Modulation of tumor plasticity by senescent cells: Deciphering basic mechanisms and survival pathways to unravel therapeutic options.

Senescence is a cellular state in which the cell loses its proliferative capacity, often irreversibly. Physiologically, it occurs due to a limited capacity of cell division associated with telomere shortening, the so-called replicative senescence. It can also be induced early due to DNA damage, oncogenic activation, oxidative stress, or damage to other cellular components (collectively named induced senescence). Tumor cells acquire the ability to bypass replicative senescence, thus ensuring the replicative immortality, a hallmark of cancer. Many anti-cancer therapies, however, can lead tumor cells to induced senescence. Initially, this response leads to a slowdown in tumor growth. However, the longstanding accumulation of senescent cells (SnCs) in tumors can promote neoplastic progression due to the enrichment of numerous molecules and extracellular vesicles that constitutes the senescence-associated secretory phenotype (SASP). Among other effects, SASP can potentiate or unlock the tumor plasticity and phenotypic transitions, another hallmark of cancer. This review discusses how SnCs can fuel mechanisms that underlie cancer plasticity, like cell differentiation, stemness, reprogramming, and epithelial-mesenchymal transition. We also discuss the main molecular mechanisms that make SnCs resistant to cell death, and potential strategies to target SnCs. At the end, we raise open questions and clinically relevant perspectives in the field.

Subcellular structure, heterogeneity, and plasticity of senescent cells.

Cellular senescence is a state of permanent growth arrest. It can be triggered by telomere shortening (replicative senescence) or prematurely induced by stresses such as DNA damage, oncogene overactivation, loss of tumor suppressor genes, oxidative stress, tissue factors, and others. Advances in techniques and experimental designs have provided new evidence about the biology of senescent cells (SnCs) and their importance in human health and disease. This review aims to describe the main aspects of SnCs phenotype focusing on alterations in subcellular compartments like plasma membrane, cytoskeleton, organelles, and nuclei. We also discuss the heterogeneity, dynamics, and plasticity of SnCs' phenotype, including the SASP, and pro-survival mechanisms. We advance on the multiple layers of phenotypic heterogeneity of SnCs, such as the heterogeneity between inducers, tissues and within a population of SnCs, discussing the relevance of these aspects to human health and disease. We also raise the main challenges as well alternatives to overcome them. Ultimately, we present open questions and perspectives in understanding the phenotype of SnCs from the perspective of basic and applied questions.

The rational modulation of autophagy sensitizes colorectal cancer cells to 5-fluouracil and oxaliplatin.

Colorectal cancer (CRC) is the third most common and deadliest cancer globally. Regimens using 5-fluorouracil (5FU) and Oxaliplatin (OXA) are the first-line treatment for CRC, but tumor recurrence is frequent. It is plausible to hypothesize that differential cellular responses are triggered after treatments depending on the genetic background of CRC cells and that the rational modulation of cell tolerance mechanisms like autophagy may reduce the regrowth of CRC cells. This study proposes investigating the cellular mechanisms triggered by CRC cells exposed to 5FU and OXA using a preclinical experimental design mimicking one cycle of the clinical regimen (i.e., 48 h of treatment repeated every 2 weeks). To test this, we treated CRC human cell lines HCT116 and HT29 with the 5FU and OXA, combined or not, for 48 h, followed by analysis for two additional weeks. Compared to single-drug treatments, the co-treatment reduced tumor cell regrowth, clonogenicity and stemness, phenotypes associated with tumor aggressiveness and poor prognosis in clinics. This effect was exerted by the induction of apoptosis and senescence only in the co-treatment. However, a week after treatment, cells that tolerated the treatment had high levels of autophagy features and restored the proliferative phenotype, resembling tumor recurrence. The pharmacologic suppression of early autophagy during its peak of occurrence, but not concomitant with chemotherapeutics, strongly reduced cell regrowth. Overall, our experimental model provides new insights into the cellular mechanisms that underlie the response and tolerance of CRC cells to 5FU and OXA, suggesting optimized, time-specific autophagy inhibition as a new avenue for improving the efficacy of current treatments.

Ex vivo gene therapy for lysosomal storage disorders: future perspectives.

INTRODUCTION: Lysosomal storage disorders (LSD) are a group of monogenic rare diseases caused by pathogenic variants in genes that encode proteins related to lysosomal function. These disorders are good candidates for gene therapy for different reasons: they are monogenic, most of lysosomal proteins are enzymes that can be secreted and cross-correct neighboring cells, and small quantities of these proteins are able to produce clinical benefits in many cases. Ex vivo gene therapy allows for autologous transplant of modified cells from different sources, including stem cells and hematopoietic precursors. AREAS COVERED: Here, we summarize the main gene therapy and genome editing strategies that are currently being used as ex vivo gene therapy approaches for lysosomal disorders, highlighting important characteristics, such as vectors used, strategies, types of cells that are modified and main results in different disorders. EXPERT OPINION: Clinical trials are already ongoing, and soon approved therapies for LSD based on ex vivo gene therapy approaches should reach the market.

Energetic and electronic properties of NH3, NO2 and SO2 interacting with GaN nanotube: a DFT study.

In this work, the interaction of GaN nanotube (GaNNT) with common air pollutants of industrialized cities, such as NH3, NO2 and SO2 in different configurations was studied. For this study, the single-walled (10,0) GaNNT was used. The analysis was done via the density functional theory implemented in the SIESTA simulation software. The analysis of the results shows that the air pollutants alter the properties of nanotubes when they interact with them. The stability analysis shows that the most stable configurations are those in which adsorption occurs through a chemical process. The systems remain semiconductors, but in the case of NO2 and SO2 molecules interacting with GaNNT, there was a significant reduction in the energy gap. Our results also indicate that GaNNT is a promising material to detect and remove NH3 and NO2 molecules from the environment; however, it may be not applicable to detect or remove SO2, because the latter interacts strongly with the nanotube, which prevents the GaNNT from being reused.

Molecular markers associated with the outcome of tamoxifen treatment in estrogen receptor-positive breast cancer patients: scoping review and in silico analysis.

Tamoxifen (TMX) is used as adjuvant therapy for estrogen receptor-positive (ER+) breast cancer cases due to its affinity and inhibitory effects. However, about 30% of cases show drug resistance, resulting in recurrence and metastasis, the leading causes of death. A literature review can help to elucidate the main cellular processes involved in TMX resistance. A scoping review was performed to find clinical studies investigating the association of expression of molecular markers profiles with long-term outcomes in ER+ patients treated with TMX. In silico analysis was performed to assess the interrelationship among the selected markers, evaluating the joint involvement with the biological processes. Forty-five studies were selected according to the inclusion and exclusion criteria. After clustering and gene ontology analysis, 23 molecular markers were significantly associated, forming three clusters of strong correlation with cell cycle regulation, signal transduction of proliferative stimuli, and hormone response involved in morphogenesis and differentiation of mammary gland. Also, it was found that overexpression of markers in selected clusters is a significant indicator of poor overall survival. The proposed review offered a better understanding of independent data from the literature, revealing an integrative network of markers involved in cellular processes that could modulate the response of TMX. Analysis of these mechanisms and their molecular components could improve the effectiveness of TMX.

Cancer Cell Fitness Is Dynamic.

Several phenotypes that impact the capacity of cancer cells to survive and proliferate are dynamic. Here we used the number of cells in colonies as an assessment of fitness and devised a novel method called Dynamic Fitness Analysis (DynaFit) to measure the dynamics in fitness over the course of colony formation. DynaFit is based on the variance in growth rate of a population of founder cells compared with the variance in growth rate of colonies with different sizes. DynaFit revealed that cell fitness in cancer cell lines, primary cancer cells, and fibroblasts under unhindered growth conditions is dynamic. Key cellular mechanisms such as ERK signaling and cell-cycle synchronization differed significantly among cells in colonies after 2 to 4 generations and became indistinguishable from randomly sampled cells regarding these features. In the presence of cytotoxic agents, colonies reduced their variance in growth rate when compared with their founder cell, indicating a dynamic nature in the capacity to survive and proliferate in the presence of a drug. This finding was supported by measurable differences in DNA damage and induction of senescence among cells of colonies. The presence of epigenetic modulators during the formation of colonies stabilized their fitness for at least four generations. Collectively, these results support the understanding that cancer cell fitness is dynamic and its modulation is a fundamental aspect to be considered in comprehending cancer cell biology and its response to therapeutic interventions. SIGNIFICANCE: Cancer cell fitness is dynamic over the course of the formation of colonies. This dynamic behavior is mediated by asymmetric mitosis, ERK activity, cell-cycle duration, and DNA repair capacity in the absence or presence of a drug.

Maternal feeding associated to post-weaning diet affects metabolic and behavioral parameters in female offspring.

Genetic and environmental factors related to maternal diet may predispose offspring to serious diseases. However, consequences of a maternal diet intervention during gestation and lactation, and its association with caloric restriction after weaning on the progeny are not completely known. In this context, the goal of the present study was to investigate how different maternal diets, control (CONT), hypercaloric (HD) or restrictive (RD) diets during gestation and lactation, may affect the metabolism and behavior of the offspring that was also submitted to RD. Experimental groups were abbreviated accordingly maternal/offspring diets: CONT/CONT, CONT/RD, RD/CONT, RD/RD, HD/CONT, HD/RD. Our results showed that glucose serum concentration is increased in mice from dams fed a HD. However, offspring from RD-fed dams showed lower insulin and leptin levels than the other groups, indicating a maternal diet effect. Moreover, animals from RD/CONT group showed a higher adipocyte area in comparison to both HD/CONT and CON/CONT. Offspring from RD-fed dams exhibited a decrease in lateral area locomotion in the open field test. Evaluation of anxiety-like behavior and recognition memory showed no significant difference among groups. Thus, maternal RD provides a beneficial response in metabolic parameters, but its effects on behavior is not completely clarified.

Do FHIT gene alterations play a role in human solid tumors?

The fragile histidine triad (FHIT) gene encloses an active common chromosomal fragile site, FRA3B. This gene is known to be associated with genomic instability, apoptosis and DNA damage. FHIT disturbances have been related to carcinogenesis in different types of human tumor. Despite this, there are some controversies about the exact role of the FHIT gene in relation to tumor biology. Several pieces of evidence support the hypothesis that FHIT acts as a tumor suppressor gene. A loss or decrease in the Fhit protein expression appears to be related to tumor progression, poor prognostic factors and lower survival rates. The most frequent causes of FHIT expression changes are gene mutations, epigenetic alteration and loss of heterozygosity. This literature review aims to clarify the involvement of the FHIT gene in carcinogenesis, tumor progression and clinical outcome in prevalent solid malignancies, such as breast, lung, cervical, esophageal, gastric and colorectal cancers.

Design and Objectives of the South American Youth/Child Cardiovascular and Environmental (SAYCARE) Study.

OBJECTIVE: The purpose of this paper is to introduce the overarching study design of the South American Youth/Child Cardiovascular and Environmental (SAYCARE) study, which is an observational multicenter feasibility study held in seven South American cities: Buenos Aires (Argentina), Lima (Peru), Medellin (Colombia), Montevideo (Uruguay), Santiago (Chile), and São Paulo and Teresina (Brazil). Children and adolescents (3-17 years of age) were studied. METHODS: The data management systems, quality assurance monitoring activities, standardized operating procedure manuals, and training and study management are addressed in this paper. Various quality controls to ensure the collection of valid and reliable data are also discussed. RESULTS AND CONCLUSIONS: Data were obtained from 237 preschoolers and schoolchildren and 258 adolescents during the validation phase measurements. The results of the SAYCARE study are expected to provide higher accuracy in the assessment of cardiovascular disease risk factors, including eating behaviors, body composition, physical activity, sedentary behaviors, lipid profiles and cardiovascular health biomarkers, oral health, social conditions, environmental factors and home environment, and their determinants in children and adolescents from ages 3 to 17 in seven South American cities.

Vinblastine and antihelmintic mebendazole potentiate temozolomide in resistant gliomas.

Glioblastoma (GBM) is a very aggressive tumor that has not had substantial therapeutic improvement since the introduction of temozolomide (TMZ) in combination with radiotherapy. Combining TMZ with other chemotherapeutic agents is a strategy that could be further explored for GBM. To search for molecular predictors of TMZ resistance, the TCGA (The Cancer Genome Atlas) database was utilized to assess the impact of specific genes on TMZ response. Patients whose tumors expressed low levels of FGFR3 and AKT2 responded poorly to TMZ. Combination treatment of vinblastine (VBL) plus mebendazole (MBZ) with TMZ was more effective in reducing cell number in most cultures when compared to TMZ alone, especially in cells with low expression levels of FGFR3 and AKT2. Cell cycle distribution and nuclear morphometric analysis indicated that the triple combination of TMZ, VBL and MBZ (TVM) was able to induce polyploidy and senescence, in addition to increasing the Notch3 RNA level in patient-derived gliomas. Thus, this set of data suggests that the triple combination of TMZ, VBL and MBZ may be a considerable therapeutic alternative for the TMZ-tolerant gliomas that harbor low expression of FGFR3/AKT2.

Low Dose of Doxorubicin Potentiates the Effect of Temozolomide in Glioblastoma Cells.

Glioblastoma (GBM) is an aggressive brain tumor with temozolomide (TMZ)-based chemotherapy as the main therapeutic strategy. Doxorubicin (DOX) is not used in gliomas due to its low bioavailability in the brain; however, new delivery strategies and low doses may be effective in the long term, especially as part of a drug cocktail. Our aim was to evaluate the chronic effects of low doses of DOX and TMZ in GBM. Human U87-ATCC cells and a primary GBM culture were chronically treated with TMZ (5 µM) and DOX (1 and 10 nM) alone or combined. DOX resulted in a reduction in the number of cells over a period of 35 days and delayed the cell regrowth. In addition, DOX induced cell senescence and reduced tumor sphere formation and the proportion of NANOG- and OCT4-positive cells after 7 days. Low doses of TMZ potentiated the effects of DOX on senescence and sphere formation. This combined response using low doses of DOX may pave the way for its use in glioma therapy, with new technologies to overcome its low blood-brain barrier permeability.

A guide for the analysis of long-term population growth in cancer.

Although cancer is a chronic disease, most of the in vitro experiments to assess the effectiveness of intervention are performed in hours or a few days. Moreover, none of the available methodologies to measure cell proliferation are adapted to provide information about the growth kinetic during and after treatment. Thus, the objective of this work is to provide a guide to assess long-term changes in cell population size to be used mainly in cancer research. Cumulative population doubling (CPD) graphs based on cell counting for in vitro or tumor volume for in vivo assays were used to calculate four parameters: relative end CPD (RendCPD), to quantify the end point analysis of proliferation; relative area under curve (rAUC), to describe the global chronic effect of a treatment; relative time to cross a threshold (RTCT), to indicate the delay in cell population recovery produced by a treatment; and relative proliferation rate (RPR), to describe the relative regrowth velocity of the cells that survived after treatment. These parameters describe not only the acute and chronic effects of a treatment but also the behavior of cells that are not eliminated by the treatment, providing crucial information about the growth kinetic of the surviving population. Moreover, the proposed analysis allowed the grouping of independent CPD experiments quantified at different time points and even the direct comparison of in vitro and in vivo experiments. Therefore, this new way to analyze long-term outcomes provides a global view of the effectiveness of an intervention, as an important tool for long-term studies.

Analysis of the safety of mesenchymal stromal cells secretome for glioblastoma treatment.

BACKGROUND AIMS: The purpose of this study was to investigate whether the secretome of human adipose-derived stem cells (hADSC) affects human glioblastoma (GBM) cancer stem cell (CSC) subpopulation or has any influence on drug resistance and cell migration, evaluating the safety of hADSCs for novel cancer therapies. METHODS: hADSCs were maintained in contact with fresh culture medium to produce hADSCs conditioned medium (CM). GBM U87 cells were cultured with CM and sphere formation, expression of genes related to resistance and CSCs-MGMT, OCT4, SOX2, NOTCH1, MSI1-and protein expression of OCT4 and Nanog were analyzed. The influence of hADSC CM on GBM resistance to temozolomide (TMZ) was evaluated by measuring cumulative population doubling and hADSC CM influence on tumor cell migration was analyzed using transwell assay. RESULTS: hADSC CM did not alter CSC-related features such as sphere-forming capacity and expression of genes related to CSC. hADSC CM treatment alone did not change proliferation rate of U87 cells and, most important, did not alter the response of tumor cells to TMZ. However, hADSC CM secretome increased the migration capacity of glioblastoma cells. DISCUSSION: hADSC CM neither induced an enrichment of CSCs in U87 cells population nor interfered in the response to TMZ in culture. Nevertheless, paracrine factors released by hADSCs were able to modulate glioblastoma cells migration. These findings provide novel information regarding the safety of using hADSCs against cancer and highlight the importance of considering hADSC-tumor cells interactions in tumor microenvironment in the design of novel cell therapies.

Sensitization of glioma cells by X-linked inhibitor of apoptosis protein knockdown.

OBJECTIVE: Glioblastomas are a kind of cancer with high resistance to treatments, requiring more efficient alternatives of treatment. X-linked inhibitor of apoptosis (XIAP) is highly expressed in gliomas and, due to its inhibition of caspases, can participate in resistance to therapy. Here we test the sensitization of glioma cells with XIAP gene knockdown (KD) to drugs used in chemotherapy. METHODS: We silenced XIAP expression in U87MG glioblastoma using stable shRNA, and cells were treated with taxol, BCNU, temozolomide, cisplatin, etoposide, resveratrol (Rsv), vincristine and doxorubicin. We analyzed cell viability, cell cycle, apoptosis and senescence. RESULTS: XIAP KD cells were more sensitive to etoposide, Rsv, vincristine and doxorubicin compared to wild-type (WT) cells. Doxorubicin 1 µM and vincristine 100 nM induced higher activation of caspases after 24 h and doxorubicin induced a higher degree of senescence induction in XIAP KD cells in relation to WT cells. Phospho-p53 and phospho-H2Ax Western blot indicate subsequent DNA damage as an important effector of doxorubicin-induced death. CONCLUSIONS: This study suggests that XIAP inhibitors may sensitize gliomas to certain drugs and induce death and that the mechanisms of sensitization involve apoptosis, senescence and p53 signaling.

Body mass index is inversely correlated with the expanded CAG repeat length in SCA3/MJD patients.

Spinocerebellar ataxia type 3, also known as Machado-Joseph disease (SCA3/MJD), is an autosomal dominant neurodegenerative disorder with no current treatment. We aimed to evaluate the body mass index (BMI) of patients with SCA3/MJD and to assess the correlations with clinical, molecular, biochemical, and neuroimaging findings. A case-control study with 46 SCA3/MJD patients and 42 healthy, non-related control individuals with similar age and sex was performed. Clinical evaluation was done with the ataxia scales SARA and NESSCA. Serum insulin, insulin-like growth factor 1 (IGF-1) and magnetic resonance imaging normalized volumetries of cerebellum and brain stem were also assessed. BMI was lower in SCA3/MJD patients when compared to controls (p = 0.01). BMI was associated with NESSCA, expanded CAG repeat number (CAG)n, age of onset, age, disease duration, and serum insulin levels; however, in the linear regression model, (CAG)n was the only variable independently associated with BMI, in an inverse manner (R = -0.396, p = 0.015). In this report, we present evidence that low BMI is not only present in SCA3/MJD, but is also directly related to the length of the expanded CAG repeats, which is the causative mutation of the disease. This association points that weight loss might be a primary disturbance of SCA3/MJD, although further detailed analyses are necessary for a better understanding of the nutritional deficit and its role in the pathophysiology of SCA3/MJD.

Serum insulin-like system alterations in patients with spinocerebellar ataxia type 3.

Spinocerebellar ataxias (SCAs) constitute a group of autosomal dominant neurodegenerative disorders with no current treatment. The insulin/insulin-like growth factor 1 (IGF-1) system (IIS) has been shown to play a role in the neurological dysfunction of SCAs and other polyglutamine disorders. We aimed to study the biomarker profile of serum IIS components in SCA3. We performed a case-control study with 46 SCA3 patients and 42 healthy individuals evaluating the peripheral IIS profile (insulin, IGF-1, IGFBP1 and 3) and the correlation with clinical, molecular, and neuroimaging findings. SCA3 patients presented lower insulin and IGFBP3 levels and higher insulin sensitivity (HOMA2), free IGF-I, and IGFBP1 levels when compared with controls. IGFBP-1 levels were directly associated with CAG expanded repeat length; IGF-1 was associated with the volumetries of specific brainstem regions on magnetic resonance imaging (MRI). Insulin levels and sensitivity were related to age at onset of symptoms. Our findings indicate an involvement of IIS components in SCA3 neurobiology and IGFBP-1 as a potential biomarker of the disease.

X-linked adrenoleukodystrophy: clinical course and minimal incidence in South Brazil.

UNLABELLED: X-linked adenoleukodystrophy is a genetic disease that affects the degradation of very long-chain fatty acids. In male patients, common pictures are the cerebral form (CALD), myeloneuropathy (AMN), and Addison-only. OBJECTIVE: To describe the clinical course of affected male patients from South Brazil between 1993 and 2007. METHODS: Affected male patients and their maternal lineages were studied from a clinical, neurological and biochemical standpoint. RESULTS: Eighty-three male patients from 30 families were biochemically evaluated: 51 were affected. 27/51 (54%) presented the cerebral form; 11/51 had AMN (22%); 5 had Addison-only (10%), and 8 (16%) were asymptomatic. Between 2002 and 2006, the minimal incidence was 1:35,000 males in our State (South Brazil). Forty-three affected individuals were followed for 5.4+/-3.7 years. Of 10 boys detected at early stages, three developed CALD. These three boys and another five CALD at baseline were referred to hematopoietic stem cell transplantation. Seven transplants were carried out, 5 with good clinical evolution after 2.2 years post-transplant. The non-transplanted case was later defined as a stable cerebral form. DISCUSSION: Among the present families, the observed cases were comparable to the 50% expected by Mendelian segregation. Based on the natural history, the number of cases that developed CALD was similar to the expected. Transplants were successful in 70% of cases. The occurrence of a stable cerebral form pointed to an urgent need for better markers of active cerebral disease.