Cytoplasmic p27Kip1 promotes tumorigenesis via suppression of RhoB activity.

The cell cycle inhibitor p27Kip1 is a tumor suppressor via the inhibition of CDK complexes in the nucleus. However, p27 also plays other functions in the cell and may acquire oncogenic roles when located in the cytoplasm. Activation of oncogenic pathways such as Ras or PI3K/AKT causes the relocalization of p27 in the cytoplasm, where it can promote tumorigenesis by unclear mechanisms. Here, we investigated how cytoplasmic p27 participates in the development of non-small cell lung carcinomas. We provide molecular and genetic evidence that the oncogenic role of p27 is mediated, at least in part, by binding to and inhibiting the GTPase RhoB, which normally acts as a tumor suppressor in the lung. Genetically modified mice revealed that RhoB expression is preferentially lost in tumors in which p27 is absent and maintained in tumors expressing wild-type p27 or p27CK- , a mutant that cannot inhibit CDKs. Moreover, although the absence of RhoB promoted tumorigenesis in p27-/- animals, it had no effect in p27CK- knock-in mice, suggesting that cytoplasmic p27 may act as an oncogene, at least in part, by inhibiting the activity of RhoB. Finally, in a cohort of lung cancer patients, we identified a subset of tumors harboring cytoplasmic p27 in which RhoB expression is maintained and these characteristics were strongly associated with decreased patient survival. Thus, monitoring p27 localization and RhoB levels in non-small cell lung carcinoma patients appears to be a powerful prognostic marker for these tumors. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Extended-depth of field random illumination microscopy, EDF-RIM, provides super-resolved projective imaging.

The ultimate aim of fluorescence microscopy is to achieve high-resolution imaging of increasingly larger biological samples. Extended depth of field presents a potential solution to accelerate imaging of large samples when compression of information along the optical axis is not detrimental to the interpretation of images. We have implemented an extended depth of field (EDF) approach in a random illumination microscope (RIM). RIM uses multiple speckled illuminations and variance data processing to double the resolution. It is particularly adapted to the imaging of thick samples as it does not require the knowledge of illumination patterns. We demonstrate highly-resolved projective images of biological tissues and cells. Compared to a sequential scan of the imaged volume with conventional 2D-RIM, EDF-RIM allows an order of magnitude improvement in speed and light dose reduction, with comparable resolution. As the axial information is lost in an EDF modality, we propose a method to retrieve the sample topography for samples that are organized in cell sheets.

Apelin-VEGF-C mRNA delivery as therapeutic for the treatment of secondary lymphedema.

Secondary lymphedema (LD) corresponds to a severe lymphatic dysfunction leading to the accumulation of fluid and fibrotic adipose tissue in a limb. Here, we identified apelin (APLN) as a powerful molecule for regenerating lymphatic function in LD. We identified the loss of APLN expression in the lymphedematous arm compared to the normal arm in patients. The role of APLN in LD was confirmed in APLN knockout mice, in which LD is increased and associated with fibrosis and dermal backflow. This was reversed by intradermal injection of APLN-lentivectors. Mechanistically, APLN stimulates lymphatic endothelial cell gene expression and induces the binding of E2F8 transcription factor to the promoter of CCBE1 that controls VEGF-C processing. In addition, APLN induces Akt and eNOS pathways to stimulate lymphatic collector pumping. Our results show that APLN represents a novel partner for VEGF-C to restore lymphatic function in both initial and collecting vessels. As LD appears after cancer treatment, we validated the APLN-VEGF-C combination using a novel class of nonintegrative RNA delivery LentiFlash® vector that will be evaluated for phase I/IIa clinical trial.

p57Kip2 acts as a transcriptional corepressor to regulate intestinal stem cell fate and proliferation.

p57Kip2 is a cyclin/CDK inhibitor and a negative regulator of cell proliferation. Here, we report that p57 regulates intestinal stem cell (ISC) fate and proliferation in a CDK-independent manner during intestinal development. In the absence of p57, intestinal crypts exhibit an increased proliferation and an amplification of transit-amplifying cells and of Hopx+ ISCs, which are no longer quiescent, while Lgr5+ ISCs are unaffected. RNA sequencing (RNA-seq) analyses of Hopx+ ISCs show major gene expression changes in the absence of p57. We found that p57 binds to and inhibits the activity of Ascl2, a transcription factor critical for ISC specification and maintenance, by participating in the recruitment of a corepressor complex to Ascl2 target gene promoters. Thus, our data suggest that, during intestinal development, p57 plays a key role in maintaining Hopx+ ISC quiescence and repressing the ISC phenotype outside of the crypt bottom by inhibiting the transcription factor Ascl2 in a CDK-independent manner.

In vitro models of intestinal epithelium: Toward bioengineered systems.

The intestinal epithelium, the fastest renewing tissue in human, is a complex tissue hosting multiple cell types with a dynamic and multiparametric microenvironment, making it particularly challenging to recreate in vitro. Convergence of recent advances in cellular biology and microfabrication technologies have led to the development of various bioengineered systems to model and study the intestinal epithelium. Theses microfabricated in vitro models may constitute an alternative to current approaches for studying the fundamental mechanisms governing intestinal homeostasis and pathologies, as well as for in vitro drug screening and testing. Herein, we review the recent advances in bioengineered in vitro intestinal models.

p27 controls autophagic vesicle trafficking in glucose-deprived cells via the regulation of ATAT1-mediated microtubule acetylation.

The cyclin-dependent kinase inhibitor p27Kip1 (p27) has been involved in promoting autophagy and survival in conditions of metabolic stress. While the signaling cascade upstream of p27 leading to its cytoplasmic localization and autophagy induction has been extensively studied, how p27 stimulates the autophagic process remains unclear. Here, we investigated the mechanism by which p27 promotes autophagy upon glucose deprivation. Mouse embryo fibroblasts (MEFs) lacking p27 exhibit a decreased autophagy flux compared to wild-type cells and this is correlated with an abnormal distribution of autophagosomes. Indeed, while autophagosomes are mainly located in the perinuclear area in wild-type cells, they are distributed throughout the cytoplasm in p27-null MEFs. Autophagosome trafficking towards the perinuclear area, where most lysosomes reside, is critical for autophagosome-lysosome fusion and cargo degradation. Vesicle trafficking is mediated by motor proteins, themselves recruited preferentially to acetylated microtubules, and autophagy flux is directly correlated to microtubule acetylation levels. p27-/- MEFs exhibit a marked reduction in microtubule acetylation levels and restoring microtubule acetylation in these cells, either by re-expressing p27 or with deacetylase inhibitors, restores perinuclear positioning of autophagosomes and autophagy flux. Finally, we find that p27 promotes microtubule acetylation by binding to and stabilizing α-tubulin acetyltransferase (ATAT1) in glucose-deprived cells. ATAT1 knockdown results in random distribution of autophagosomes in p27+/+ MEFs and impaired autophagy flux, similar to that observed in p27-/- cells. Overall, in response to glucose starvation, p27 promotes autophagy by facilitating autophagosome trafficking along microtubule tracks by maintaining elevated microtubule acetylation via an ATAT1-dependent mechanism.

Functional Versatility of the CDK Inhibitor p57Kip2.

The cyclin/CDK inhibitor p57Kip2 belongs to the Cip/Kip family, with p21Cip1 and p27Kip1, and is the least studied member of the family. Unlike the other family members, p57Kip2 has a unique role during embryogenesis and is the only CDK inhibitor required for embryonic development. p57Kip2 is encoded by the imprinted gene CDKN1C, which is the gene most frequently silenced or mutated in the genetic disorder Beckwith-Wiedemann syndrome (BWS), characterized by multiple developmental anomalies. Although initially identified as a cell cycle inhibitor based on its homology to other Cip/Kip family proteins, multiple novel functions have been ascribed to p57Kip2 in recent years that participate in the control of various cellular processes, including apoptosis, migration and transcription. Here, we will review our current knowledge on p57Kip2 structure, regulation, and its diverse functions during development and homeostasis, as well as its potential implication in the development of various pathologies, including cancer.

p27 controls Ragulator and mTOR activity in amino acid-deprived cells to regulate the autophagy-lysosomal pathway and coordinate cell cycle and cell growth.

Autophagy is a catabolic process whereby cytoplasmic components are degraded within lysosomes, allowing cells to maintain energy homeostasis during nutrient depletion. Several studies reported that the CDK inhibitor p27Kip1 promotes starvation-induced autophagy by an unknown mechanism. Here we find that p27 controls autophagy via an mTORC1-dependent mechanism in amino acid-deprived cells. During prolonged starvation, a fraction of p27 is recruited to lysosomes, where it interacts with LAMTOR1, a component of the Ragulator complex required for mTORC1 activation. Binding of p27 to LAMTOR1 prevents Ragulator assembly and mTORC1 activation, promoting autophagy. Conversely, p27-/- cells exhibit elevated mTORC1 signalling as well as impaired lysosomal activity and autophagy. This is associated with cytoplasmic sequestration of TFEB, preventing induction of the lysosomal genes required for lysosome function. LAMTOR1 silencing or mTOR inhibition restores autophagy and induces apoptosis in p27-/- cells. Together, these results reveal a direct coordinated regulation between the cell cycle and cell growth machineries.

Human adipose stromal-vascular fraction self-organizes to form vascularized adipose tissue in 3D cultures.

Native human subcutaneous adipose tissue (AT) is well organized into unilocular adipocytes interspersed within dense vascularization. This structure is completely lost under standard culture conditions and may impair the comparison with native tissue. Here, we developed a 3-D model of human white AT reminiscent of the cellular architecture found in vivo. Starting with adipose progenitors derived from the stromal-vascular fraction of human subcutaneous white AT, we generated spheroids in which endogenous endothelial cells self-assembled to form highly organized endothelial networks among stromal cells. Using an optimized adipogenic differentiation medium to preserve endothelial cells, we obtained densely vascularized spheroids containing mature adipocytes with unilocular lipid vacuoles. In vivo study showed that when differentiated spheroids were transplanted in immune-deficient mice, endothelial cells within the spheroids connected to the recipient circulatory system, forming chimeric vessels. In addition, adipocytes of human origin were still observed in transplanted mice. We therefore have developed an in vitro model of vascularized human AT-like organoids that constitute an excellent tool and model for any study of human AT.

Fabrication of 3D scaffolds reproducing intestinal epithelium topography by high-resolution 3D stereolithography.

The small intestine is a complex tissue with a crypt/villus architecture and high tissue polarity. Maintenance of tissue integrity and function is supported by a constant renewal of the epithelium, with proliferative cells located in the crypts and differentiated cells migrating upward to the top of villi. So far, most in vitro studies have been limited to 2D surfaces or 3D organoid cultures that do not fully recapitulate the tissue 3D architecture, microenvironment and cell compartmentalization found in vivo. Here, we report the development of a 3D model that reproduces more faithfully the architecture of the intestinal epithelium in vitro. We developed a new fabrication process combining a photopolymerizable hydrogel that supports the growth of intestinal cell lines with high-resolution stereolithography 3D printing. This approach offers the possibility to create artificial 3D scaffolds matching the dimensions and architecture of mouse intestinal crypts and villi. We demonstrate that these 3D culture models support the growth and differentiation of Caco-2 cells for 3 weeks. These models may constitute a complementary approach to organoid cultures to study intestinal homeostasis by allowing guided self-organization and controlled differentiation, as well as for in vitro drug screening and testing.

p27Kip1 regulates the microtubule bundling activity of PRC1.

Cytokinesis begins in anaphase with the formation of the central spindle. PRC1 is a microtubule associated protein that plays an essential role in central spindle formation by crosslinking antiparallel microtubules. We have identified PRC1 as a novel binding partner for p27Kip1 (p27). p27 is a cyclin-CDK inhibitor that causes cell cycle arrest in G1. However, p27 has also been involved in the regulation of G2/M progression and cytokinesis, as well as of other cellular processes, including actin and microtubule cytoskeleton dynamics. We found that p27 interferes with the ability of PRC1 to bind to microtubules, without affecting PRC1 dimerization or its capacity to interact with other partners such as KIF4. In this way, p27 inhibited microtubule bundling by PRC1 in vitro and prevented the extensive microtubule bundling phenotype caused by PRC1 overexpression in cells in culture. Finally, co-expression of p27 or a p27 mutant that does not bind cyclin-CDKs inhibited multinucleation induced by PRC1 overexpression. Together, our results suggest that p27 may participate in the regulation of mitotic progression in a CDK-independent manner by modulating PRC1 activity.