Biology subjects

Rojo, F.

Publications and source records attributed to Rojo, F..

4 recordsLinked to original sources

bioRxiv2025

The ISPpu9 insertion sequence of Pseudomonas putida KT2440 generates various circular intermediates enabling modular transposition

Pseudomonas putida KT2440 contains seven copies of an insertion sequence (IS) belonging to the IS110 family, designated ISPpu9, which are inserted into repetitive extragenic palindromic sequences. Five of these copies include a small RNA, ssr9, located downstream of the transposase gene. Additionally, three separate copies of ssr9 are also present. When transferred to a different P. putida strain, ISPpu9 inserted at a specific target site highly similar to that of the KT2440 strain. Ssr9 was not needed for transposition. Circular intermediates were detected containing either the transposase, the transposase along with ssr9, or ssr9 alone. This finding explains the presence of these distinct modules in KT2440 chromosome. Minicircle formation required the transposase and short sequences located within the IS ends, which were identified. Unlike other ISs of this family, minicircle formation in ISPpu9 did not produce a hybrid promoter to enhance transcription of the ISPpu9 transposase. Instead, transcription occurred efficiently from its native promoter. In contrast, minicircles formed by ISPpu10, another IS of the same family also present in KT2440, generated a hybrid promoter significantly stronger than its weak native transposase promoter. This suggests that hybrid promoters might only emerge when the native transposase promoter is inherently weak.

molecular biology↗

bioRxiv2022

Distinct GSDMB protein isoforms and protease cleavage processes differentially control pyroptotic cell death and mitochondrial damage in cancer cells.

The formation of Gasdermin (GSDM) pores, leading to pyroptosis or other context-dependent consequences, is directly involved in multiple diseases. Gasdermin-B (GSDMB) plays complex and controversial roles in pathologies, with pyroptosis-dependent and independent functions. GSDMB is promising oncologic therapeutic target since it exhibits either antitumor function, when immunocyte-mediated Granzyme-A (GZMA) cleaves GSDMB releasing its cytotoxic N-terminal domain, or pro-tumoral activities (invasion, metastasis, and drug resistance). However, it is still unknown the precise regulatory mechanisms of GSDMB pyroptosis as well as the differential effects of the four translated GSDMB variants (GSDMB1-4, that differ in the alternative usage of exons 6-7) in this process. Here, we first prove that exon 6 translation (in the interdomain protein linker) is essential for pyroptosis, and therefore, GSDMB isoforms lacking this exon (GSDMB1-2) cannot provoke cancer cell death. Consistently, in large series of breast cancer samples GSDMB2 expression, and not of exon6-containing variants (GSDMB3-4), associates with unfavourable clinical-pathological parameters. Moreover, cellular, and biochemical analyses combined with confocal, live cell imaging, and electron microscopy studies, demonstrated that diverse GSDMB N-terminal constructs containing exon-6 induce mitochondrial damage (increased mitochondrial ROS, membrane potential loss and mitochondrial DNA release) together with pyroptotic membrane cell lysis. While exon-6 residues are not required for membrane or mitochondrial localization, we also identified other key residues for N-terminal domain cytotoxicity. Additionally, we demonstrated that all GSDMB variants share the cleavage sites for GZMA, Neutrophil Elastase (identified in this study) and caspases. Interestingly, whereas Neutrophil Elastase and caspases produce N-terminal fragments in all GSDMB isoforms with no pyroptotic activity, thus acting as a potential inhibitory mechanism, GZMA cleavage activates pyroptosis in an isoform-dependent way. Summarizing, our results have important implications for understanding the complex roles of GSDMB isoforms in cancer and other pathologies and for the future design of GSDMB-targeted therapies.

molecular biology↗

bioRxiv2017

Shallow whole genome sequencing for robust copy number profiling of formalin-fixed paraffin-embedded breast cancers

Pathology archives with linked clinical data are an invaluable resource for translational research, with the limitation that most cancer samples are formalin-fixed paraffin-embedded (FFPE) tissues. Therefore, FFPE tissues are an important resource for genomic profiling studies but are under-utilised due to the low amount and quality of extracted nucleic acids. We profiled the copy number landscape of 356 breast cancer patients using DNA extracted FFPE tissues by shallow whole genome sequencing. We generated a total of 491 sequencing libraries from 2 kits and obtained data from 98.4% of libraries with 86.4% being of good quality. We generated libraries from as low as 3.8ng of input DNA and found that the success was independent of input DNA amount and quality, processing site and age of the fixed tissues. Since copy number alterations (CNA) play a major role in breast cancer, it is imperative that we are able to use FFPE archives and we have shown in this study that sWGS is a robust method to do such profiling.

genomics↗

bioRxiv2017

Targeting stromal remodeling and cancer stem cell plasticity to overcome chemoresistance in triple negative breast cancer

The cellular and molecular basis of stromal cell recruitment, activation and crosstalk in carcinomas is poorly understood, limiting the development of targeted anti-stromal therapies. In mouse models of triple negative breast cancer (TNBC), Hh ligand produced by neoplastic cells reprogrammed cancer-associated fibroblast (CAF) gene expression, driving tumor growth and metastasis. Hh-activated CAFs upregulated expression of FGF5 and production of fibrillar collagen, leading to FGFR and FAK activation in adjacent neoplastic cells, which then acquired a stem-like, drug-resistant phenotype. Treatment with smoothened inhibitors (SMOi) reversed these phenotypes. Stromal treatment of TNBC patient-derived xenograft (PDX) models with SMOi downregulated the expression of cancer stem cell markers and sensitized tumors to docetaxel, leading to markedly improved survival and reduced metastatic burden. In the phase I clinical trial EDALINE, 3 of 12 patients with metastatic TNBC derived clinical benefit from combination therapy with the SMOi Sonidegib and docetaxel chemotherapy, with one patient experiencing a complete response. Markers of pathway activity correlated with response. These studies identify Hh signaling to CAFs as a novel mediator of cancer stem cell plasticity and an exciting new therapeutic target in TNBC.\n\nSIGNIFICANCECompared to other breast cancer subtypes, TNBCs are associated with significantly worse patient outcomes. Standard of care systemic treatment for patients with non-BRCA1/2 positive TNBC is cytotoxic chemotherapy. However, the failure of 70% of treated TNBCs to attain complete pathological response reflects the relative chemoresistance of these tumors. New therapeutic strategies are needed to improve patient survival and quality of life. Here, we provide new insights into the dynamic interactions between heterotypic cells within a tumor. Specifically, we establish the mechanisms by which CAFs define cancer cell phenotype and demonstrate that the bidirectional CAF-cancer cell crosstalk can be successfully targeted in mice and humans using anti-stromal therapy.

cancer biology↗