Biology subjects

Oliveira, M. J.

Publications and source records attributed to Oliveira, M. J..

8 recordsLinked to original sources

bioRxiv2026

CD44-CD206 axis drives dendritic cell loss and immunosuppression in immaturely glycosylated bladder tumours

Antigen-presenting-cell dysfunction drives immune exclusion, but how tumour glycans disable these cells remains incompletely defined. Immature O-glycosylation, marked by Tn and sialyl-Tn antigens, drives myeloid dysfunction in bladder cancer. Transcriptomics and human tumour profiling linked this state to reduced representation of antigen-presenting myeloid cells and attenuated lymphoid-effector programmes. In glycoengineered models, immature O-glycans promoted immunoregulatory macrophage phenotypes and contact-dependent death of monocyte-derived dendritic cells (moDCs), while surviving cells showed impaired maturation and T-cell activation. Tumour-cell CD44 standard isoform was required for DC death, which was accompanied by inflammasome-associated proteins and gasdermin D cleavage, consistent with pyroptosis. Receptor-discovery approaches converged on CD206, which bound immaturely glycosylated CD44. CD206 blockade restored DC viability. In immunocompetent mice, immature glycosylation accelerated tumour growth and depleted conventional DCs, whereas CD206 blockade restrained tumour progression and restored local and nodal myeloid and lymphoid immunity. These findings identify CD44-CD206 as a targetable glycoimmune checkpoint that promotes immune exclusion.

immunology↗

bioRxiv2024

Unveiling the Mechanisms to Bypass KRAS Inhibition: In Vitro Insights into the Influence of Fibroblast-Secretome

Novel KRAS-targeted therapies unlocked new treatment options for previously untreatable patients. However, in colorectal cancer (CRC), resistance to KRAS-targeted therapy develops rapidly, making it imperative to understand its underlying mechanisms. Cancer-associated fibroblasts (CAFs) induce therapy resistance by generating and maintaining cancer stem cells (CSCs). Additionally, CAFs secretome can modulate KRAS mutant CRC cells proteomic profile, independently of mutant KRAS. Hence, we investigated whether CAF-derived factors could induce resistance to KRAS inhibition by promoting a KRAS-independent stem-like phenotype. Evaluation of KRAS-mutant CRC cell lines (HCT15, HCT116, and SW480) revealed unique basal stem cell marker expression levels. Silencing KRAS lead to up-regulation of CD24, down- regulation of CD49f and CD104, and reduced stemness. However, CAF-secreted factors attenuated these effects, restoring stem cell markers expression and increasing stemness. RNA sequencing showed that CAF-secreted factors upregulate pro-tumorigenic pathways in KRAS-silenced cells, including cell cycle control, epithelial-mesenchymal transition (EMT), NOTCH, and immune regulation, leading to increased cell cycling and exit from quiescence. Overall, we provide mechanistic insights illuminating the role of fibroblasts in counteracting KRAS silencing-induced growth inhibition and enhancing stemness. Our results show that the limited success of KRAS-targeted therapies is not only derived from cell-intrinsic factors but also dependent on external factors derived from the tumor microenvironment, thus opening avenues to improve therapy responses in CRC.

cancer biology↗

bioRxiv2023

Multilevel Plasticity and Altered Glycosylation Drive Aggressiveness in Hypoxic and Glucose-Deprived Bladder Cancer Cells

Bladder tumours with aggressive characteristics often present with microenvironmental niches marked by low oxygen levels (hypoxia) and limited glucose supply due to inadequate vascularization. The molecular mechanisms facilitating cellular adaptation to these stimuli remain largely elusive. Employing a multi-omics approach, we discovered that hypoxic and glucose- deprived cancer cells enter a quiescent state supported by mitophagy, fatty acid {beta}-oxidation, and amino acid catabolism, concurrently enhancing their invasive capabilities. Reoxygenation and glucose restoration efficiently reversed cell quiescence without affecting cellular viability, highlighting significant molecular plasticity in adapting to microenvironmental challenges. Furthermore, cancer cells exhibited substantial perturbation of protein O-glycosylation, leading to simplified glycophenotypes with shorter glycosidic chains. Exploiting glycoengineered cell models, we established that immature glycosylation contributes to reduced cell proliferation and increased invasion. Our findings collectively indicate that hypoxia and glucose deprivation trigger cancer aggressiveness, reflecting an adaptive escape mechanism underpinned by altered metabolism and protein glycosylation, providing grounds for clinical intervention.

biochemistry↗

bioRxiv2023

Pro-inflammatory polarization and colorectal cancer modulate alternative and intronic polyadenylation in primary human macrophages

Macrophages are essential cells of the immune system that alter their inflammatory profile depending on their microenvironment. Alternative polyadenylation in the 3UTR (3UTR-APA) and intronic polyadenylation (IPA) are mechanisms that modulate gene expression, in particular in cancer and activated immune cells. Yet, how polarization and colorectal cancer (CRC) cells microenvironment affect 3UTR-APA and IPA in primary human macrophages remains unknown. Here, primary human monocytes were isolated from healthy donors, differentiated and polarized into a pro-inflammatory state and ChrRNA-Seq and 3RNA-Seq were performed to quantify gene expression and characterize new 3UTR-APA and IPA mRNA isoforms. Our results show that polarization of human macrophages from naive to a pro-inflammatory state causes a marked increase both in proximal polyA site selection in the 3UTR and in IPA events, in genes relevant for macrophage functions. Additionally, we found a negative correlation between differential gene expression and IPA during pro-inflammatory polarization of primary human macrophages. As macrophages are abundant immune cells in the CRC microenvironment that either promote or abrogate cancer progression, we investigated how indirect exposure to CRC cells affects macrophage gene expression and 3UTR-APA and IPA mRNA events. Co-culture with CRC cells alters the inflammatory phenotype of macrophages, increases the expression of pro-tumoral genes and induce 3UTR-APA alterations. Notably, some of these gene expression differences were also found in tumour-associated macrophages of CRC patients, indicating that they are physiological relevant. Upon macrophage pro-inflammatory polarization SRSF12 is the pre-mRNA processing gene that is most upregulated. After SRSF12 knockdown in M1 macrophages there is a global downregulation of gene expression, in particular in genes involved in gene expression regulation and in immune responses. Our results reveal new 3UTR-APA and IPA mRNA isoforms produced during pro-inflammatory polarization of primary human macrophages and CRC co-culture that may be used in the future as diagnostic or therapeutic tools.

molecular biology↗

bioRxiv2022

Modulation of fibroblasts phenotype by colorectal cancer cells-secreted factors is mostly independent of oncogenic KRAS

KRAS mutations have been shown to extend their oncogenic effects beyond the cancer cell, influencing the tumor microenvironment components. Herein, we studied the impact of mutant KRAS on the modulation of cancer-associated fibroblasts (CAFs) pro-tumorigenic properties. To do so, we challenged CCD-18Co normal-like colon fibroblasts with control media (DMEM alone and DMEM+rhTGF{beta}1 media), and conditioned media from control and KRAS silenced colorectal cancer (CRC) cells. Two mutant KRAS CRC cell lines-HCT116 and LS174T, were used. Major pro-tumorigenic fibroblasts phenotypic features, such as -SMA expression, TGF{beta}1 and HGF production, extracellular matrix components and metalloproteinases expression, collagen contraction and migration capacities, were analyzed upon fibroblast challenging with cells conditioned media. Our results showed that the mutant KRAS CRC cells-secreted factors are capable of turning normal-like fibroblasts into CAF-like by modulating -SMA expression, TGF{beta}1 and HGF production and migration capacity, though in a cell line-specific manner. In this scenario, oncogenic KRAS showed to play a secondary role, regulating only discrete features in each cancer cell-educated fibroblasts. For instance, in HCT116, KRAS impairs fibroblasts migration and in LS174T it promotes -SMA expression. In summary, our work suggests that mutant KRAS does not play a major role in controlling the CRC cell secreted factors that modulate fibroblasts behavior. This KRAS-independent modulation of fibroblast pro-tumorigenic features is likely to negatively impact the response to KRAS inhibitors, thus standing as a putative mechanism of resistance to KRAS-inhibition, with possible therapeutical relevance.

cancer biology↗

bioRxiv2022

Mutant KRAS-associated proteome is mainly controlled by exogenous factors

KRAS signaling has been extensively studied, yet the clarification between KRAS-autonomous and non-autonomous mechanisms are still less explored. Understanding how KRAS signaling and effects are affected by exogenous stimuli can provide valuable insights not only to understand resistance mechanisms that justify pathway inhibition failure, but also to uncover novel therapeutic targets for mutant KRAS patients. Hence, aiming at understanding KRAS-autonomous versus non autonomous mechanisms, we studied the response of two mutant KRAS colorectal cancer cell lines (HCT116 and LS174T) - control and KRAS silenced- to TGF{beta}1-activated fibroblasts secretome. By performing a total proteome analysis, we observed that TGF{beta}1-activated fibroblast-secreted factors triggered cell line-specific proteome alterations and that mutant KRAS governs approximately 1/3 of those alterations. Moreover, the analysis of the impact of exogenous factors on the modulation of KRAS proteome revealed that, in both cell lines, more than 2/3 of the KRAS-associated proteome is controlled in a KRAS-non-autonomous manner and dependent on the exogenous factors. This work highlights the context-dependency of KRAS-associated signaling and reinforces the importance of establishing more integrative models resembling the complexity of the tumor microenvironment to study KRAS-associated signals.

cancer biology↗

bioRxiv2021

Mutant KRAS modulates colorectal cancer cells invasive response to fibroblast-secreted factors through the HGF/C-MET axis

Genetic alterations influence the malignant potential of cancer cells, and so does the tumor microenvironment. Herein, we combined the study of KRAS oncogenic effects in colorectal cancer cells with the influence of fibroblasts-derived factors. Results revealed that mutant KRAS regulates cell fate through both autonomous and non-autonomous signaling mechanisms. Specifically, processes such as proliferation and cell-cell aggregation were autonomously controlled by mutant KRAS independently of the stimulation with fibroblasts conditioned media. However, cancer cell invasion revealed to be a KRAS-dependent non-autonomous effect, resulting from the cooperation between fibroblasts-derived HGF and mutant KRAS regulation of C-MET expression. C-MET downregulation upon KRAS silencing rendered cells less responsive to HGF and thus less invasive. Yet, in one cell line, KRAS inhibition triggered invasion upon stimulation with fibroblasts conditioned media. Inhibition of PIK3CA oncogene did not promoted invasion, thus showing a KRAS-specific effect. Moreover, the invasive capacity also depended on the HGF-C-MET axis. Overall, our study awards oncogenic KRAS an important role in modulating the response to fibroblast-secreted factors either by promoting or impairing invasion, and depicts the HGF-C-MET axis as a putative therapeutic target to impair the invasive properties of mutant KRAS cancer cells. SignificanceTargeting mutant KRAS cancers is an urgent clinical need. HGF-C-MET axis inhibition arises as a possible strategy to target mutant KRAS CRC, both primary and metastatic tumors. Additional informationO_ST_ABSFinancial supportC_ST_ABSThis work was supported through FEDER funds through the Operational Programme for Competitiveness Factors (COMPETE 2020), Programa Operacional de Competitividade e Internacionalizacao (POCI), Programa Operacional Regional do Norte (Norte 2020), European Regional Development Fund (ERDF), and by National Funds through the Portuguese Foundation for Science and Technology (FCT) (PTDC/MED-ONC/31354/2017). PDC is a PhD student from Doctoral Program in Pathology and Molecular Genetics from the Institute of Biomedical Sciences Abel Salazar (ICBAS, University of Porto) and she is funded through a PhD fellowship (SFRH/BD/131156/2017) awarded by the FCT. FM is a PhD student from Doctoral Program in Biomedicine from the Faculty of Medicine of the University of Porto and she is funded through a PhD fellowship (SFRH/BD/143669/2019) awarded by the FCT. SM is a PhD student from Doctoral Program in Biomedicine from the Faculty of Medicine of the University of Porto and she is funded through a PhD fellowship (SFRH/BD/143642/2019) awarded by the FCT. AR is a junior researcher hired by IPATIMUP under the CaTCh project funded by FEDER and FCT (POCI-01-0145-FEDER-031354). ALM is a PhD student from Doctoral Program in Biomedicine from the Faculty of Medicine of the University of Porto and she is funded through a PhD fellowship (2020.08932.BD) awarded by the FCT. MJO is principal researcher at INEB. SV is hired by IPATIMUP under norma transitoria do DL n.{o} 57/2016 alterada pela lei n.{o} 57/2017.

cancer biology↗

bioRxiv2021

Metabolomics, Transcriptomics and Functional Glycomics Reveals Bladder Cancer Cells Plasticity and Enhanced Aggressiveness Facing Hypoxia and Glucose Deprivation

Bladder cancer constitutes one of the deadliest genitourinary diseases, especially when diagnosed at late stages. These tumours harbour microenvironmental niches characterized by low levels of oxygen (hypoxia) and limited glucose supply due to poor vascularization. However, the synergic contribution of these features to disease development is poorly understood. Here, we demonstrated that cells with distinct histopathological and molecular backgrounds responded similarly to such stimuli. Cancer cells arrested proliferation, significantly increased invasive capacity in vitro and enhanced tolerance to cisplatin-based chemotherapy. Reoxygenation and access to glucose restored basal proliferation and invasion levels without triggering stress-induced apoptosis, denoting significant cellular plasticity in adapting to microenvironmental cues. Whole transcriptomics showed major molecular reprogramming, supporting main functional alterations. Metabolomics evidenced fatty acids {beta}-oxidation as main bioenergetic pathway rather than anaerobic glycolysis generally adopted by hypoxic cells. Joint pathway analysis also suggested relevant alterations in mucin-type O-glycan biosynthesis. Glycomics confirmed a major antagonization of O-glycosylation pathways, leading to simple cell glycophenotypes characterized by the accumulation of immature short-chain O-glycans such as Tn and STn antigens at the cell surface. Glycoengineered models reflecting simple cell glycophenotypes were developed and functional studies in vitro and in vivo showed that Tn and STn overexpression decreased proliferation and promoted chemoresistance, reinforcing their close link with tumour aggressiveness. Collectively, we have demonstrated that hypoxia and glucose deprivation trigger more aggressive cell behaviours, in what appears to be an escape mechanism from microenvironmental stress. We propose that, altered glycosylation may be used to target these subpopulations, paving the way for precision oncology.

cancer biology↗