Biology subjects

Dolcet, X.

Publications and source records attributed to Dolcet, X..

7 recordsLinked to original sources

bioRxiv2025

Endometrial cancer progression driven by PTEN-deficiency requires miR-424(322)~503.

Endometrial cancer is the most frequent type of cancer in the female reproductive tract. Loss-of-function alterations in PTEN, leading to enhanced PI3K/AKT activation, are among the most frequent molecular alterations in endometrial cancer. Increased PI3K/AKT signaling resulting from PTEN loss promotes cellular proliferation and confers resistance to TGF{beta}-mediated apoptosis, a key regulator of endometrial homeostasis. In this study, we have analyzed the role of miRNAs in driving these altered cellular responses. A comprehensive transcriptomic analysis of miRNA expression revealed the upregulation of several miRNAs caused by PTEN deficiency and/or TGF{beta} stimulation. The miR-424(322)[~]503 cluster drew our attention due to its involvement in regulating apoptosis and proliferation. However, miR-424(322)[~]503 cluster has a paradoxical role in cancer, exhibiting either oncogenic and tumor suppressive functions depending on cell type or context. To ascertain the function of miR-424(322)[~]503 in endometrial carcinogenesis caused by PTEN deficiency, we generated a double Pten/miR-424(322)[~]503 knock-out mice. We demonstrate that loss of miR-424(322)[~]503 impairs proliferation of both wild type or Pten deficient endometrial organoids by interfering with growth factor and PI3K/AKT signaling. Furthermore, the absence of miR-424(322)[~]503 restores TGF{beta}-induced apoptosis, which is otherwise compromised by PTEN deficiency. In vivo, Pten/miR-424(322)[~]503 knock-out mice exhibit reduced endometrial cancer progression compared to Pten deficient mice through a cell-autonomous mechanism.

cancer biology↗

bioRxiv2024

Cytoplasmic expression of the cell cycle regulator cyclin D1 in radial glial progenitor cells modulates brain cortex development

During nervous system development, the interplay between cell cycle regulation and neurogenesis is fundamental to achieve the correct timing for neuronal differentiation. However, the molecular players regulating this transition are poorly understood. Among these, the cell-cycle regulatory cyclins and their cyclin-dependent kinases (Cdks) play a pivotal role. In the present work we uncover an unknown function of cyclin D1 (Ccnd1) during cortex development which is independent of cell cycle regulation and that relies on its cytoplasmic localization and membrane association. We show that Ccnd1 is localized in the cytoplasm of the radial glial process (RGP) of neuron progenitors in different regions of the developing brain, including the cortex. Cytoplasmic Ccnd1 is enriched at the distal tip of the RGP, adjacent to the meningeal basement membrane, and overlaps with {beta}1-integrin at the plasma membrane. CCND1 knock-out animals show an abnormal cortical layering in which the distribution of Tbr2+ and Ctip2+ cells are affected without displaying proliferation defects. This is consistent with a cytoplasmic function of Ccnd1 as overexpression by in utero electroporation of a dominant negative Ccnd1, unable to activate Cdks, and targeted to the cytoplasmic membranes, reproduces some of these Tbr2 and Ctip2 defects. Finally, we provide evidence that cytoplasmic Ccnd1 affects neuron morphology and that it is required for the proper detachment of the RGP from the meningeal basement membrane by a mechanism involving the phosphorylation of the integrin effector protein paxillin. Hence, we propose that Ccnd1 has an important cytoplasmic function for cortical development independently of cell cycle regulation. Significant StatementA key developmental step during nervous system formation is the transition from proliferating progenitors to postmitotic neurons. However, the molecular mechanisms regulating this process are not fully understood. Cyclin D1 (Ccnd1) is a canonical regulator of cell cycle in the cell nucleus. Surprisingly, we show that Ccnd1 is also located in the radial glial process of neuron progenitors and associated to the plasma membrane in different regions of the developing mouse brain. We uncover a novel function for this cytoplasmic Ccnd1 and show that it is required for proper cortical layering, independent of cell cycle regulation. Mechanistically, we provide evidence that this function is mediated by the integrin effector paxillin. We propose therefore that cytoplasmic Ccnd1 is important for cortex development independent of cell cycle regulation.

neuroscience↗

bioRxiv2024

Multiple endocrine defects in adult-onset Sprouty1/2/4 triple knockout mice

Genes of the Sprouty family (Spry1-4) are feedback inhibitors of receptor tyrosine kinases, especially of Ret and the FGF receptors. As such, they play distinct and overlapping roles in embryo morphogenesis and are considered to be tumor suppressors in adult life. Genetic experiments in mice have defined in great detail the role of these genes during embryonic development, however their function in adult mice is less clearly established. Here we generate adult-onset, whole body Spry1/2/4 triple knockout mice. Tumor incidence in triple mutant mice is comparable to that of wild type littermates of up to one year of age, indicating that Sprouty loss per se is not sufficient to initiate tumorigenesis. On the other hand, triple knockout mice do not gain weight as they age, show less visceral fat, and have lower plasma glucose levels than wild type littermates, despite showing similar food intake and slightly reduced motor function. They also show alopecia, eyelid inflammation, and mild hyperthyroidism. Finally, triple knockout mice present phosphaturia and hypophosphatemia, suggesting exacerbated signaling downstream of FGF23. In conclusion, triple knockout mice develop a series of endocrine abnormalities but do not show increased tumor incidence.

physiology↗

bioRxiv2023

In vivo intra-uterine delivery of TAT-fused Cre recombinase and CRISPR/Cas9 editing unveil histopathology of Pten/p53-deficient endometrial cancers.

Pten and p53 are two of the most frequently mutated tumor suppressor genes in endometrial cancer. However, the functional consequences and histopathological manifestation of concomitant p53 and Pten loss of function alterations in the development of endometrial cancer is still controversial. Here, we demonstrate that simultaneous Pten and p53 deletion is sufficient to cause epithelial to mesenchymal transition phenotype in endometrial organoids. By a novel TAT-fused Cre intravaginal delivery method, we achieved local ablation of both p53 and Pten specifically in the uterus. These mice developed high-grade endometrial carcinomas and a high percentage of uterine carcinosarcomas resembling those found in humans. To further demonstrate that carcinosarcomas arise from epithelium, double Pten/p53 deficient epithelial cells were mixed with wild type stromal and myometrial cells and subcutaneously transplanted to Scid mice. All xenotransplants resulted in the development of uterine carcinosarcomas displaying high nuclear pleomorphism and metastatic potential. Accordingly, in vivo CRISPR/Cas9 disruption of Pten and p53 also triggered the development of metastatic carcinosarcomas. Our results unfadingly demonstrate that simultaneous deletion of p53 and Pten in endometrial epithelial cells is enough to trigger epithelial to mesenchymal transition that is consistently translated to the formation of uterine carcinosarcomas in vivo.

pathology↗

bioRxiv2022

Transient, DNA-free in vivo CRISPR/Cas9 genome edition for flexible modelling of endometrial carcinogenesis

The CRISPR/Cas9 technology has emerged as a powerful tool to generate mouse models of disease. Endometrial cancer is the most common malignancy of the female genital tract. In the present study, we have developed a pipeline for the generation of somatically engineered mouse models of endometrial cancer by in vivo electroporation-mediated delivery of Cas9 ribonucleoprotein into the uterine cavity. By using mT/mG dual-fluorescent reporter mice, we show that this system allows an efficient genomic edition specifically in epithelial endometrial cells. As a proof of its applicability for endometrial cancer modeling, we designed Cas9 ribonucleoprotein targeting Pten, the most frequently tumor suppressor gene mutated in this type of cancer. Pten-targeting ribonucleoprotein delivery into the uterine cavity caused loss of expression of PTEN protein in epithelial endometrial cells that resulted in the development of endometrial lesions. We also validated this technique for gene edition in other important endometrial driver genes such as p53 or Fbxw7. By co-targeting LoxP sites of mT/mG reporter mice and Pten, we demonstrate the generation of differentially edited cell populations that may be a useful tool to model tumoral heterogeneity. Moreover, the combination of CRISPR/Cas9 technology in mT/mG dual-reporter mice and light-sheet microscopy represents an interesting approach for in vivo cancer cell tracing. This methodology opens a new door for future rapid, flexible, customizable and multiplexable in vivo modeling of endometrial cancer.

cancer biology↗

bioRxiv2022

The ERK5/NF-κB signaling pathway targets endometrial cancer proliferation and survival

Endometrial cancer (EC) is the most common type of gynaecologic cancer in women of developed countries. Despite surgery combined with chemo-/radiotherapy regimens, overall survival of patients with high-risk EC tumors is poor, indicating a need for novel molecular therapies. The MEK5-ERK5 pathway is activated in response to growth factors and to different forms of stress, including oxidative stress and cytokines. Previous evidence support a role for the MEK5-ERK5 pathway in the pathology of several cancers. We have investigated the role of ERK5 in EC. In silico analysis of the PanCancer Atlas dataset showed alterations in components of the MEK5-ERK5 pathway in 48% of EC patients. Here, we show that ERK5 inhibition decreased EGF-induced EC cell proliferation, and that depletion of MEK5 resulted in EC impaired proliferation and reduced tumor growth capacity in nude mice. Pharmacologic or genetic silencing of ERK5 impaired NF-kB pathway in EC cells and xenografts. Furthermore, we found a positive correlation between ERK5 and p65/RELA protein levels in human EC tumor samples. Mechanistically, impairment of ERK5 resulted in downregulation of NEMO/IKK{gamma} expression, leading to impaired p65/RELA activity and to apoptosis in EC cells and xenografts, which was rescued by NEMO/IKK{gamma} overexpression. Notably, ERK5 inhibition, MEK5 depletion or NF-kB inhibition sensitized EC cells to standard EC chemotherapy (paclitaxel/carboplatin) toxicity, whereas ERK5 inhibition synergized with paclitaxel to reduce tumor xenograft growth in mice. Together, our results suggest that the ERK5-NEMO-NF-{kappa}B pathway mediates EC cell proliferation and survival. We propose the ERK5/NF-{kappa}B axis as new target for EC treatment.

cancer biology↗

bioRxiv2022

A dominant negative mutation uncovers cooperative control of caudal Wolffian Duct development by Sprouty genes

The Wolffian Ducts (WD) are paired epithelial tubules central to the development of the mammalian genitourinary tract. Outgrowths from the WD known as the ureteric buds (UB) generate the collecting ducts of the kidney. Later during development, the caudal portion of the WD will form the vas deferens, epididymis and seminal vesicle in males, and will degenerate in females. While the genetic pathways controlling the development of the UB are firmly established, less is known about those governing development of WD portions caudal to the UB. Sprouty proteins are inhibitors of receptor tyrosine kinase (RTK) signaling in vivo. We have recently shown that homozygous mutation of a conserved tyrosine (Tyr53) of Spry1 results in UB defects indistinguishable from that of Spry1 null mice. Here we show that heterozygosity for the Spry1 Y53A allele causes caudal WD developmental defects consisting on ectopically branched seminal vesicles in males and persistent WD in females, without affecting kidney development. Detailed analysis reveals that this phenotype also occurs in Spry1+/- mice but with a much lower penetrance, indicating that removal of tyrosine 53 generates a dominant negative mutation in vivo. Supporting this notion, concomitant deletion of one allele of Spry1 and Spry2 also recapitulates the genital phenotype of Spry1Y53A/+ mice with high penetrance. Mechanistically, we show that unlike the effects of Spry1 in kidney development, these caudal WD defects are independent of Ret signaling, but can be completely rescued by lowering the genetic dosage of Fgf10. In conclusion, mutation of tyrosine 53 of Spry1 generates a dominant-negative allele that uncovers fine-tuning of caudal WD development by Sprouty genes.

developmental biology↗