were elucidated by 1D and 2D NMR spectroscopy and mass spectrometry, and as a result some corrections are made to their previous 13C NMR assignments. Cytotoxicity of 3 -Ecoumaroyl lupeol (dioslupecin A) and kolavic acid 15-methyl ester was evaluated against HCT116 human colorectal cancer cells although biological activity was not evident

2021Journal article

Open access

Targeting Neuroinflammation and Neurodegeneration in Parkinson’s Disease

Publication . Oliveira, Sara; Rodrigues, Cecília Maria Pereira; Amaral, Joana São José Dias; Carvalho, Félix Dias

Parkinson’s disease (PD) is the second most common neurodegenerative disease worldwide, being mostly characterized by motor features correlating with dopaminergic neuronal degeneration in the substantia nigra pars compacta (SN) and striatum. Although predominantly sporadic, approximately 10% of all cases are classified as heritable forms of PD, with mutations in the leucine-rich repeat kinase 2 (LRRK2) gene being the most frequent known cause of familial PD. Necroptosis is a caspase-independent form of regulated cell death mediated by the strenuous action of receptor-interacting protein 3 (RIP3) and pseudokinase mixed lineage domain-like protein (MLKL), being also dependent on RIP1 kinase activity, according with the cellular context. Importantly, in the past few years, activation of necroptosis has been linked to PD, while necroptosis inhibition results in disease improvement, unveiling an alternative approach for therapeutic intervention. In turn, microRNAs (miRNAs or miRs), including miR-335, are small non-coding RNAs, whose deregulation has been associated with neurodegeneration and neuroinflammation in different neurodegenerative conditions, such as PD. Identification of deregulated miRNAs may serve as biomarkers for disease detection and prognosis prediction, can help in understanding the complex mechanism of neurodegenerative disease development, and their use in therapy may tackle several issues of aging and neurodegeneration. The main objectives of this thesis were to extend the current knowledge on therapeutic approaches in PD, mostly focusing on the discovery of novel small molecule inhibitors of necroptosis, as well as miRNAs that could represent promising therapeutic strategies and/or biomarkers of disease. In our first work, we phenotypically screened a library of 21 newly synthetized small compounds to identify novel inhibitors of necroptosis, using the BV2 murine microglial cell line treated with the well-known pan-caspase inhibitor, zVAD-fmk (zVAD). We identified one hit – compound Oxa12 – that strongly inhibited zVAD-induced necroptosis. Importantly, Oxa12 counteracted zVAD- and LPS-induced inflammation, by attenuating TNF-α and IL-1β expression. Moreover, Oxa12 negatively regulated JNK and p38 signalling pathways and NF-кB activation, thus suggesting an overall reduction of necroptosis-driven inflammation. In our second study, as a proof of concept, we evaluated the protective effect of Oxa12 in vivo, using the sub-acute 1-methyl-1-4-phenyl-1,2,3,6-tetrahydripyridine hydrochloride (MPTP) PD-related mouse model. Importantly, we observed that Oxa12 protected from MPTP-induced dopaminergic neuronal loss in the SN and striatum. A combination of the analysis of whether Oxa12 was included in the CNS drug property space, using a CNS-multiparameter optimization (MPO) score and metabolic data indicated that Oxa12 has a good brain exposure and an estimated half-life of 13 minutes, turning this compound into a novel addition to the chemical toolbox of CNS-targeting anti-necroptotic compounds. Overall, Oxa12 is a good drug candidate for further optimization to attenuate PD pathogenesis. Finally, we investigated the expression profile of a selected panel of miRNAs, previously linked to cell death and/or inflammatory pathways, in the serum of idiopathic PD (iPD) patients and patients carrying a mutation in the LRRK2 gene (LRRK2-PD), and aged-matched healthy controls, and investigated its value as molecular marker of disease pathogenesis. We determined a differential pattern of miRNA expression between PD patients and age- and gender-matched healthy individuals, with a downregulation of miR-146a, miR-335-3p and miR-335-5p in PD patients in comparison with controls, and no significant alteration in miR-21, miR-34a, miR-34c and miR-155. Interestingly, miR-155 was significantly upregulated in the serum of LRRK2-PD patients versus iPD patients. Since miR-146a role in neurodegenerative diseases with an inflammatory component was already better known, and in silico studies pointed LRRK2 as a putative target of miR-335, we decided to dissect the protective role of miR-335 in experimental models of PD, during inflammatory and/or neurodegenerative events. Our results clearly demonstrated that miR-335 is downregulated in various PD-mimicking conditions, including LPS-stimulated and/or LRRK2wt-overexpressing BV2 microglia cells. These results were further confirmed in the serum of MPTP-injected mice. In terms of mechanism, we confirmed that miR-335 directly targets LRRK2 mRNA. More importantly, miR-335 overexpression clearly counteracted proinflammatory gene expression induced by LPS and LRRK2wt, in BV2 and N9 microglia cells. Further, miR-335 also reduced LPS-induced RIP1 and RIP3 proteins and activation of ERK1/2 and NF-кB cascade. In SH-SY5Y neuroblastoma cells, miR-335 reduced α-synuclein-triggered proinflammatory gene expression. In conclusion, we identified one compound – Oxa12 – that strongly inhibits necroptosis in vitro, in the murine L929 fibroblast and BV2 microglia cell lines and protected from MPTP-driven dopaminergic neuronal loss in vivo, being therefore considered a strong drug candidate for further optimization to slow or attenuate PD pathogenesis. In addition, we identified different miRNA signatures between PD patients, including iPD and LRRK2-PD patients and healthy controls, and further unravelled novel roles for miR-335 in microglia and neuronal cells that halt chronic neuroinflammation effects induced by a classical inflammatory stimulus or even LRRK2wt overexpression. Overall, further characterization of the synergistic function of necroptosis inhibition and miRNA-based strategies may highlight their therapeutic potential for several neurodegenerative diseases with a strong neuroinflammatory component, such as PD.

2021-10Doctoral thesis

Open access

Adiponectin, Leptin, and IGF-1 are useful diagnostic and stratification biomarkers of NAFLD

Publication . Marques, Vanda; Afonso, Marta; Bierig, Nina; Duarte-Ramos, Filipa; Santos-Laso, Álvaro; Jimenez-Agüero, Raul; Eizaguirre, Emma; Bujanda, Luis; Pareja, Maria J.; Luís, Rita; Costa, Adília; Machado, Mariana; Alonso, Cristina; Arretxe, Enara; Alustiza, José M.; Krawczyk, Marcin; Lammert, Frank; Tiniakos, Dina G.; Flehmig, Bertram; Cortez-Pinto, Helena; Banales, Jesus M.; Castro, Rui E.; Normann, Andrea; Rodrigues, Cecília M. P.

Background: Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease where liver biopsy remains the gold standard for diagnosis. Here we aimed to evaluate the role of circulating adiponectin, leptin, and insulin-like growth factor 1 (IGF-1) levels as non-invasive NAFLD biomarkers and assess their correlation with the metabolome. Materials and Methods: Leptin, adiponectin, and IGF-1 serum levels were measured by ELISA in two independent cohorts of biopsy-proven obese NAFLD patients and healthy-liver controls (discovery: 38 NAFLD, 13 controls; validation: 194 NAFLD, 31 controls) and correlated with clinical data, histology, genetic parameters, and serum metabolomics. Results: In both cohorts, leptin increased in NAFLD vs. controls (discovery: AUROC 0.88; validation: AUROC 0.83; p < 0.0001). The leptin levels were similar between obese and non-obese healthy controls, suggesting that obesity is not a confounding factor. In the discovery cohort, adiponectin was lower in non-alcoholic steatohepatitis (NASH) vs. non-alcoholic fatty liver (AUROC 0.87; p < 0.0001). For the validation cohort, significance was attained for homozygous for PNPLA3 allele c.444C (AUROC 0.63; p < 0.05). Combining adiponectin with specific serum lipids improved the assay performance (AUROC 0.80; p < 0.0001). For the validation cohort, IGF-1 was lower with advanced fibrosis (AUROC 0.67, p < 0.05), but combination with international normalized ratio (INR) and ferritin increased the assay performance (AUROC 0.81; p < 0.01). Conclusion: Serum leptin discriminates NAFLD, and adiponectin combined with specific lipids stratifies NASH. IGF-1, INR, and ferritin distinguish advanced fibrosis.

2021Journal article

Open access