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- The Bridge Between Nanotechnology and Chronobiology: Circadian Control of Cancer Therapy by Gene Delivery SystemsPublication . Neves, Ana Raquel Bastos; Costa, Diana Rita Barata; Paixão, Telma Alexandra Quintela; Fan, Donglei EmmaCancer is nowadays the second leading cause of death worldwide, making it a significant public health concern. Europe, following Asia, is, according to the Global Cancer Observatory (GLOBOCAN), the second continent where the incidence, mortality, and prevalence are highest. Statistics indicate that the number of cancer-related deaths in Europe is projected to increase 36.4% by 2050 compared to 2022. In particular, glioblastoma (GB), designated by the World Health Organization (WHO) as a grade 4 astrocytoma, represents the most prevalent (more than 50% of the total cases) and highly aggressive type of primary brain cancer in adults. This cancer is characterized by a high inter- and intratumourally genetic heterogeneity, aggressiveness, angiogenesis, invasiveness, resistance to current standard treatment protocols, poor patient prognosis (12-15 months), and low survival rate. Molecularly, several altered signalling pathways (p53 tumour suppressor pathway inclusive), gene mutations (tumour suppressor p53 gene (TP53), epidermal growth factor receptor (EGFR), isocitrate dehydrogenase (IDH)-1/2, etc.), and methylation of O6-methylguanine-DNA methyltransferase (MGMT) promoter contribute to its hyperproliferation, growth, and chemotherapeutic resistance. For these reasons, actual care treatments, as the classical Stupp protocol, face several challenges. This protocol focuses on first removing the tumour by surgical resection, without compromising the normal neurological function, followed by radiotherapy and chemotherapy with Temozolomide (TMZ), whose effectiveness depends on the MGMT gene promotor methylation status. However, after being subjected to this, patients prognosis remains very poor, and 90% of them have a tumour recurrence within two years. Thus, the development of new and more effective therapeutic approaches is essential to fight this disease. Innovative therapeutic strategies as nanotechnology, gene therapy, and chronotherapy, have been developed and explored to improve the treatment efficacy of GB. Gene therapy consists of delivering exogenous nucleic acids to cells to activate, silence, modify, or edit certain genes and correct genetic defects that may contribute to disease progression. In this case, correcting abnormal gene expression, since several mutations have been associated with glioblastoma, seems a promising approach to treat it. However, delivering nucleic acids to cells is difficult unless delivery systems are used. Nanotechnology, with the use of non-viral vectors (cell-penetrating peptides (CPPs), liposomes, polymers, exosomes, dendrimers, etc.), leads to ground-breaking possibilities for a precise delivery of anticancer drugs and nucleic acids to specific sites, overcoming several physiological barriers, as the blood-brain barrier (BBB). Targeting tumour-specific receptors using ligands at the delivery systems surface can minimize side effects and improve therapeutic responses. Additionally, in the past few years, the study of cancer cells circadian rhythms, normally disrupted, has shown that the circadian clock as a significant role in cancer development and therapeutic efficacy. Circadian rhythm is defined as the approximately 24 h oscillation of physiological and metabolic processes, which are synchronized with the Earth's diurnal cycle. Several studies indicated that rhythms could modulate drug’s effectiveness and their side effects, and delivery systems cellular uptake and efficacy. Studies have shown that some chemotherapy drugs are better tolerated or more effective at certain times of the day. Accordingly, the main object of this doctoral thesis was to design and develop a delivery system, functionalized to target glioblastoma cells, and to evaluate the influence of circadian rhythms on the efficacy of targeting, internalization, cargo release, and, ultimately, therapeutic effect for a precise cancer therapy strategy. This approach focused on the use of a non-viral CPP, namely the WRAP5 peptide, for the construction of a delivery system bearing a transferrin (Tf) receptor ligand sequence (TfR) to co-deliver the anticancer TMZ drug and a plasmid DNA (pDNA) coding for p53 to glioblastoma cells. This system was designed to present an improved brain cell targeting ability and cellular uptake and to penetrate the brain barriers easily. The first step consisted of acquiring its physicochemical properties (size, polydisperse index, surface charge, and complexation capacity), morphology, and biocompatibility. Results revealed these properties to be under the influence of the N/P ratio, which can be optimized to improve complexes desired characteristics. These formulations demonstrated appropriate physicochemical characteristics for in vitro applications, and confocal microscopy using U87 glioblastoma cells confirmed their ability to internalize into cells and deliver the pDNA into the nucleus. Following nuclear localization, successful transcription and translation of the TP53 were observed. The resulting complexes significantly reduced the viability of glioblastoma cells. In a three-dimensional (3D) 9-day U87 spheroid model, generated by two different protocols, complexes were shown to have some effect on the spheroids morphology and size after a single dose treatment. Moreover, complexes have been revealed to be biocompatible with several non-cancerous cells and with zebrafish Danio rerio embryos. Experiments with other two glioma cell lines (SNB19 and U373) also highlighted the complexes ability for internalization and p53 expression levels increase. In these cell lines, apoptosis activation by the intrinsic pathway was implicated. This thesis also aimed to elucidate the influence of circadian core clock components in the performance of developed peptide nanocomplexes, namely, TfR expression, complexes internalization, and p53 expression promotion. The obtained results demonstrated that, at specific time-points, the highest circadian activity of Period circadian regulator 2 (PER2) and TfR led to higher cellular uptake of complexes, TP53 expression induction, and consequently p53 expression. In summary, our comprehensive dataset provides strong evidence supporting the high potential of TMZ/TP53 co-delivery WRAP5 complexes for targeted cellular transfection, p53 expression, and for effectively triggering apoptotic pathways, holding promising therapeutic value toward glioblastoma. Moreover, aligning the timing of complexes administration with the circadian rhythms of GB cells may significantly enhance cellular uptake and gene/protein expression. This chronobiologically optimized approach offers a promising possibility for developing more precise and impactful treatment strategies against glioblastoma. Future preclinical research should prioritize the study of delivery systems bioavailability, tumour targeting, and pharmacokinetics at specific times of day in patient-derived 3D models and in vivo xenograft models. Additionally, it will be relevant to study the temporal expression of glioblastoma biomarkers and the effects of their knockout on survival and therapeutic outcomes. Understanding how brain barrier dynamics, such as permeability, for example, oscillate with circadian changes and impact delivery systems uptake, will bring noticeable improvements to this strategy. Ultimately, this research should instigate complexes clinical translation toward more effective glioblastoma therapy.
- Obesogens-induced deregulation of periprostatic adipose tissue: a driven force in the onset and progression of prostate cancer?Publication . Feijó, Mariana Pombal ; Socorro, Sílvia Cristina da Cruz Marques; Correia, Sara Carina de Lima; Kiss-Tóth, EndreProstate cancer (PCa) is a hormone-dependent cancer whose development and progression are strongly influenced by the tumour microenvironment and exogenous factors, such as environmental influences. The periprostatic adipose tissue (PPAT), by its anatomical proximity and functional crosstalk with prostate cells, emerged as a key driver of tumour growth, particularly in obesity, with the secretome of “obese” PPAT being associated with enhanced tumour aggressiveness. On the other hand, epidemiological and experimental studies have implicated endocrine-disrupting chemicals (EDCs) as environmental risk factors for PCa. Given the hormone dependency of PCa, it is predictable that it is a cancer highly susceptible to the influence of environmental exposures, namely EDCs and specifically those with obesogenic properties (i.e. obesogens), which are capable of disrupting both endocrine and metabolic pathways. Notably, based on their mechanisms of action and the cellular and molecular alterations they induce, obesogens may promote tumorigenesis either directly by acting on prostate cells or indirectly by inducing adipose tissue dysfunction. However, the extent to which obesogenic compounds drive these alterations and the consequent impact on prostate tumorigenesis remain largely unknown. Moreover, despite the well-established effects of obesogens on adipose tissue, no study has characterised their actions on PPAT. Addressing these gaps is critical to understanding how environmental factors intersect with adipose tissue biology, influencing interorgan communication between the prostate and adipose tissue and PCa development. Based on this scientific rationale, this doctoral thesis hypothesises that obesogen-induced PPAT dysfunction represents a driving force in the initiation and progression of PCa. Tributyltin (TBT) is a well-characterised obesogenic EDC and a potent regulator of adipogenesis, widely used in experimental settings to investigate the effects of obesogens. Therefore, using the obesogen model TBT this thesis aimed to: (i) characterise the morphological and secretory alterations of PPAT following TBT exposure; (ii) assess the impact of TBT-induced PPAT dysregulation on prostate cell fate, metabolism, oxidative and inflammatory status, and response to chemotherapeutic drugs; and (iii) identify the molecular targets and signalling pathways mediating the crosstalk between dysregulated PPAT and prostate cells. First, it was demonstrated that in vivo exposure to TBT (50 μg/kg) besides increasing rat body weight, enhanced PPAT somatic index and altered its functional phenotype. TBT treatment promoted a shift in rat prostate cells toward a glycolytic and lipogenic metabolic profile and stimulated oncogenic signalling pathways, including increased phosphorylated/total protein kinase B (pAKT/AKT) ratio and androgen receptor expression. Moreover, macrophage infiltration and a shift in macrophage polarisation towards a pro-inflammatory phenotype were observed both in the prostate and PPAT of TBT-exposed animals, suggesting that TBT can perturb the local prostate-PPAT immune status, contributing to an environment permissive to prostate carcinogenesis. These findings confirmed the effects of TBT on prostate cells, supporting the hypothesis and the investigation into the contribution of PPAT-mediated effects in altering prostate cell behaviour. Culture of the PPAT from rats exposed to TBT clearly demonstrated that TBT induced a dysregulation of the PPAT secretome. TBT-treated PPAT (TBT-PPAT) displayed increased leptin/adiponectin ratio and C-C motif chemokine ligand 7 (CCL7) levels. This adipokine/chemokine profile induced by TBT mimics that observed in obesity and is concurrent with a metabolic reprogramming associated with enhanced glucose, free fatty acids, and lipid peroxidation. Importantly, ex vivo exposure of rat PPAT to TBT (100 nM) recapitulated the findings obtained in vivo concerning the features of its secretome, which are capable of having an impact on prostate cell fate. The results obtained in the subsequent preclinical approaches using co-cultures and conditioned media (CM) assays confirmed the ability of TBT-PPAT enhance the viability, proliferation and migration, as well as apoptosis resistance, in all the studied prostate cell line models, namely non-neoplastic prostate epithelial cells (PNT1A), and androgen-sensitive (22Rv1) and androgen-insensitive (DU145 and PC3) PCa cells. Notably, the TBT-PPAT secretome increased the expression of CCL7 receptor, the C-C motif receptor 3 (CCR3), in prostate cells, which, together with the enhanced CCL7 secretion observed in our experimental setting, raised curiosity about the role of the CCL7-CCR3 axis underlying the pro-tumorigenic effects of TBT-PPAT. The use of a CCR3 antagonist significantly reduced TBT-PPAT induced migration across all cell lines, allowing to implicate the CCL7 and CCR3 in the observed responses of prostate cells. Other molecular targets beyond the CCL7-CCR3 axis were also highlighted. It is the case of tribbles homolog 1 (TRIB1), a pseudokinase involved in tumorigenesis and lipid homeostasis that was overexpressed across all studied cell models exposed to TBT-PPAT-CM. Cell fate alterations observed upon TBT treatment were accompanied by metabolic changes with distinct outcomes in non-neoplastic and neoplastic cell lines: in PNT1A, enhanced fatty acid β-oxidation and synthesis indicate a plausible shift toward a cancer-like metabolic profile; in 22Rv1, the unaltered metabolic and oxidative status suggests the activation of alternative signalling pathways sustaining TBT-PPAT effects; in DU145 and PC3, the distinct metabolic responses observed underscore the differential responsiveness of androgen-insensitive PCa cell subtypes to adipose-derived cues. The investigation in the present thesis was extended to a clinically relevant setting. Human PPAT obtained from patients submitted to radical prostatectomy or prostatic adenomectomy (Millin’s procedure) was treated ex vivo with TBT (100 nM) to confirm if the human tissues resemble the pro-tumorigenic cues identified in controlled experimental models. This approach demonstrated that human PPAT is a target of obesogenic dysregulation and showed that the secretome of obesogen-dysregulated PPAT can significantly enhance the viability of prostate cells. Moreover, the presence of human TBT-PPAT reduced the sensitivity of PCa cells to docetaxel and cabazitaxel, suggesting that obesogenic dysregulation contributes to PCa resistance to taxane-based chemotherapy. Overall, the scientific evidence gathered in this thesis identifies PPAT as a key target of obesogenic EDCs, which disrupt PPAT function and its crosstalk with prostate cells, thereby contributing to the initiation and progression of PCa. These findings open new avenues for developing interventions aimed at modulating PPAT activity to counteract its tumour-promoting effects and emphasise obesity as a critical modulator of PCa aggressiveness.