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 of 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.

Endometrial Carcinoma: Specific Targeted Pathways.

Endometrial cancer (EC) is the most common gynecologic malignancy in the western world with more than 280,000 cases per year worldwide. Prognosis for EC at early stages, when primary surgical resection is the most common initial treatment, is excellent. Five-year survival rate is around 70 %.Several molecular alterations have been described in the different types of EC. They occur in genes involved in important signaling pathways. In this chapter, we will review the most relevant altered pathways in EC, including PI3K/AKT/mTOR, RAS-RAF-MEK-ERK, Tyrosine kinase, WNT/ß-Catenin, cell cycle, and TGF-ß signaling pathways. At the end of the chapter, the most significant clinical trials will be briefly discussed.This information is important to identify specific targets for therapy.

Hypoxia-independent gene expression signature associated with radiosensitisation of prostate cancer cell lines by histone deacetylase inhibition.

BACKGROUND: Histone deacetylase inhibitors (HDACis) like vorinostat are promising radiosensitisers in prostate cancer, but their effect under hypoxia is not known. We investigated gene expression associated with radiosensitisation of normoxic and hypoxic prostate cancer cells by vorinostat. METHODS: Cells were exposed to vorinostat under normoxia or hypoxia and subjected to gene expression profiling before irradiation and clonogenic survival analysis. RESULTS: Pretreatment with vorinostat led to radiosensitisation of the intrinsically radioresistant DU 145 cells, but not the radiosensitive PC-3 and 22Rv1 cells, and was independent of hypoxia status. Knockdown experiments showed that the sensitisation was not caused by repression of hypoxia-inducible factor HIF1 or tumour protein TP53. Global deregulation of DNA repair and chromatin organisation genes was associated with radiosensitisation under both normoxia and hypoxia. A radiosensitisation signature with expression changes of 56 genes was generated and valid for both conditions. For eight signature genes, baseline expression also correlated with sensitisation, showing potential as pretreatment biomarker. The hypoxia independence of the signature was confirmed in a clinical data set. CONCLUSIONS: Pretreatment with HDACi may overcome radioresistance of hypoxic prostate tumours by similar mechanisms as under normoxia. We propose a gene signature to predict radiosensitising effects independent of hypoxia status.

Long-term estradiol exposure is a direct mitogen for insulin/EGF-primed endometrial cells and drives PTEN loss-induced hyperplasic growth.

Loss of tumor-suppressor PTEN is the most common alteration in endometrial carcinoma. However, the relationship between loss of PTEN, growth factors [eg, insulin/insulin-like growth factor (IGF)-1], epidermal growth factor (EGF), and hyperestrogenism in the development of endometrial carcinoma is still controversial. By using three-dimensional (3D) cultures of PTEN(+/+) and PTEN(+/-) endometrial epithelial cells, we investigated the effects of EGF, insulin/IGF, and estradiol in endometrial cell proliferation. We have previously demonstrated that 3D cultures of endometrial cells require EGF and insulin/IGF to proliferate. Herein, we demonstrate that, in the presence of EGF and insulin/IGF, long-term estradiol treatment directly induces proliferation of 3D cultures. Moreover, we show that the mitogenic effects of estradiol require the presence of insulin/IGF and EGF, because withdrawal of such factors completely abolishes estradiol-induced proliferation. In the presence of EGF and insulin/IGF, PTEN(+/-) and PTEN(+/+) spheroids display a similar rate of proliferation. However, the addition of estradiol causes an exaggerated proliferation of PTEN(+/-) cultures, leading to formation of complex structures, such as those observed in endometrial hyperplasia or carcinoma. In summary, we demonstrate that EGF and insulin/IGF prime endometrial epithelial cells to direct the mitogenic effects of estradiol. Furthermore, PTEN deficiency results in enhanced responsiveness to this combination, leading to the development of hyperplasia of endometrial cells in culture.

Arginine transport is impaired in C57Bl/6 mouse macrophages as a result of a deletion in the promoter of Slc7a2 (CAT2), and susceptibility to Leishmania infection is reduced.

Host genetic factors play a crucial role in immune response. To determine whether the differences between C57Bl/6 and BALB-C mice are due only to the production of cytokines by T-helper 1 cells or T-helper 2 cells, we obtained bone marrow-derived macrophages from both strains and incubated them with these cytokines. Although the induction of Nos2 and Arg1 was similar in the 2 strains, infectivity to Leishmania major differed, as did macrophage uptake of arginine, which was higher in BALB-C macrophages. The levels of interferon γ- and interleukin 4-dependent induction of the cationic amino acid transporter SLC7A2 (also known as "cationic amino acid transporter 2," or "CAT2") were decreased in macrophages from C57Bl/6 mice. This reduction was a result of a deletion in the promoter of one of the 4 AGGG repeats. These results demonstrate that the availability of arginine controls critical aspects of macrophage activation and reveal a factor for susceptibility to Leishmania infection.

Sprouty1 is a broad mediator of cellular senescence.

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.

PKN1 Kinase: A Key Player in Adipocyte Differentiation and Glucose Metabolism.

Adipocyte dysfunction is the driver of obesity and correlates with insulin resistance and the onset of type 2 diabetes. Protein kinase N1 (PKN1) is a serine/threonine kinase that has been shown to contribute to Glut4 translocation to the membrane and glucose transport. Here, we evaluated the role of PKN1 in glucose metabolism under insulin-resistant conditions in primary visceral adipose tissue (VAT) from 31 patients with obesity and in murine 3T3-L1 adipocytes. In addition, in vitro studies in human VAT samples and mouse adipocytes were conducted to investigate the role of PKN1 in the adipogenic maturation process and glucose homeostasis control. We show that insulin-resistant adipocytes present a decrease in PKN1 activation levels compared to nondiabetic control counterparts. We further show that PKN1 controls the adipogenesis process and glucose metabolism. PKN1-silenced adipocytes present a decrease in both differentiation process and glucose uptake, with a concomitant decrease in the expression levels of adipogenic markers, such as PPARγ, FABP4, adiponectin and CEBPα. Altogether, these results point to PKN1 as a regulator of key signaling pathways involved in adipocyte differentiation and as an emerging player of adipocyte insulin responsiveness. These findings may provide new therapeutic approaches for the management of insulin resistance in type 2 diabetes.

Metformin exhibits antineoplastic effects on Pten-deficient endometrial cancer by interfering with TGF-ß and p38/ERK MAPK signalling.

Metformin is a widespread antidiabetic agent that is commonly used as a treatment against type 2 diabetes mellitus patients. Regarding its therapeutic potential, multiple studies have concluded that Metformin exhibits antineoplastic activity on several types of cancer, including endometrial carcinoma. Although Metformin's antineoplastic activity is well documented, its cellular and molecular anticancer mechanisms are still a matter of controversy because a plethora of anticancer mechanisms have been proposed for different cancer cell types. In this study, we addressed the cellular and molecular mechanisms of Metformin's antineoplastic activity by using both in vitro and in vivo studies of Pten-loss driven carcinoma mouse models. In vivo, Metformin reduced endometrial neoplasia initiated by Pten-deficiency. Our in vitro studies using Pten-deficient endometrial organoids focused on both cellular and molecular levels in Metformin's tumor suppressive action. At cellular level, we showed that Metformin is involved in not only the proliferation of endometrial epithelial cells but also their regulation via a variety of mechanisms of epithelial-to-mesenchymal transition (EMT) as well as TGF-ß-induced apoptosis. At the molecular level, Metformin was shown to affect the TGF-ß signalling., a widely known signal that plays a pivotal role in endometrial carcinogenesis. In this respect, Metformin restored TGF-ß-induced apoptosis of Pten-deficient endometrial organoids through a p38-dependent mechanism and inhibited TGF-ß-induced EMT on no-polarized endometrial epithelial cells by inhibiting ERK/MAPK signalling. These results provide new insights into the link between the cellular and molecular mechanism for Metformin's antineoplastic activity in Pten-deficient endometrial cancers.

In Vivo Intra-Uterine Delivery of TAT-Fused Cre Recombinase and CRISPR/Cas9 Editing System in Mice Unveil Histopathology of Pten/p53-Deficient Endometrial Cancers.

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.

Oxidative stress-induced FAK activation contributes to uterine serous carcinoma aggressiveness.

Uterine serous carcinoma (USC) is an aggressive form of endometrial cancer (EC), characterized by its high propensity for metastases. In fact, while endometrioid endometrial carcinoma (EEC), which accounts for 85% of EC, presents a good prognosis, USC is the most frequently fatal. Herein, we used for the first time a peptide-based tyrosine-kinase-activity profiling approach to quantify the changes in tyrosine kinase activation between USC and EEC. Among the tyrosine kinases highly activated in USC, we identified focal adhesion kinase (FAK). We conducted mechanistic studies using cellular models. In a USC cell line, targeting FAK either by inhibitors PF-573228 and defactinib (VS-6063) or by gene silencing limits 3D cell growth and reduces cell migration. Moreover, results from our studies suggest that oxidative stress is increased in USC tumors compared to EEC ones. Reactive oxygen species (ROS) induce tyrosine phosphorylation of FAK and a concomitant tyrosine phosphorylation of paxillin, a mediator of FAK signal transduction. Mechanistically, by tracking hundreds of individual cells per condition, we show that ROS increased cell distance and migration velocity, highlighting the role of ROS-FAK-PAX signaling in cell migration. Both defactinib and ROS scavenger N-acetylcysteine (NAC) revert this effect, pointing toward ROS as potential culprits for the increase in USC cell motility. A proof of concept of the role of FAK in controlling cell growth was obtained in in vivo experiments using cancer-tissue-originated spheroids (CTOS) and a patient-derived orthotopic xenograft model (orthoxenograft/PDOX). Defactinib reduces cell proliferation and protein oxidation, supporting a pro-tumoral antioxidant role of FAK, whereas antioxidant NAC reverts FAK inhibitor effects. Overall, our data points to ROS-mediated FAK activation in USC as being responsible for the poor prognosis of this tumor type and emphasize the potential of FAK inhibition for USC treatment.

Inhibition of activated receptor tyrosine kinases by Sunitinib induces growth arrest and sensitizes melanoma cells to Bortezomib by blocking Akt pathway.

Despite the use of multiple therapeutic strategies, metastatic melanoma remains a challenge for oncologists. Thus, new approaches using combinational treatment may be used to try to improve the prognosis of this disease. In this report, we have analyzed the expression of receptor tyrosine kinases (RTKs) in melanoma specimens and in four metastatic melanoma cell lines. Both melanoma specimens and cell lines expressed RTKs, suggesting that they may represent eventual targets for multitargeted tyrosine kinase inhibitor, Suntinib. Sunitinib reduced the proliferation of two melanoma cell lines (M16 and M17) and increased apoptosis in one of them (M16). Moreover, the two metastatic melanoma cell lines harbored an activated receptor (PDGFRα and VEGFR, respectively), and Sunitinib suppressed the phosphorylation of the RTKs and their downstream targets Akt and ribosomal protein S6, in these two cell lines. Similar results were obtained when either PDGFRα or VEGFR2 expression was silenced by lentiviral-mediated short-hairpin RNA delivery in M16 and M17, respectively. To evaluate the interaction between Sunitinib and Bortezomib, median dose effect analysis using MTT assay was performed, and combination index was calculated. Bortezomib synergistically enhanced the Sunitinib-induced growth arrest in Sunitinib-sensitive cells (combination index < 1). Moreover, LY294002, a PI3K inhibitor, sensitized melanoma cells to Bortezomib treatment, suggesting that downregulation of phospho-Akt by Sunitinib mediates the synergy obtained by Bortezomib + Sunitinib cotreatment. Altogether, our results suggest that melanoma cells harboring an activated RTK may be clinically responsive to pharmacologic RTK inhibition by Sunitinib, and a strategy combining Sunitinib and Bortezomib, may provide therapeutic benefit.

KSR1 is overexpressed in endometrial carcinoma and regulates proliferation and TRAIL-induced apoptosis by modulating FLIP levels.

The Raf/MEK/extracellular signal-regulated kinase (ERK) pathway participates in many processes altered in development and progression of cancer in human beings such as proliferation, transformation, differentiation, and apoptosis. Kinase suppressor of Ras 1 (KSR1) can interact with various kinases of the Raf/MEK/extracellular signal-regulated kinase pathway to enhance its activation. The role of KSR1 in endometrial carcinogenesis was investigated. cDNA and tissue microarrays demonstrated that expression of KSR1 was up-regulated in endometrial carcinoma. Furthermore, inhibition of KSR1 expression by specific small hairpin RNA resulted in reduction of both proliferation and anchorage-independent cell growth properties of endometrial cancer cells. Because inhibition of apoptosis has a pivotal role in endometrial carcinogenesis, the effects of KSR1 in regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis were investigated. KSR1 knock-down sensitized resistant endometrial cell lines to both TRAIL- and Fas-induced apoptosis. Sensitization to TRAIL and agonistic anti-Fas antibody was caused by down-regulation of FLIP (FLICE-inhibitory protein). Also investigated was the molecular mechanism by which KSR1 regulates FLIP protein levels. It was demonstrated that KSR1 small hairpin RNA did not affect FLIP transcription or degradation. Rather, FLIP down-regulation was caused by Fas-associated death domain protein-dependent inhibition of FLIP translation triggered after TRAIL stimulation in KSR1-silenced cells. Re-expression of heterologous KSR1 in cells with down-regulated endogenous KSR1 restored FLIP protein levels and TRAIL resistance. In conclusion, KSR1 regulates endometrial sensitivity to TRAIL by regulating FLIP levels.

Immunohistochemical features of post-radiation vaginal recurrences of endometrioid carcinomas of the endometrium: role for proteins involved in resistance to apoptosis and hypoxia.

AIMS: Endometrioid carcinoma of the endometrium (EEC) is treated with surgery and radiotherapy. Post-radiation recurrences are associated with increased risk of metastases. Comparison of the expression of genes important in the development and progression of EEC, and others involved in resistance to apoptosis and hypoxia and adaptation to radiation, was performed between post-radiation vaginal recurrences (PVRs) and primary EECs. We tried to reproduce the results by exposing an EEC cell line to hypoxia and radiation. METHODS AND RESULTS: Immunohistochemistry and tissue microarrays were used to compare 24 PVRs with 82 primary EECs. PVRs exhibited increased expression of p53 (P < 0.0001), cytoplasmic FLICE-inhibitory protein (FLIP) (P < 0.0001), and Ki67 (P < 0.0001), and nuclear staining for FLIP, nuclear factor kappaB (NF-κB) family members (p50, P < 0.0001; c-Rel, P = 0.0077; RelB, P = 0.0157), and ß-catenin (P = 0.0001). Differences regarding p50, hypoxia-inducible factor 1α (HIF-1α), and cytoplasmic FLIP were statistically significant when PVRs and primary EECs were matched for histological grade. Exposure of the EEC cell line to hypoxia induced nuclear expression of ß-catenin, FLIP, and HIF-1α, as well as increased NF-κB activity. No changes in FLIP, HIF-1α or NF-κB were seen when cells were exposed to radiation. Nuclear expression of ß-catenin was seen at 3 Gy, but not at 1 Gy. CONCLUSIONS: Genes involved in resistance to hypoxia are expressed in PVRs, and may play a role in the development of post-radiation recurrences.

Metabolomic Analysis Points to Bioactive Lipid Species and Acireductone Dioxygenase 1 (ADI1) as Potential Therapeutic Targets in Poor Prognosis Endometrial Cancer.

Metabolomic profiling analysis has the potential to highlight new molecules and cellular pathways that may serve as potential therapeutic targets for disease treatment. In this study, we used an LC-MS/MS platform to define, for the first time, the specific metabolomic signature of uterine serous carcinoma (SC), a relatively rare and aggressive variant of endometrial cancer (EC) responsible for 40% of all endometrial cancer-related deaths. A metabolomic analysis of 31 ECs (20 endometrial endometrioid carcinomas (EECs) and 11 SCs) was performed. Following multivariate statistical analysis, we identified 232 statistically different metabolites among the SC and EEC patient samples. Notably, most of the metabolites identified (89.2%) were lipid species and showed lower levels in SCs when compared to EECs. In addition to lipids, we also documented metabolites belonging to amino acids and purine nucleotides (such as 2-Oxo-4-methylthiobutanoic acid, synthesised by acireductone dioxygenase 1 (ADI1) enzyme), which showed higher levels in SCs. To further investigate the role of ADI1 in SC, we analysed the expression protein levels of ADI1 in 96 ECs (67 EECs and 29 SCs), proving that the levels of ADI1 were higher in SCs compared to EECs. We also found that ADI1 mRNA levels were higher in p53 abnormal ECs compared to p53 wild type tumours. Furthermore, elevated ADI1 mRNA levels showed a statistically significant negative correlation with overall survival and progression-free survival among EEC patients. Finally, we tested the ability of ADI1 to induce migration and invasion capabilities in EC cell lines. Altogether, these results suggest that ADI1 could be a potential therapeutic target in poor-prognosis SCs and other Ecs with abnormal p53 expression.

Lack of extracellular matrix switches TGF-ß induced apoptosis of endometrial cells to epithelial to mesenchymal transition.

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-ß. It is well known that TGF-ß 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-ß remain controversial. Here, we have demonstrated that the lack of extracellular matrix and consequent loss of cell polarity inhibits TGF-ß-induced apoptosis, observed when endometrial epithelial cells are polarized in presence of extracellular matrix. Rather, in absence of extracellular matrix, TGF-ß-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-ß-induced apoptosis. On the other hand, we demonstrate that TGF-ß-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-ß to tumor-associated phenotype such as epithelial-to-mesenchymal transition. These results may be important for understanding the dual role of TGF-ß in normal versus tumoral cells.

Nuclear factor-κB2/p100 promotes endometrial carcinoma cell survival under hypoxia in a HIF-1α independent manner.

Endometrial carcinoma (EC) is a common female cancer, treated mainly by surgery and adjuvant radiotherapy. Relapse following treatment is associated with increased risk of metastases. Hypoxia, a common microenvironment in solid tumors, correlates with malignant progression, rendering tumors resistant to ionizing therapy. Hence, we assessed here the immunohistochemical expression of hypoxia-inducible factor-1α (HIF-1α) and members of the NF-κB family in 82 primary EC and 10 post-radiation recurrences of EC. Post-radiation recurrences were highly hypoxic, with a higher expression of HIF-1α and also RelA (p65) and p52 when compared with primary EC. We next investigated the effects of hypoxia on EC cell lines. We found that EC cell lines are highly resistant to hypoxia-induced apoptosis. We thus focused on the molecular mechanisms involved in conferring hypoxic cell death resistance. We show that in addition to the classical NF-κB, hypoxia activates the alternative NF-κB pathway. To characterize the upstream kinases involved in the activation of these pathways, we used lentiviral-mediated knockdown and mouse embryonic fibroblasts lacking IKKα and IKKß kinases. Both IKKα and IKKß kinases are required for RelA (p65) and p100 accumulation, whereas p52 processing under hypoxia is IKKα dependent. Furthermore, Ishikawa endometrial cell line harboring either RelA (p65) or p52 short-hairpin RNA was sensitive to hypoxia-induced cell death, indicating that, in addition to the known prosurvival role of RelA (p65) under hypoxia, alternative NF-κB pathway also enhances hypoxic survival of EC cells. Interestingly, although HIF-1α controlled classical NF-κB activation pathway and survival under hypoxia through RelA (p65) nuclear accumulation, the alternative pathway was HIF-1α independent. These findings have important clinical implications for the improvement of EC prognosis before radiotherapy.

A novel three-dimensional culture system of polarized epithelial cells to study endometrial carcinogenesis.

Development of three-dimensional (3D) cultures that mimic in vivo tissue organization has a pivotal role in the investigation of the involvement of cell adhesion and polarity genes in the pathogenesis of epithelial cancers. Here we describe a novel 3D culture model with primary mouse endometrial epithelial cells. In this model, isolated endometrial epithelial cells develop single-lumened, polarized glandular structures resembling those observed in endometrial tissue. Our in vitro 3D culture model of endometrial glands requires the use of serum-free defined medium with only epidermal growth factor and insulin as growth supplements and 3% Matrigel as reconstituted extracellular matrix. Under these culture conditions, glands of epithelial cells displaying typical apicobasal polarity and proper positioning of tight and adherent junctions are formed by hollowing as early as 7 to 8 days in culture. Addition of the phosphatidylinositol 3-kinase inhibitor LY294002 completely inhibits bromodeoxyuridine incorporation and cyclinD1 expression, confirming that in vitro growth of endometrial glands depends on phosphatidylinositol 3-kinase/Akt signaling. To prove that our culture method is a good model to study endometrial carcinogenesis, we knocked down E-cadherin or phosphatase and tensin homolog expression by lentivirus-delivered short hairpin RNAs. Down-regulation of E-cadherin resulted in complete loss of epithelial cell polarity and glandular formation, whereas phosphatase and tensin homolog down-regulation resulted in increased proliferation of glandular epithelial cells. These properties indicate that our 3D culture model is suitable to study the effect of growth factors, drugs, and gene alterations in endometrial carcinogenesis and to study normal endometrial biology/physiology.

Randomized Clinical Trial to Evaluate the Morphological Changes in the Adventitial Vasa Vasorum Density and Biological Markers of Endothelial Dysfunction in Subjects with Moderate Obesity Undergoing a Very Low-Calorie Ketogenic Diet.

Weight loss after bariatric surgery decreases the earlier expansion of the adventitial vasa vasorum (VV), a biomarker of early atheromatous disease. However, no data are available regarding weight loss achieved by very low calorie ketogenic diets (VLCKD) on VV and lipid-based atherogenic indices. A randomized clinical trial was performed to examine changes in adventitial VV density in 20 patients with moderate obesity who underwent a 6-month very low calorie ketogenic diet (VLCKD, 600-800 kcal/day), and 10 participants with hypocaloric diet based on the Mediterranean Diet (MedDiet, estimated reduction of 500 kcal on the usual intake). Contrast-enhanced carotid ultrasound was used to assess the VV. Body composition analysis was also used. The atherogenic index of plasma (log (triglycerides to high-density lipoprotein cholesterol ratio)) and the triglyceride-glucose index were calculated. Serum concentrations of soluble intercellular adhesion molecule 1 (sICAM-1), and soluble vascular cell adhesion molecule 1 (sVCAM-1) were measured. The impact of weight on quality of life-lite (IWQOL-Lite) questionnaire was administered. Participants of intervention groups displayed a similar VV values. Significant improvements of BMI (-5.3 [-6.9 to -3.6] kg/m2, p < 0.001), total body fat (-7.0 [-10.7 to -3.3] %, p = 0.003), and IWQOL-Lite score (-41.4 [-75.2 to -7.6], p = 0.027) were observed in VLCKD group in comparison with MedDiet group. Although after a 6-months follow-up period VV density (mean, right and left sides) did not change significantly in any group, participants in the VLCKD exhibited a significantly decrease both in their atherogenic index of plasma and serum concentration of sICAM-1. A 6-month intervention with VLCKD do not impact in the density of the adventitial VV in subjects with moderate obesity, but induces significant changes in markers of endothelial dysfunction and CV risk.

CK2beta is expressed in endometrial carcinoma and has a role in apoptosis resistance and cell proliferation.

Protein kinase CK2 (CK2) is a serine/threonine kinase that participates in important cellular processes. We have recently demonstrated that CK2 plays a role in resistance to TRAIL/Fas-induced apoptosis in endometrial carcinoma (EC) by regulating FLIP. Here, we assessed the immunohistochemical expression of CK2beta in EC and checked its role in cell proliferation and anchorage-independent cell growth. CK2beta immunostaining was assessed in two tissue microarrays, one constructed from paraffin-embedded blocks of 95 ECs and another from 70 samples of normal endometrium. CK2beta expression was correlated with histological type; grade and stage; cell proliferation (Ki-67) and apoptotic index; immunostaining for cyclin D1, PTEN, AKT, beta-catenin, and FLIP. Moreover, the Ishikawa EC cell line was subjected to down-regulation of CK2 by shRNA. CK2beta expression was frequent in EC (nuclear, 100%; cytoplasmic, 87.5%). The staining was more intense in EC than in normal endometrium (P = 0.000), and statistically correlated with AKT, PTEN, beta-catenin, and FLIP. In EC, CK2beta expression correlated with cell proliferation. Knock-down of CK2beta blocked colony formation of EC in soft agar, and also resulted in decreased expression of cyclin D1 and ERK phosphorylation. The results confirm that CK2beta is widely expressed in EC, and suggest a role in cell proliferation and anchorage-independent cell growth.

The influence of sleep apnea syndrome and intermittent hypoxia in carotid adventitial vasa vasorum.

Subjects with sleep apnea-hypopnea syndrome (SAHS) show an increased carotid intima-media thickness. However, no data exist about earlier markers of atheromatous disease, such as the proliferation and expansion of the adventitial vasa vasorum (VV) to the avascular intima in this setting. Our aim was to assess carotid VV density and its relationship with sleep parameters in a cohort of obese patients without prior vascular events. A total of 55 subjects evaluated for bariatric surgery were prospectively recruited. A non-attended respiratory polygraphy was performed. The apnea-hypopnea index (AHI) and the cumulative percentage of time spent with oxygen saturation below 90% (CT90) were assessed. Serum concentrations of soluble intercellular adhesion molecule 1, P-selectin, lipocalin-2 and soluble vascular cell adhesion molecule 1 (sVCAM-1) were measured. Contrast-enhanced carotid ultrasound was used to assess the VV density. Patients with SAHS (80%) showed a higher adventitial VV density (0.801±0.125 vs. 0.697±0.082, p = 0.005) and higher levels of sVCAM-1 (745.2±137.8 vs. 643.3±122.7 ng/ml, p = 0.035) than subjects with an AHI lower than 10 events/hour. In addition, a positive association exist between mean VV density and AHI (r = 0.445, p = 0.001) and CT90 (r = 0.399, p = 0.005). Finally, in the multiple linear regression analysis, female sex, fasting plasma glucose and AHI (but not CT90) were the only variables independently associated with the mean adventitial VV density (R2 = 0.327). In conclusion, a high VV density is present in obese subjects with SAHS, and chronic intermittent hypoxia is pointed as an independent risk factor for the development of this early step of atheromatous disease.