Targeting cholesterol impairs cell invasion of all breast cancer types.

BACKGROUND: Breast cancer clinical outcome relies on its intrinsic molecular subtype and mortality is almost exclusively due to metastasis, whose mechanism remains unclear. We recently revealed the specific contribution of plasma membrane cholesterol to the invasion of malignant MCF10CAIa but not premalignant MCF10AT and normal MCF10A cell lines in 2D, through invadopodia formation and extracellular matrix (ECM) degradation. In the present study, we address the impact of breast cancer subtypes, mutations and aggressiveness on cholesterol implication in breast cancer cell invasion and 3D spheroid invasion and growth. METHODS: We used nine breast cancer cell lines grouped in four subtypes matching breast tumor classification. Four of these cell lines were also used to generate 3D spheroids. These cell lines were compared for cell invasion in 2D and 3D, spheroid growth in 3D, gelatin degradation, cortactin expression, activation and subcellular distribution as well as cell surface cholesterol distribution and lipid droplets. The effect of plasma membrane cholesterol depletion on all these parameters was determined in parallel and systematically compared with the impact of global matrix metalloproteinase (MMP) inhibition. RESULTS: The six invasive cell lines in 2D were sensitive to partial cholesterol depletion, independently of their subtype, aggressiveness or mutation. Nevertheless, the effect was stronger in the three cell lines able to degrade gelatin. 3D spheroid invasion was also reduced after cholesterol depletion in all breast cancer subtypes tested. Notably, targeting cholesterol was more powerful than MMP inhibition in reducing invasion in both 2D and 3D culture models. Moreover, cholesterol depletion in the six invasive cell lines impaired cortactin distribution in the perinuclear region where invadopodia localized. Breast cancer cell line aggressiveness relied on cholesterol-enriched domains at the ECM-free side and intracellular lipid droplets. Furthermore, the three gelatin-degrading cell lines were characterized by increased cholesterol-enriched submicrometric domains at their ECM-contact side. CONCLUSION: Together, our data suggest cell surface cholesterol combined with lipid droplet labeling as a breast cancer cell aggressiveness marker. They also open the way to test other cholesterol-targeting drugs in more complex models to further evaluate whether cholesterol could represent a strategy in breast cancer therapy.

Effects of estradiol, progesterone or cAMP on expression of PGRMC1 and progesterone receptor in a xenograft model of human endometrium and in endometrial cell culture.

Estradiol and progesterone are key regulators of the menstrual cycle. In the human endometrium, progesterone induces morphological changes required for blastocyst implantation. Dysregulated response to progesterone can lead to endometrial pathologies including uterine bleeding and endometriosis. Besides the canonical nuclear progesterone receptor (encoded by the PGR gene), alternative response pathways include Progesterone Receptor Membrane Component 1 (PGRMC1), suspected to be involved in pathogenesis of endometrial diseases. We previously reported the spatiotemporal profile of PGRMC1 expression in the human endometrium along the menstrual cycle, highlighting progressive increase and decrease during the proliferative and secretory phases, respectively. Here we directly addressed its regulation by estradiol and progesterone, with systematic comparison with regulation of PGR expression. We found a direct correlation between expression of both genes during the proliferative and secretory phases in the cycling endometrium, but not during the menstrual phase. In a xenograft model mimicking the cycle phases, estradiol significantly increased and progesterone significantly decreased PGR expression but changes were not significant for PGRMC1. Finally, we did not find any significant effect of the ovarian steroids on expression of PGR or PGRMC1 in primary culture of endometrial stromal cells, except for a small increase in PGR expression by estradiol. Altogether, our experiments do not allow a major advance in our understanding of the mechanisms of cyclic variation of PGRMC1 expression, in particular regarding potential regulation by the ovarian steroids.

BRAFV600E Expression in Thyrocytes Causes Recruitment of Immunosuppressive STABILIN-1 Macrophages.

Papillary thyroid carcinoma (PTC) is the most frequent histological subtype of thyroid cancers (TC), and BRAFV600E genetic alteration is found in 60% of this endocrine cancer. This oncogene is associated with poor prognosis, resistance to radioiodine therapy, and tumor progression. Histological follow-up by anatomo-pathologists revealed that two-thirds of surgically-removed thyroids do not present malignant lesions. Thus, continued fundamental research into the molecular mechanisms of TC downstream of BRAFV600E remains central to better understanding the clinical behavior of these tumors. To study PTC, we used a mouse model in which expression of BRAFV600E was specifically switched on in thyrocytes by doxycycline administration. Upon daily intraperitoneal doxycycline injection, thyroid tissue rapidly acquired histological features mimicking human PTC. Transcriptomic analysis revealed major changes in immune signaling pathways upon BRAFV600E induction. Multiplex immunofluorescence confirmed the abundant recruitment of macrophages, among which a population of LYVE-1+/CD206+/STABILIN-1+ was dramatically increased. By genetically inactivating the gene coding for the scavenger receptor STABILIN-1, we showed an increase of CD8+ T cells in this in situ BRAFV600E-dependent TC. Lastly, we demonstrated the presence of CD206+/STABILIN-1+ macrophages in human thyroid pathologies. Altogether, we revealed the recruitment of immunosuppressive STABILIN-1 macrophages in a PTC mouse model and the interest to further study this macrophage subpopulation in human thyroid tissues.

Role of Extracellular Vesicles in Thyroid Physiology and Diseases: Implications for Diagnosis and Treatment.

Extracellular vesicles are spherical subcellular structures delimited by a lipid bilayer and released by most cells in the human body. They are loaded with a myriad of molecules (i.e., nucleic acids and proteins) depending on their cell of origin and provide the ability to transmit a message to surrounding or distant target cells. In several organs, including the thyroid, abundant recent literature reports that extracellular vesicles are responsible for intercellular communication in physiological and pathological processes, and that their utilization as a potential biomarker of pathological states (i.e., cancer, autoimmune diseases) or as therapeutic delivery vehicles promise clinical options. In this review, we present the current knowledge and understanding regarding the role of extracellular vesicles in developing thyroid diseases and diagnosis.

Spatiotemporal expression pattern of Progesterone Receptor Component (PGRMC) 1 in endometrium from patients with or without endometriosis or adenomyosis.

The endometrium plays a crucial role in reproduction and, in humans, is cyclically remodeled under hormonal control. Estradiol favors tissue proliferation whereas progesterone inhibits tissue growth and induces morphological changes. Endometriosis is often associated with fertility issues and with exacerbated estrogen and reduced progesterone concentration or response in the eutopic endometrium. However, underlying mechanisms remain unclear. Progesterone Receptor Membrane Component (PGRMC) 1 is a protein able to modulate progesterone response and its murine knockout reduced fertility. However, the precise spatiotemporal pattern of PGRMC1 expression in the human endometrium is still poorly characterized. We investigated variations of eutopic endometrial PGRMC1 expression by combining RT-qPCR, immunofluorescence and in situ hybridization. We found that PGRMC1 expression progressively increases during the proliferative phase and decreases during the secretory phase. However, immunolabeling and identification of mRNA-containing cells were regularly heterogeneous in samples, according to tissue depth, with a gradient extending from the surface epithelium towards the basalis. There was no significant difference in PGRMC1 mRNA amounts between patients with or without endometriosis or adenomyosis, for any phase of the menstrual cycle, but cells with strong or moderate PGRMC1 immunolabeling were reduced during the proliferative phase in endometriotic patients. In conclusion, although the cyclical variation of PGRMC1 expression globally follows fluctuation of ovarian steroids, further work is required to precisely characterize hormonal control and identify the additional levels of regulation responsible for local adjustment of PGRMC1 concentration. This is particularly important in the light of recent studies emphasizing the correlation between adequate PGRMC1 amounts and fertility.

Two miRNAs enriched in plasma extracellular vesicles are potential biomarkers for thyroid cancer.

Differential diagnosis of thyroid cancer and benign nodules is still one of the most challenging issues in the field of endocrinology. To overcome overdiagnosis of papillary thyroid carcinomas (PTC) and the consecutive overtreatment of multinodular diseases, the search for easily accessible, sensitive and accurate biomarkers is critical. Several micro-RNAs (miRNAs) freely circulating in peripheral blood or enclosed in extracellular vesicles (EVs) have been proposed as potential biomarkers from non-invasive liquid biopsies. However, protocols are rarely comparable and conflicting data exist in the literature. In this work, we aimed to assess the diagnostic value of six micro-RNAs by comparing their expression in thyroid tissue to their abundance in bulk plasma and in plasma-EVs, before and after thyroid surgery. Plasma-EVs were isolated using a sequential density- and size-based fractionation, followed by in-depth characterization, confirming EV purity. Micro-RNA levels were measured by RT-qPCR in thyroid tissue, plasma and plasma-EVs. Among the six candidates, only miR-146b-5p and miR-21a-5p displayed a significant differential abundance in purified plasma-derived EVs from patients with PTC and benign disease. However, no difference could be demonstrated in bulk plasma through our cohort of patients. Overall, our work supports the use of a well-defined protocol of plasma-EV miRNAs purification for biomarker discovery, rather than the use of freely circulating miRNAs in bulk plasma. Our work also demonstrates that standardized pre-analytical and analytical procedures as well as optimized EV-miRNAs detection methods are essential.

BRAFV600E Induction in Thyrocytes Triggers Important Changes in the miRNAs Content and the Populations of Extracellular Vesicles Released in Thyroid Tumor Microenvironment.

Papillary thyroid cancer (PTC) is the most common endocrine malignancy for which diagnosis and recurrences still challenge clinicians. New perspectives to overcome these issues could come from the study of extracellular vesicle (EV) populations and content. Here, we aimed to elucidate the heterogeneity of EVs circulating in the tumor and the changes in their microRNA content during cancer progression. Using a mouse model expressing BRAFV600E, we isolated and characterized EVs from thyroid tissue by ultracentrifugations and elucidated their microRNA content by small RNA sequencing. The cellular origin of EVs was investigated by ExoView and that of deregulated EV-microRNA by qPCR on FACS-sorted cell populations. We found that PTC released more EVs bearing epithelial and immune markers, as compared to the healthy thyroid, so that changes in EV-microRNAs abundance were mainly due to their deregulated expression in thyrocytes. Altogether, our work provides a full description of in vivo-derived EVs produced by, and within, normal and cancerous thyroid. We elucidated the global EV-microRNAs signature, the dynamic loading of microRNAs in EVs upon BRAFV600E induction, and their cellular origin. Finally, we propose that thyroid tumor-derived EV-microRNAs could support the establishment of a permissive immune microenvironment.

Sox9 is involved in the thyroid differentiation program and is regulated by crosstalk between TSH, TGFß and thyroid transcription factors.

While the signaling pathways and transcription factors involved in the differentiation of thyroid follicular cells, both in embryonic and adult life, are increasingly well understood, the underlying mechanisms and potential crosstalk between the thyroid transcription factors Nkx2.1, Foxe1 and Pax8 and inductive signals remain unclear. Here, we focused on the transcription factor Sox9, which is expressed in Nkx2.1-positive embryonic thyroid precursor cells and is maintained from embryonic development to adulthood, but its function and control are unknown. We show that two of the main signals regulating thyroid differentiation, TSH and TGFß, modulate Sox9 expression. Specifically, TSH stimulates the cAMP/PKA pathway to transcriptionally upregulate Sox9 mRNA and protein expression, a mechanism that is mediated by the binding of CREB to a CRE site within the Sox9 promoter. Contrastingly, TGFß signals through Smad proteins to inhibit TSH-induced Sox9 transcription. Our data also reveal that Sox9 transcription is regulated by the thyroid transcription factors, particularly Pax8. Interestingly, Sox9 significantly increased the transcriptional activation of Pax8 and Foxe1 promoters and, consequently, their expression, but had no effect on Nkx2.1. Our study establishes the involvement of Sox9 in thyroid follicular cell differentiation and broadens our understanding of transcription factor regulation of thyroid function.

Development of a 3D atlas of the embryonic pancreas for topological and quantitative analysis of heterologous cell interactions.

Generating comprehensive image maps, while preserving spatial three-dimensional (3D) context, is essential in order to locate and assess quantitatively specific cellular features and cell-cell interactions during organ development. Despite recent advances in 3D imaging approaches, our current knowledge of the spatial organization of distinct cell types in the embryonic pancreatic tissue is still largely based on two-dimensional histological sections. Here, we present a light-sheet fluorescence microscopy approach to image the pancreas in three dimensions and map tissue interactions at key time points in the mouse embryo. We demonstrate the utility of the approach by providing volumetric data, 3D distribution of three main cellular components (epithelial, mesenchymal and endothelial cells) within the developing pancreas, and quantification of their relative cellular abundance within the tissue. Interestingly, our 3D images show that endocrine cells are constantly and increasingly in contact with endothelial cells forming small vessels, whereas the interactions with mesenchymal cells decrease over time. These findings suggest distinct cell-cell interaction requirements for early endocrine cell specification and late differentiation. Lastly, we combine our image data in an open-source online repository (referred to as the Pancreas Embryonic Cell Atlas).

Effects of an acute exercise bout in hypoxia on extracellular vesicle release in healthy and prediabetic subjects.

The purpose of this study is to investigate exosome-like vesicle (ELV) plasma concentrations and markers of multivesicular body (MVB) biogenesis in skeletal muscle in response to acute exercise. Seventeen healthy [body mass index (BMI): 23.5 ± 0.5 kg·m-2] and 15 prediabetic (BMI: 27.3 ± 1.2 kg·m-2) men were randomly assigned to two groups performing an acute cycling bout in normoxia or hypoxia ([Formula: see text] 14.0%). Venous blood samples were taken before (T0), during (T30), and after (T60) exercise, and biopsies from m. vastus lateralis were collected before and after exercise. Plasma ELVs were isolated by size exclusion chromatography, counted by nanoparticle tracking analysis (NTA), and characterized according to international standards, followed by expression analyses of canonical ELV markers in skeletal muscle. In the healthy normoxic group, the total number of particles in the plasma increased during exercise from T0 to T30 (+313%) followed by a decrease from T30 to T60 (-53%). In the same group, an increase in TSG101, CD81, and HSP60 protein expression was measured after exercise in plasma ELVs; however, in the prediabetic group, the total number of particles in the plasma was not affected by exercise. The mRNA content of TSG101, ALIX, and CD9 was upregulated in skeletal muscle after exercise in normoxia, whereas CD9 and CD81 were downregulated in hypoxia. ELV plasma abundance increased in response to acute aerobic exercise in healthy subjects in normoxia, but not in prediabetic subjects, nor in hypoxia. Skeletal muscle analyses suggested that this tissue did not likely play a major role of the exercise-induced increase in circulating ELVs.

Modelling of Epithelial Growth, Fission and Lumen Formation During Embryonic Thyroid Development: A Combination of Computational and Experimental Approaches.

Organogenesis is the phase of embryonic development leading to the formation of fully functional organs. In the case of the thyroid, organogenesis starts from the endoderm and generates a multitude of closely packed independent spherical follicular units surrounded by a dense network of capillaries. Follicular organisation is unique and essential for thyroid function, i.e. thyroid hormone production. Previous in vivo studies showed that, besides their nutritive function, endothelial cells play a central role during thyroid gland morphogenesis. However, the precise mechanisms and biological parameters controlling the transformation of the multi-layered thyroid epithelial primordium into a multitude of single-layered follicles are mostly unknown. Animal studies used to improve understanding of organogenesis are costly and time-consuming, with recognised limitations. Here, we developed and used a 2-D vertex model of thyroid growth, angiogenesis and folliculogenesis, within the open-source Chaste framework. Our in silico model, based on in vivo images, correctly simulates the differential growth and proliferation of central and peripheral epithelial cells, as well as the morphogen-driven migration of endothelial cells, consistently with our experimental data. Our simulations further showed that reduced epithelial cell adhesion was critical to allow endothelial invasion and fission of the multi-layered epithelial mass. Finally, our model also allowed epithelial cell polarisation and follicular lumen formation by endothelial cell abundance and proximity. Our study illustrates how constant discussion between theoretical and experimental approaches can help us to better understand the roles of cellular movement, adhesion and polarisation during thyroid embryonic development. We anticipate that the use of in silico models like the one we describe can push forward the fields of developmental biology and regenerative medicine.

Detection of alternative lengthening of telomeres mechanism on tumor sections.

The vast majority of adult cancer cells achieve cellular immortality by activating a telomere maintenance mechanism (TMM). While this is mostly achieved by the de-silencing of hTERT telomerase gene expression, an alternative homologous recombination-based and telomerase-independent mechanism, known as ALT (Alternative Lengthening of Telomeres), is frequently activated in a subset of tumors, including paediatric cancers. Being absent from normal cells, the ALT mechanism offers interesting perspectives for new targeted cancer therapies. To date, however, the development of better translationally applicable tools for ALT detection in tumor sections is still needed. Here, using a newly derived ALT-positive cancer cell mouse xenograft model, we extensively examined how the previously known ALT markers could be used as reliable tools for ALT diagnosis in tumor sections. We found that, together with the detection of ultra-bright telomeric signals (UBS), an ALT hallmark, native telomeric FISH, that detects single-stranded C-rich telomeric DNA, provides a very sensitive and robust tool for ALT diagnosis in tissues. We applied these assays to paediatric tumor samples and readily identified three ALT-positive tumors for which the TMM was confirmed by the gold-standard C-circle amplification assay. Although the latter offers a robust assay for ALT detection in the context of research laboratories, it is more difficult to set up in histopathological laboratories and could therefore be conveniently replaced by the combination of UBS detection and native telomeric FISH.

Protection of Cystinotic Mice by Kidney-Specific Megalin Ablation Supports an Endocytosis-Based Mechanism for Nephropathic Cystinosis Progression.

BACKGROUND: Deletions or inactivating mutations of the cystinosin gene CTNS lead to cystine accumulation and crystals at acidic pH in patients with nephropathic cystinosis, a rare lysosomal storage disease and the main cause of hereditary renal Fanconi syndrome. Early use of oral cysteamine to prevent cystine accumulation slows progression of nephropathic cystinosis but it is a demanding treatment and not a cure. The source of cystine accumulating in kidney proximal tubular cells and cystine's role in disease progression are unknown. METHODS: To investigate whether receptor-mediated endocytosis by the megalin/LRP2 pathway of ultrafiltrated, disulfide-rich plasma proteins could be a source of cystine in proximal tubular cells, we used a mouse model of cystinosis in which conditional excision of floxed megalin/LRP2 alleles in proximal tubular cells of cystinotic mice was achieved by a Cre-LoxP strategy using Wnt4-CRE. We evaluated mice aged 6-9 months for kidney cystine levels and crystals; histopathology, with emphasis on swan-neck lesions and proximal-tubular-cell apoptosis and proliferation (turnover); and proximal-tubular-cell expression of the major apical transporters sodium-phosphate cotransporter 2A (NaPi-IIa) and sodium-glucose cotransporter-2 (SGLT-2). RESULTS: Wnt4-CRE-driven megalin/LRP2 ablation in cystinotic mice efficiently blocked kidney cystine accumulation, thereby preventing lysosomal deformations and crystal deposition in proximal tubular cells. Swan-neck lesions were largely prevented and proximal-tubular-cell turnover was normalized. Apical expression of the two cotransporters was also preserved. CONCLUSIONS: These observations support a key role of the megalin/LRP2 pathway in the progression of nephropathic cystinosis and provide a proof of concept for the pathway as a therapeutic target.

Pancreatic acinar differentiation is guided by differential laminin deposition.

Endothelial cells play multiple roles during pancreas organogenesis. First, they are required to instruct endoderm-derived pancreatic progenitor cells to initiate branching morphogenesis. Later, blood vessels promote ß-cell differentiation but also limit acinar development. In this work, we show how endothelial cells might signal to pancreatic progenitors and spatially regulate acinar differentiation. Using an ex vivo culture system of undifferentiated E12.5 pancreata, we demonstrate that embryonic endothelial progenitor cells and their conditioned medium prevent the expression of two members of the pro-acinar transcriptional PTF1L-complex. This effect is not mediated by SPARC, a protein abundantly released in the medium conditioned by endothelial progenitors. On the contrary, heterotrimeric laminin-α1ß1γ1, also produced by endothelial progenitor cells, can repress acinar differentiation when used on its own on pancreatic explants. Lastly, we found that laminin-α1 is predominantly found in vivo around the pancreatic trunk cells, as compared to the tip cells, at E14.5. In conclusion, we propose that expression or deposition of laminin-α1ß1γ1 around the trunk cells, where blood vessels are predominantly localized, prevent acinar differentiation of these cells. On the contrary, transient decreased expression or deposition of laminin-α1ß1γ1 around the tip cells would allow PTF1L-complex formation and acinar differentiation.

Vps34/PI3KC3 deletion in kidney proximal tubules impairs apical trafficking and blocks autophagic flux, causing a Fanconi-like syndrome and renal insufficiency.

Kidney proximal tubular cells (PTCs) are highly specialized for ultrafiltrate reabsorption and serve as paradigm of apical epithelial differentiation. Vps34/PI3-kinase type III (PI3KC3) regulates endosomal dynamics, macroautophagy and lysosomal function. However, its in vivo role in PTCs has not been evaluated. Conditional deletion of Vps34/PI3KC3 in PTCs by Pax8-Cre resulted in early (P7) PTC dysfunction, manifested by Fanconi-like syndrome, followed by kidney failure (P14) and death. By confocal microscopy, Vps34∆/∆ PTCs showed preserved apico-basal specification (brush border, NHERF-1 versus Na+/K+-ATPase, ankyrin-G) but basal redistribution of late-endosomes/lysosomes (LAMP-1) and mis-localization to lysosomes of apical recycling endocytic receptors (megalin, cubilin) and apical non-recycling solute carriers (NaPi-IIa, SGLT-2). Defective endocytosis was confirmed by Texas-red-ovalbumin tracing and reduced albumin content. Disruption of Rab-11 and perinuclear galectin-3 compartments suggested mechanistic clues for defective receptor recycling and apical biosynthetic trafficking. p62-dependent autophagy was triggered yet abortive (p62 co-localization with LC3 but not LAMP-1) and PTCs became vacuolated. Impaired lysosomal positioning and blocked autophagy are known causes of cell stress. Thus, early trafficking defects show that Vps34 is a key in vivo component of molecular machineries governing apical vesicular trafficking, thus absorptive function in PTCs. Functional defects underline the essential role of Vps34 for PTC homeostasis and kidney survival.

Highly Aggressive Metastatic Melanoma Cells Unable to Maintain Telomere Length.

Unlimited replicative potential is one of the hallmarks of cancer cells. In melanoma, hTERT (telomerase reverse transcriptase) is frequently overexpressed because of activating mutations in its promoter, suggesting that telomerase is necessary for melanoma development. We observed, however, that a subset of melanoma metastases and derived cell lines had no telomere maintenance mechanism. Early passages of the latter displayed long telomeres that progressively shortened and fused before cell death. We propose that, during melanoma formation, oncogenic mutations occur in precursor melanocytes with long telomeres, providing cells with sufficient replicative potential, thereby bypassing the need to re-activate telomerase. Our data further support the emerging idea that long telomeres promote melanoma formation. These observations are important when considering anticancer therapies targeting telomerase.

Thyroid follicle development requires Smad1/5- and endothelial cell-dependent basement membrane assembly.

Thyroid follicles, the functional units of the thyroid gland, are delineated by a monolayer of thyrocytes resting on a continuous basement membrane. The developmental mechanisms of folliculogenesis, whereby follicles are formed by the reorganization of a non-structured mass of non-polarized epithelial cells, are largely unknown. Here we show that assembly of the epithelial basement membrane is crucial for folliculogenesis and is controlled by endothelial cell invasion and by BMP-Smad signaling in thyrocytes. Thyroid-specific Smad1 and Smad5 double-knockout (Smad1/5(dKO)) mice displayed growth retardation, hypothyroidism and defective follicular architecture. In Smad1/5(dKO) embryonic thyroids, epithelial cells remained associated in large clusters and formed small follicles. Although similar follicular defects are found in Vegfa knockout (Vegfa(KO)) thyroids, Smad1/5(dKO) thyroids had normal endothelial cell density yet impaired endothelial differentiation. Interestingly, both Vegfa(KO) and Smad1/5(dKO) thyroids displayed impaired basement membrane assembly. Furthermore, conditioned medium (CM) from embryonic endothelial progenitor cells (eEPCs) rescued the folliculogenesis defects of both Smad1/5(dKO) and Vegfa(KO) thyroids. Laminin α1, ß1 and γ1, abundantly released by eEPCs into CM, were crucial for folliculogenesis. Thus, epithelial Smad signaling and endothelial cell invasion promote folliculogenesis via assembly of the basement membrane.

Proliferation-Independent Initiation of Biliary Cysts in Polycystic Liver Diseases.

Biliary cysts in adult patients affected by polycystic liver disease are lined by cholangiocytes that proliferate, suggesting that initiation of cyst formation depends on proliferation. Here, we challenge this view by analyzing cyst-lining cell proliferation and differentiation in Cpk mouse embryos and in livers from human fetuses affected by Autosomal Recessive Polycystic Kidney Disease (ARPKD), at early stages of cyst formation. Proliferation of fetal cholangiocyte precursors, measured by immunostaining in human and mouse livers, was low and did not differ between normal and ARPKD or Cpk livers, excluding excessive proliferation as an initiating cause of liver cysts. Instead, our analyses provide evidence that the polycystic livers exhibit increased and accelerated differentiation of hepatoblasts into cholangiocyte precursors, eventually coalescing into large biliary cysts. Lineage tracing experiments, performed in mouse embryos, indicated that the cholangiocyte precursors in Cpk mice generate cholangiocytes and periportal hepatocytes, like in wild-type animals. Therefore, contrary to current belief, cyst formation in polycystic liver disease does not necessarily depend on overproliferation. Combining our prenatal data with available data from adult livers, we propose that polycystic liver can be initiated by proliferation-independent mechanisms at a fetal stage, followed by postnatal proliferation-dependent cyst expansion.

Imprinting of the COPD airway epithelium for dedifferentiation and mesenchymal transition.

In chronic obstructive pulmonary disease (COPD), epithelial changes and subepithelial fibrosis are salient features in conducting airways. Epithelial-to-mesenchymal transition (EMT) has been recently suggested in COPD, but the mechanisms and relationship to peribronchial fibrosis remain unclear. We hypothesised that de-differentiation of the COPD respiratory epithelium through EMT could participate in airway fibrosis and thereby, in airway obstruction. Surgical lung tissue and primary broncho-epithelial cultures (in air-liquid interface (ALI)) from 104 patients were assessed for EMT markers. Cell cultures were also assayed for mesenchymal features and for the role of transforming growth factor (TGF)-ß1. The bronchial epithelium from COPD patients showed increased vimentin and decreased ZO-1 and E-cadherin expression. Increased vimentin expression correlated with basement membrane thickening and airflow limitation. ALI broncho-epithelial cells from COPD patients also displayed EMT phenotype in up to 2 weeks of culture, were more spindle shaped and released more fibronectin. Targeting TGF-ß1 during ALI differentiation prevented vimentin induction and fibronectin release. In COPD, the airway epithelium displays features of de-differentiation towards mesenchymal cells, which correlate with peribronchial fibrosis and airflow limitation, and which are partly due to a TGF-ß1-driven epithelial reprogramming.

Validation of housekeeping gene and impact on normalized gene expression in clear cell renal cell carcinoma: critical reassessment of YBX3/ZONAB/CSDA expression.

BACKGROUND: YBX3/ZONAB/CSDA is an epithelial-specific transcription factor acting in the density-based switch between proliferation and differentiation. Our laboratory reported overexpression of YBX3 in clear cell renal cell arcinoma (ccRCC), as part of a wide study of YBX3 regulation in vitro and in vivo. The preliminary data was limited to 5 cases, of which only 3 could be compared to paired normal tissue, and beta-Actin was used as sole reference to normalize gene expression. We thus decided to re-evaluate YBX3 expression by real-time-PCR in a larger panel of ccRCC samples, and their paired healthy tissue, with special attention on experimental biases such as inter-individual variations, primer specificity, and reference gene for normalization. RESULTS: Gene expression was measured by RT-qPCR in 16 ccRCC samples, each compared to corresponding healthy tissue to minimize inter-individual variations. Eight potential housekeeping genes were evaluated for expression level and stability among the 16-paired samples. Among tested housekeeping genes, PPIA and RPS13, especially in combination, proved best suitable to normalize gene expression in ccRCC tissues as compared to classical reference genes such as beta-Actin, GAPDH, 18S or B2M. Using this pair as reference, YBX3 expression level among a collection of 16 ccRCC tumors was not significantly increased as compared to normal adjacent tissues. However, stratification according to Fuhrman grade disclosed higher YBX3 expression levels in low-grade tumors and lower in high-grade tumors. Immunoperoxidase confirmed homogeneous nuclear staining for YBX3 in low-grade but revealed nuclear heterogeneity in high-grade tumors. CONCLUSIONS: This paper underlines that special attention to reference gene products in the design of real-time PCR analysis of tumoral tissue is crucial to avoid misleading conclusions. Furthermore, we found that global YBX3/ZONAB/CSDA mRNA expression level may be considered within a "signature" of RCC grading.