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During nervous system development, the interplay between cell cycle regulation and neurogenesis is fundamental to achieving the correct timing for neuronal differentiation. However, the molecules 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 pallium. Cytoplasmic CCND1 is enriched at the distal tip of the RGP, adjacent to the meningeal basement membrane, and overlaps with β1-integrin at the plasma membrane. Ccnd1 knock-out embryos 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.

Colorectal cancer (CRC) is a leading cause of cancer-related morbidity and mortality worldwide, with molecular subtypes and signaling pathways playing critical roles in its progression. The miR-424(322) ~ 503 cluster, comprising miR-424 and miR-503, has been implicated in various malignancies, exhibiting dual roles as tumor suppressors or oncogenes depending on the context. However, its function in CRC remains poorly understood. This study investigates the role of the miR-424(322) ~ 503 cluster in CRC driven by PTEN deficiency using genetically modified mouse models. Our findings reveal that the loss of miR-424(322) ~ 503 significantly exacerbates CRC progression in PTEN-deficient mice. Double knockout (dKO) mice lacking both PTEN and miR-424(322) ~ 503 exhibited a higher number and larger size of colorectal lesions compared to PTEN-deficient counterparts. Histological analysis demonstrated increased severity of dysplasia and adenocarcinoma development in dKO mice. Mechanistically, while Wnt/β-catenin signaling remained unaltered, transcriptomic analyses highlighted dysregulation of MAPK and TGFβ pathways, alongside epithelial-to-mesenchymal transition (EMT)-related gene signatures. Protein-level validation confirmed hyperactivation of MAPK (ERK1/2 and p38) and TGFβ signaling, as well as elevated cyclin D1 expression in dKO colonic tissues. These results underscore the tumor-suppressive role of the miR-424(322) ~ 503 cluster in CRC by modulating key oncogenic pathways such as MAPK and TGFβ. Our study provides novel insights into the interplay between PTEN loss and miRNA regulation in CRC pathogenesis.

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 TGFβ-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 TGFβ 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 TGFβ-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.

Endothelial cell activation leading to leukocyte recruitment and adhesion plays an essential role in the initiation and progression of atherosclerosis. Vitamin D has cardioprotective actions, while its deficiency is a risk factor for the progression of cardiovascular damage. Our aim was to assess the role of basal levels of vitamin D receptor (VDR) on the early leukocyte recruitment and related endothelial cell-adhesion-molecule expression, as essential prerequisites for the onset of atherosclerosis. Knockdown of VDR in endothelial cells (shVDR) led to endothelial cell activation, characterized by upregulation of VCAM-1, ICAM-1 and IL-6, decreased peripheral blood mononuclear cell (PBMC) rolling velocity and increased PBMC rolling flux and adhesion to the endothelium. shVDR cells showed decreased IκBα levels and accumulation of p65 in the nucleus compared to shRNA controls. Inhibition of NF-κB activation with super-repressor IκBα blunted all signs of endothelial cell activation caused by downregulation of VDR in endothelial cells. In vivo, deletion of VDR led to significantly larger aortic arch and aortic root lesions in apoE-/- mice, with higher macrophage content. apoE-/-VDR-/-mice showed higher aortic expression of VCAM-1, ICAM-1 and IL-6 when compared to apoE-/-VDR+/+ mice. Our data demonstrate that lack of VDR signaling in endothelial cells leads to a state of endothelial activation with increased leukocyte-endothelial cell interactions that may contribute to the more severe plaque accumulation observed in apoE-/-VDR-/- mice. The results reveal an important role for basal levels of endothelial VDR in limiting endothelial cell inflammation and atherosclerosis.

The extracellular matrix and the correct establishment of epithelial cell polarity plays a critical role in epithelial cell homeostasis and cell polarity. In addition, loss of tissue structure is a hallmark of carcinogenesis. In this study, we have addressed the role of extracellular matrix in the cellular responses to TGF-β. It is well known that TGF-β is a double-edged sword: it acts as a tumor suppressor in normal epithelial cells, but conversely has tumor-promoting effects in tumoral cells. However, the factors that determine cellular outcome in response to TGF-β remain controversial. Here, we have demonstrated that the lack of extracellular matrix and consequent loss of cell polarity inhibits TGF-β-induced apoptosis, observed when endometrial epithelial cells are polarized in presence of extracellular matrix. Rather, in absence of extracellular matrix, TGF-β-treated endometrial epithelial cells display features of epithelial-to-mesenchymal transition. We have also investigated the molecular mechanism of such a switch in cellular response. On the one hand, we found that the lack of Matrigel results in increased AKT signaling which is sufficient to inhibit TGF-β-induced apoptosis. On the other hand, we demonstrate that TGF-β-induced epithelial-to-mesenchymal transition requires ERK and SMAD2/3 activation. In summary, we demonstrate that loss of cell polarity changes the pro-apoptotic function of TGF-β to tumor-associated phenotype such as epithelial-to-mesenchymal transition. These results may be important for understanding the dual role of TGF-β in normal versus tumoral cells.

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.

To evaluate senescence mechanisms, including senescence-associated secretory phenotype (SASP), in the motor neuron disease model hSOD1-G93A, we quantified the expression of p16 and p21 and senescence-associated β-galactosidase (SA-β-gal) in nervous tissue. As SASP markers, we measured the mRNA levels of Il1a, Il6, Ifna and Ifnb. Furthermore, we explored whether an alteration of alternative splicing is associated with senescence by measuring the Adipor2 cryptic exon inclusion levels, a specific splicing variant repressed by TAR DNA-binding protein (TDP-43; encoded by Tardbp). Transgenic mice showed an atypical senescence profile with high p16 and p21 mRNA and protein in glia, without the canonical increase in SA-β-gal activity. Consistent with SASP, there was an increase in Il1a and Il6 expression, associated with increased TNF-R and M-CSF protein levels, with females being partially protected. TDP-43 splicing activity was compromised in this model, and the senolytic drug Navitoclax did not alter the disease progression. This lack of effect was reproduced in vitro, in contrast to dasatinib and quercetin, which diminished p16 and p21. Our findings show a non-canonical profile of senescence biomarkers in the model hSOD1-G93A.

Genes of the Sprouty family (Spry1-4) restrain signaling by certain receptor tyrosine kinases. Consequently, these genes participate in several developmental processes and function as tumor suppressors in adult life. Despite these important roles, the biology of this family of genes still remains obscure. Here we show that Sprouty proteins are general mediators of cellular senescence. Induction of cellular senescence by several triggers in vitro correlates with upregulation of Sprouty protein levels. More importantly, overexpression of Sprouty genes is sufficient to cause premature cellular senescence, via a conserved N-terminal tyrosine (Tyrosine 53 of Sprouty1). Accordingly, fibroblasts from knockin animals lacking that tyrosine escape replicative senescence. In vivo, heterozygous knockin mice display delayed induction of cellular senescence during cutaneous wound healing and upon chemotherapy-induced cellular senescence. Unlike other functions of this family of genes, induction of cellular senescence appears to be independent of activation of the ERK1/2 pathway. Instead, we show that Sprouty proteins induce cellular senescence upstream of the p38 pathway in these in vitro and in vivo paradigms.

Phosphatase and TENsin homolog (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, it is demonstrated that simultaneous Pten and p53 deletion is sufficient to cause epithelial to mesenchymal transition phenotype in endometrial organoids. By a novel intravaginal delivery method using HIV1 trans‐activator of transcription cell penetrating peptide fused with a Cre recombinase protein (TAT‐Cre), local ablation of both p53 and Pten is achieved 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 are 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. The 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.