Cláudia Aparecida Rainho

Purpose: This study investigates the ability of Nanoskin, a bacterial cellulose (BC) biofilm, to transfer human corneal epithelial cells (CECs) onto wounded rabbit corneas, evaluating integration and re-epithelialization of the ocular surface. Methods: Primary cultures of human mesenchymal stem cells (MSCs) were established and differentiated into CECs, which were then seeded onto Nanoskin BC or soft contact lenses. Wounded rabbit corneas were covered with human CECs on BC biofilms or contact lenses and maintained in organ culture for 5 days. The control group was left untreated. Integration and reepithelialization were assessed using immunofluorescence (IF) staining with human anti-nuclei antibodies. Results: Wounded rabbit corneas successfully re-epithelialized with both scaffolds. IF staining confirmed the presence of human epithelial cells in the regenerating rabbit corneal epithelium for both Nanoskin BC and contact lens groups, confirming effective cell transfer. Morphometric analysis revealed that control wounded corneas had significantly fewer epithelial cells (P < 0.001) compared to those treated with cell transfer. Conclusion: Our findings demonstrate the feasibility of using Nanoskin BC as a scaffold for corneal cell transplantation, highlighting its potential as a promising biomaterial for treating ocular surface diseases. Future investigations are required to evaluate the transfer efficiency of MSCs, given their superior proliferative capacity and secretion of growth factors.

Background: Pancreatic ductal adenocarcinoma (PDAC) is a highly heterogeneous malignancy, characterized by low tumor cellularity, a dense stromal response, and intricate cellular and molecular interactions within the tumor microenvironment (TME). Although bulk omics technologies have enhanced our understanding of the molecular landscape of PDAC, the specific contributions of non-malignant immune and stromal components to tumor progression and therapeutic response remain poorly understood. Methods: We explored genome-wide DNA methylation and transcriptomic data from the Cancer Genome Atlas Pancreatic Adenocarcinoma cohort (TCGA-PAAD) to profile the immune composition of the TME and uncover gene co-expression networks. Bioinformatic analyses included DNA methylation profiling followed by hierarchical deconvolution, epigenetic age estimation, and a weighted gene co-expression network analysis (WGCNA). Results: The unsupervised clustering of methylation profiles identified two major tumor groups, with Group 2 (n = 98) exhibiting higher tumor purity and a greater frequency of KRAS mutations compared to Group 1 (n = 87) (p < 0.0001). The hierarchical deconvolution of DNA methylation data revealed three distinct TME subtypes, termed hypo-inflamed (immune-deserted), myeloid-enriched, and lymphoid-enriched (notably T-cell predominant). These immune clusters were further supported by co-expression modules identified via WGCNA, which were enriched in immune regulatory and signaling pathways. Conclusions: This integrative epigenomic–transcriptomic analysis offers a robust framework for stratifying PDAC patients based on the tumor immune microenvironment (TIME), providing valuable insights for biomarker discovery and the development of precision immunotherapies.

A differential diet with royal jelly (RJ) during early larval development in honeybees shapes the phenotype, which is probably mediated by epigenetic regulation of gene expression. Evidence indicates that small molecules in RJ can modulate gene expression in mammalian cells, such as the fatty acid 10-hydroxy-2-decenoic acid (10-HDA), previously associated with the inhibition of histone deacetylase enzymes (HDACs). Therefore, we combined computational (molecular docking simulations) and experimental approaches for the screening of potential HDAC inhibitors (HDACi) among 32 RJ-derived fatty acids. Biochemical assays and gene expression analyses (Reverse Transcriptase - quantitative Polymerase Chain Reaction) were performed to evaluate the functional effects of the major RJ fatty acids, 10-HDA and 10-HDAA (10-hydroxy-decanoic acid), in two human cancer cell lines (HCT116 and MDA-MB-231). The molecular docking simulations indicate that these fatty acids might interact with class I HDACs, specifically with the catalytic domain of human HDAC2, likewise well-known HDAC inhibitors (HDACi) such as SAHA (suberoylanilide hydroxamic acid) and TSA (Trichostatin A). In addition, the combined treatment with 10-HDA and 10-HDAA inhibits the activity of human nuclear HDACs and leads to a slight increase in the expression of HDAC-coding genes in cancer cells. Our findings indicate that royal jelly fatty acids collectively contribute to HDAC inhibition and that 10-HDA and 10-HDAA are weak HDACi that facilitate the acetylation of lysine residues of chromatin, triggering an increase in gene expression levels in cancer cells.

TP53 mutations are frequent in non-small cell lung cancer (NSCLC) and have been associated with poor outcome. The prognostic and predictive relevance of EGFR/TP53 co-mutations in NSCLC is controversial. We analyzed lung tissue specimens from 70 patients with NSCLC using next-generation sequencing to determine EGFR and TP53 status and the association between these status with baseline patient and tumor characteristics, adjuvant treatments, relapse, and progression-free (PFS) and overall survival (OS) after surgical resection. We found the EGFR mutation in 32.9% of patients (20% classical mutations and 12.9% uncommon mutations). TP53 missense mutations occurred in 25.7% and TP53/EGFR co-mutations occurred in 43.5% of patients. Stage after surgical resection was significantly associated with OS (P=0.028). We identified an association between progression-free survival and poor outcome in patients with distant metastases (P=0.007). We found a marginally significant difference in OS between genders (P=0.057) and between mutant and wild type TP53 (P=0.079). In univariate analysis, distant metastases (P=0.027), pathological stage (IIIA-IIIB vs I-II; P=0.028), and TP53 status (borderline significance between wild type and mutant; P=0.079) influenced OS. In multivariable analysis, a significant model for high risk of death and poor OS (P=0.029) selected patients in stage IIIA-IIIB, with relapse and distant metastases, non-responsive to platin-based chemotherapy and erlotinib, with tumors harboring EGFR uncommon mutations, with TP53 mutant, and with EGFR/TP53 co-mutations. Our study suggested that TP53 mutation tends to confer poor survival and a potentially negative predictive effect associated with a non-response to platinum-based chemotherapy and erlotinib in early-stage resected EGFR-mutated NSCLC.

Pancreatic cancer is one of the most lethal malignancies worldwide, it often results in therapeutic failure and unfavorable outcome because of the absence of screening strategies, difficulties in tumor early detection due to the lack of specific signs/symptoms and aggressive behavior of the disease. Early detection of this cancer is a challenge with the technology we have, its late detection results in a poor outcome and reduced quality of life for the patient. The treatment success depends on surgical intervention, which is not always possible. Thus, trying to find alterations that occur in the early stages of pancreatic cancer is needed. Taking into consideration that the accumulation of genetic and epigenetic alterations results in a complex and heterogeneous disease, each patient has a unique tumor. The heterogeneity of cancer is not restricted to inter-patient differences but, intra-tumoral heterogeneity contributes to functional properties of tumor cell subclones, including proliferative capacity and progression to aggressive phenotypes such as drug tolerance and metastatic potential. Driver mutations are responsible for malignant transformation and tumor progression across different cancer types. One of the most common driver mutations are those leading to the constitutive activation of KRAS oncogene, a GTPase enzyme responsible for cell proliferation and survival that is frequently mutated in pancreatic cancer. Gain-of-function mutation of KRAS gene leads to a protein constitutively activated, which stimulates the proliferation of tumor cells. The c.34G>T missense mutation (rs121913530) causes the substitution of glycine for cysteine in the encoded protein KRASG12C; this mutation has been related to poorer prognosis and therapy resistance in lung cancer. As KRAS occurs before the progression of pancreatic cancer to invasive carcinoma, it is a promising target for new biomarker development. We design three near-infrared probe-drug conjugate directed to the KRASG12C and to predict in silico the best orientation and conformation of the test compounds bound to the KRAS G12C (PDB code: 6OIM), the PyRx program was used, which uses AutoDock Vina as docking software. Based on these profiles and structures of the compounds, AutoDock Vina infers the possible pharmacological interactions and allows the most promising compounds to be classified according to the studied biological activity, using the empirical score function for this classification. The AMG 510 (established inhibitor) and GDP (endogenous activator) were used as a reference to establish the docking box coordinates. The binding energy of the three compounds predicted by docking was analyzed and classified according to the binding affinity described in Kcal/mol. The tested compounds show an excellent affinity for this protein based on the binding energy results obtained by docking using the crystallized KRAS G12C protein. Therefore, they could be great candidates for further chemical synthesis studies aiming to improve diagnosis, treatment, and patient survival in pancreatic cancer. Citation Format: Barbara M. Barbosa, Allana C. F. Martins, Roberto S. Gomes, Claudia A. Rainho. In silico drug design using KRASG12C as a theranostic target for pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B040.

Genetic and epigenetic changes contribute to intratumor heterogeneity and chemotherapy resistance in several tumor types. LncRNAs have been implicated, directly or indirectly, in the epigenetic regulation of gene expression. We investigated lncRNAs that potentially mediate carboplatin-resistance of cell subpopulations, influencing the progression of ovarian cancer (OC). Four carboplatin-sensitive OC cell lines (IGROV1, OVCAR3, OVCAR4, and OVCAR5), their derivative resistant cells, and two inherently carboplatin-resistant cell lines (OVCAR8 and Ovc316) were subjected to RNA sequencing and global DNA methylation analysis. Integrative and cross-validation analyses were performed using external (The Cancer Genome Atlas, TCGA dataset, n = 111 OC samples) and internal datasets (n = 39 OC samples) to identify lncRNA candidates. A total of 4255 differentially expressed genes (DEGs) and 14529 differentially methylated CpG positions (DMPs) were identified comparing sensitive and resistant OC cell lines. The comparison of DEGs between OC cell lines and TCGA-OC dataset revealed 570 genes, including 50 lncRNAs, associated with carboplatin resistance. Eleven lncRNAs showed DMPs, including the SNHG12. Knockdown of SNHG12 in Ovc316 and OVCAR8 cells increased their sensitivity to carboplatin. The results suggest that the lncRNA SNHG12 contributes to carboplatin resistance in OC and is a potential therapeutic target. We demonstrated that SNHG12 is functionally related to epigenetic mechanisms.

The common epidermal growth factor receptor (EGFR) mutations, such as the L858R point mutation in exon 21 and the in-frame deletional mutation in exon 19, have been definitively associated with response to EGFR-tyrosine kinase inhibitors (EGFR-TKI). However, the clinical outcome and response to treatment for many other rarer mutations are still unclear. In this study, we report the results of Brazilian patients in stage IB-IIIA non-small cell lung cancer (NSCLC) following complete resection with minimal residual disease and EGFR mutations treated with adjuvant chemotherapy and/or EGFR-TKIs. The frequency of EGFR mutations was investigated in 70 cases of early stage NSCLC. Mutations in exons 18 and 20, uncommon mutations in exons 19 and 21, as well as in exons 3, 7, 14, 16, 22, 27, and 28, and/or the presence of different mutations in a single tumor (complex mutations) are considered rare. EGFR mutations were detected in 23 tumors (32.9%). Fourteen cases carried rare mutations and were treated with platinum-based chemotherapy and two cases were treated with erlotinib. The clinical outcome is described case by case with references to the literature. Notably, we found two rare EGFR mutations and one of them with an unknown response to chemotherapy and/or EGFR-TKIs. We have provided complementary information concerning the clinical outcome and treatment of patients with early stage NSCLC for several rare EGFR mutations not previously or only rarely reported. Description of cases harboring rare mutations can support the decision-making process in this subset of patients.

Docetaxel (DTX) is used against breast cancer despite its side effects such as toxicity and immunosuppression. Here we investigated the cytotoxic and immunomodulatory effects of the ethanol solution extract of propolis (EEP) in combination with DTX on MCF‐7 breast cancer cells and on women's monocyte. The cytotoxic potential of EEP + DTX was assessed by MTT assay and the type of tumor cell death was evaluated by flow cytometry. The effects of EEP + DTX on the migration and invasion of MCF‐7 cells were analyzed. Cytokine production by monocytes was assessed by ELISA and the expression of cell surface markers was evaluated by flow cytometry. We also assessed the fungicidal activity of monocytes against Candida albicans and the generation of reactive oxygen species (ROS). Finally, the impact of the supernatants of treated monocytes in the viability, migration, and invasiveness of tumor cells was assessed. EEP enhanced the cytotoxicity of DTX alone against MCF‐7 cells by inducing necrosis and inhibiting their migratory ability. EEP + DTX exerted no cytotoxic effects on monocytes and stimulated HLA‐DR expression, TNF‐α, and IL‐6 production, exerted an immunorestorative action in the fungicidal activity, and reduced the oxidative stress. Our findings have practical implications and reveal new insights for complementary medicine.