Biology subjects

Dolcet, X.

Publications and source records attributed to Dolcet, X..

4 recordsLinked to original sources

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↗