Dynamic Spatiotemporal Expression Pattern of the Senescence-Associated Factor p16Ink4a in Development and Aging.

A plethora of factors have been attributed to underly aging, including oxidative stress, telomere shortening and cellular senescence. Several studies have shown a significant role of the cyclin-dependent kinase inhibitor p16ink4a in senescence and aging. However, its expression in development has been less well documented. Therefore, to further clarify a potential role of p16 in development and aging, we conducted a developmental expression study of p16, as well as of p19ARF and p21, and investigated their expression on the RNA level in brain, heart, liver, and kidney of mice at embryonic, postnatal, adult, and old ages. P16 expression was further assessed on the protein level by immunohistochemistry. Expression of p16 was highly dynamic in all organs in embryonic and postnatal stages and increased dramatically in old mice. Expression of p19 and p21 was less variable and increased to a moderate extent at old age. In addition, we observed a predominant expression of p16 mRNA and protein in liver endothelial cells versus non-endothelial cells of old mice, which suggests a functional role specifically in liver endothelium of old subjects. Thus, p16 dynamic spatiotemporal expression might implicate p16 in developmental and physiological processes in addition to its well-known function in the build-up of senescence.

Implications of the Wilms' Tumor Suppressor Wt1 in Cardiomyocyte Differentiation.

The Wilms' tumor suppressor Wt1 is involved in multiple developmental processes and adult tissue homeostasis. The first phenotypes recognized in Wt1 knockout mice were developmental cardiac and kidney defects. Wt1 expression in the heart has been described in epicardial, endothelial, smooth muscle cells, and fibroblasts. Expression of Wt1 in cardiomyocytes has been suggested but remained a controversial issue, as well as the role of Wt1 in cardiomyocyte development and regeneration after injury. We determined cardiac Wt1 expression during embryonic development, in the adult, and after cardiac injury by quantitative RT-PCR and immunohistochemistry. As in vitro model, phenotypic cardiomyocyte differentiation, i.e., the appearance of rhythmically beating clones from mouse embryonic stem cells (mESCs) and associated changes in gene expression were analyzed. We detected Wt1 in cardiomyocytes from embryonic day (E10.5), the first time point investigated, until adult age. Cardiac Wt1 mRNA levels decreased during embryonic development. In the adult, Wt1 was reactivated in cardiomyocytes 48 h and 3 weeks following myocardial infarction. Wt1 mRNA levels were increased in differentiating mESCs. Overexpression of Wt1(-KTS) and Wt1(+KTS) isoforms in ES cells reduced the fraction of phenotypically cardiomyocyte differentiated clones, which was preceded by a temporary increase in c-kit expression in Wt1(-KTS) transfected ES cell clones and induction of some cardiomyocyte markers. Taken together, Wt1 shows a dynamic expression pattern during cardiomyocyte differentiation and overexpression in ES cells reduces their phenotypical cardiomyocyte differentiation.

Novel Therapeutic Insights in Dedifferentiated Liposarcoma: A Role for FGFR and MDM2 Dual Targeting.

We aimed to evaluate the therapeutic potential of the pan-FGFR inhibitor erdafitinib to treat dedifferentiated liposarcoma (DDLPS). FGFR expression and their prognostic value were assessed in a series of 694 samples of well-differentiated/dedifferentiated liposarcoma (WDLPS/DDLPS). The effect of erdafitinib-alone or in combination with other antagonists-on tumorigenicity was evaluated in vitro and in vivo. We detected overexpression of FGFR1 and/or FGFR4 in a subset of WDLPS and DDLPS and demonstrated correlation of this expression with poor prognosis. Erdafitinib treatment reduced cell viability, inducing apoptosis and strong inhibition of the ERK1/2 pathway. Combining erdafitinib with the MDM2 antagonist RG7388 exerted a synergistic effect on viability, apoptosis, and clonogenicity in one WDLPS and two DDLPS cell lines. Efficacy of this combination was confirmed in vivo on a DDLPS xenograft. Importantly, we report the efficacy of erdafitinib in one patient with refractory DDLPS showing disease stabilization for 12 weeks. We provide evidence that the FGFR pathway has therapeutic potential for a subset of DDLPS and that an FGFR1/FGFR4 expression might constitute a powerful biomarker to select patients for FGFR inhibitor clinical trials. In addition, we show that combining erdafitinib with RG7388 is a promising strategy for patients with DDLPS that deserves further investigation in the clinical setting.

Oxytocin Controls Chondrogenesis and Correlates with Osteoarthritis.

This study investigated the relationship of oxytocin (OT) to chondrogenesis and osteoarthritis (OA). Human bone marrow and multipotent adipose-derived stem cells were cultured in vitro in the absence or presence of OT and assayed for mRNA transcript expression along with histological and immunohistochemical analyses. To study the effects of OT in OA in vivo, a rat model and a human cohort of 63 men and 19 women with hand OA and healthy controls, respectively, were used. The baseline circulating OT, interleukin-6, leptin, and oestradiol levels were measured, and hand X-ray examinations were performed for each subject. OT induced increased aggrecan, collagen (Col) X, and cartilage oligomeric matrix protein mRNA transcript levels in vitro, and the immunolabelling experiments revealed a normalization of Sox9 and Col II protein expression levels. No histological differences in lesion severity were observed between rat OA groups. In the clinical study, a multivariate analysis adjusted for age, body mass index, and leptin levels revealed a significant association between OA and lower levels of OT (odds ratio = 0.77; p = 0.012). Serum OT levels are reduced in patients with hand OA, and OT showed a stimulatory effect on chondrogenesis. Thus, OT may contribute to the pathophysiology of OA.

MDM2 amplification and fusion gene ss18-ssx in a poorly differentiated synovial sarcoma: A rare but puzzling conjunction.

The detection of specific alterations by genetic analyses has been included in the diagnostic criterions of the World Health Organization's classification of soft tissues tumors since 2013. The presence of a SS18 rearrangement is pathognomonic of synovial sarcoma (SS). MDM2 amplification is strongly correlated to well-differentiated or dedifferentiated liposarcoma (DDLPS) in the context of sarcoma. We identified one case of poorly differentiated sarcoma harboring both SS18-SSX2 fusion and MDM2 amplification. The review of the literature showed high discrepancies, concerning the incidence of MDM2 amplification in SS: from 1.4% up to 40%. Our goal was to precisely determine the specific clinico-pathological features of this case and to estimate the frequency and characteristics of the association of SS18-SSX fusion/MDM2 amplification in sarcomas. We performed a retrospective and prospective study in 96 sarcomas, (56 SS and 40 DDLPS), using FISH and/or array-CGH to detect MDM2 amplification and SS18 rearrangement. None of the 96 cases presented both genetic alterations. Among the SS, only the index case (1/57: 1.7 %) presented the double anomaly. We concluded that MDM2 amplification in SS is a very rare event. The final diagnosis of the index case was a SS with SS18-SSX2 and MDM2 amplification as a secondary alteration. If the detection of MDM2 amplification is performed first in a poorly differentiated sarcoma, that may lead to not search other anomalies such as SS18 rearrangement and therefore to an erroneous diagnosis. This observation emphasizes the strong complementarity between histomorphology, immunohistochemistry and molecular studies in sarcoma diagnosis.

Vascular PPARß/δ Promotes Tumor Angiogenesis and Progression.

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors, which function as transcription factors. Among them, PPARß/δ is highly expressed in endothelial cells. Pharmacological activation with PPARß/δ agonists had been shown to increase their angiogenic properties. PPARß/δ has been suggested to be involved in the regulation of the angiogenic switch in tumor progression. However, until now, it is not clear to what extent the expression of PPARß/δ in tumor endothelium influences tumor progression and metastasis formation. We addressed this question using transgenic mice with an inducible conditional vascular-specific overexpression of PPARß/δ. Following specific over-expression of PPARß/δ in endothelial cells, we induced syngenic tumors. We observed an enhanced tumor growth, a higher vessel density, and enhanced metastasis formation in the tumors of animals with vessel-specific overexpression of PPARß/δ. In order to identify molecular downstream targets of PPARß/δ in the tumor endothelium, we sorted endothelial cells from the tumors and performed RNA sequencing. We identified platelet-derived growth factor receptor beta (Pdgfrb), platelet-derived growth factor subunit B (Pdgfb), and the tyrosinkinase KIT (c-Kit) as new PPARß/δ -dependent molecules. We show here that PPARß/δ activation, regardless of its action on different cancer cell types, leads to a higher tumor vascularization which favors tumor growth and metastasis formation.

PGC1α Inhibits Polyamine Synthesis to Suppress Prostate Cancer Aggressiveness.

Although tumorigenesis is dependent on the reprogramming of cellular metabolism, the metabolic pathways engaged in the formation of metastases remain largely unknown. The transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) plays a pleiotropic role in the control of cancer cell metabolism and has been associated with a good prognosis in prostate cancer. Here, we show that PGC1α represses the metastatic properties of prostate cancer cells via modulation of the polyamine biosynthesis pathway. Mechanistically, PGC1α inhibits the expression of c-MYC and ornithine decarboxylase 1 (ODC1), the rate-limiting enzyme for polyamine synthesis. Analysis of in vivo metastases and clinical data from patients with prostate cancer support the proposition that the PGC1α/c-MYC/ODC1 axis regulates polyamine biosynthesis and prostate cancer aggressiveness. In conclusion, downregulation of PGC1α renders prostate cancer cells dependent on polyamine to promote metastasis. SIGNIFICANCE: These findings show that a major regulator of mitochondrial metabolism controls polyamine synthesis and prostate cancer aggressiveness, with potential applications in therapy and identification of new biomarkers.

Detection of a new melanoma in a patient treated with fingolimod.

In addition to the TRANSFORMS, FREEDOMS, INFORMS studies, very few publications have identified new cases of skin cancer in patients treated with fingolimod. Here, we present the case of a 52-year-old Caucasian patient with relapsing remitting multiple sclerosis for 19 years, with a phototype II with blue eyes, light brown hair, no personal or family history of melanoma and a low number of naevi (<10). She did not experience intense sun exposure in childhood as well as severe sunburn and did not practise sessions in ultraviolet cabins. This case is distinguished from other published cases, usually superficial spreading malignant melanoma by its unclassifiable histological character. The occurrence of skin cancers in patients with multiple sclerosis remains exceptional, but new cases have recently emerged requiring the strengthening of dermatological follow-up of such patients.

GAPDH Expression Predicts the Response to R-CHOP, the Tumor Metabolic Status, and the Response of DLBCL Patients to Metabolic Inhibitors.

Diffuse large B cell lymphoma (DLBCL) is a heterogeneous disease treated with anti-CD20-based immuno-chemotherapy (R-CHOP). We identified that low levels of GAPDH predict a poor response to R-CHOP treatment. Importantly, we demonstrated that GAPDHlow lymphomas use OxPhos metabolism and rely on mTORC1 signaling and glutaminolysis. Consistently, disruptors of OxPhos metabolism (phenformin) or glutaminolysis (L-asparaginase) induce cytotoxic responses in GAPDHlow B cells and improve GAPDHlow B cell-lymphoma-bearing mice survival, while they are low or not efficient on GAPDHhigh B cell lymphomas. Ultimately, we selected four GAPDHlow DLBCL patients, who were refractory to all anti-CD20-based therapies, and targeted DLBCL metabolism using L-asparaginase (K), mTOR inhibitor (T), and metformin (M) (called KTM therapy). Three out of the four patients presented a complete response upon one cycle of KTM. These findings establish that the GAPDH expression level predicts DLBCL patients' response to R-CHOP treatment and their sensitivity to specific metabolic inhibitors.

UBTD1 is a mechano-regulator controlling cancer aggressiveness.

Ubiquitin domain-containing protein 1 (UBTD1) is highly evolutionary conserved and has been described to interact with E2 enzymes of the ubiquitin-proteasome system. However, its biological role and the functional significance of this interaction remain largely unknown. Here, we demonstrate that depletion of UBTD1 drastically affects the mechanical properties of epithelial cancer cells via RhoA activation and strongly promotes their aggressiveness. On a stiff matrix, UBTD1 expression is regulated by cell-cell contacts, and the protein is associated with ß-catenin at cell junctions. Yes-associated protein (YAP) is a major cell mechano-transducer, and we show that UBTD1 is associated with components of the YAP degradation complex. Interestingly, UBTD1 promotes the interaction of YAP with its E3 ubiquitin ligase ß-TrCP Consequently, in cancer cells, UBTD1 depletion decreases YAP ubiquitylation and triggers robust ROCK2-dependent YAP activation and downstream signaling. Data from lung and prostate cancer patients further corroborate the in cellulo results, confirming that low levels of UBTD1 are associated with poor patient survival, suggesting that biological functions of UBTD1 could be beneficial in limiting cancer progression.

Altered VEGF Splicing Isoform Balance in Tumor Endothelium Involves Activation of Splicing Factors Srpk1 and Srsf1 by the Wilms' Tumor Suppressor Wt1.

Angiogenesis is one hallmark of cancer. Vascular endothelial growth factor (VEGF) is a known inducer of angiogenesis. Many patients benefit from antiangiogenic therapies, which however have limitations. Although VEGF is overexpressed in most tumors, different VEGF isoforms with distinct angiogenic properties are produced through alternative splicing. In podocytes, the Wilms' tumor suppressor 1 (WT1) suppresses the Serine/arginine-rich protein-specific splicing factor kinase (SRPK1), and indirectly Serine/arginine-rich splicing factor 1 (Srsf1) activity, and alters VEGF splicing. We analyzed VEGF isoforms, Wt1, Srpk1, and Srsf1 in normal and tumor endothelium. Wt1, Srpk1, Srsf1, and the angiogenic VEGF164a isoform were highly expressed in tumor endothelium compared to normal lung endothelium. Nuclear expression of Srsf1 was detectable in the endothelium of various tumor types, but not in healthy tissues. Inducible conditional vessel-specific knockout of Wt1 reduced Wt1, Srpk1, and Srsf1 expression in endothelial cells and induced a shift towards the antiangiogenic VEGF120 isoform. Wt1(-KTS) directly binds and activates both the promoters of Srpk1 and Srsf1 in endothelial cells. In conclusion, Wt1 activates Srpk1 and Srsf1 and induces expression of angiogenic VEGF isoforms in tumor endothelium.

Erratum to "Inducible Conditional Vascular-Specific Overexpression of Peroxisome Proliferator-Activated Receptor Beta/Delta Leads to Rapid Cardiac Hypertrophy".

[This corrects the article DOI: 10.1155/2016/7631085.].

Combination of blood and biphasic calcium phosphate microparticles for the reconstruction of large bone defects in dog: A pilot study.

We previously reported that biphasic calcium phosphate (BCP) microparticles embedded in a blood clot induces ectopic bone formation in mice and repairs a critical femoral defect in rat. The present pilot study aimed to evaluate in dog and in two models of large defects the efficacy of this composite named "blood for reconstruction of bone" (BRB). We show here that BRB is a cohesive biomaterial easy to prepare from dog autologous blood and to mold to fill large bone defects. First in a model of cylindrical femoral condyle defect, the BRB was compared with BCP particles alone. After 8 weeks, this revealed that the amount of mature bone was slightly and significantly higher with BRB than with BCP particles. Second, in a model consisting in a 2 cm-long critical interruptive defect of the ulna, the BRB was compared with autologous bone. After 6 months, we observed that implantation of BRB can induce the complete reconstruction of the defect and that newly formed bone exhibits high regenerative potential. Comparison with the results obtained with autologous bone grafting strongly suggests that the BRB might be an efficient biomaterial to repair large bone defects, as an alternative or in addition to autologous bone. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1842-1850, 2018.

Echocardiographic and Histological Examination of Cardiac Morphology in the Mouse.

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed in mice is high. Phenotypic characterization of these mouse models also requires the examination of cardiac function and morphology. Echocardiography and magnetic resonance imaging (MRI) are commonly used approaches to characterize cardiac function and morphology in mice. Echocardiographic and MRI equipment specialized for use in small rodents is expensive and requires a dedicated space. This protocol describes cardiac measurements in mice using a clinical echocardiographic system with a 15 MHz human vascular probe. Measurements are performed on anesthetized adult mice. At least three image sequences are recorded and analyzed for each animal in M-mode in the parasternal short-axis view. Afterwards, cardiac histological examination is performed, and cardiomyocyte diameters are determined on hematoxylin-eosin- or wheat germ agglutinin (WGA)-stained paraffin sections. Vessel density is determined morphometrically after Pecam-1 immunostaining. The protocol has been applied successfully to pharmacological studies and different genetic animal models under baseline conditions, as well as after experimental myocardial infarction by the permanent ligation of the left anterior descending coronary artery (LAD). In our experience, echocardiographic investigation is limited to anesthetized animals and is feasible in adult mice weighing at least 25 g.

Sirtuin 7: a new marker of aggressiveness in prostate cancer.

Predictive biomarkers for advanced prostate cancer (PCa) are still missing. The sirtuin 7 (SIRT7) has been linked to tumorogenesis but its role in prostate cancer is poorly documented. To determine if SIRT7 can be a biomarker for aggressive prostate cancer and plays a role in PCa aggressiveness. We analyzed the expression of SIRT7 by immunohistochemistry in 57 patients comparing healthy with adjacent cancer tissue. SIRT7 levels were significantly elevated in tumors and its expression was positively associated with the grade. We also demonstrated that the knock down of SIRT7 decreased the migration of DU145 and PC3 cells (two androgen-independent prostate cancer cell lines) whereas the overexpression of the native protein but not the mutated form increased the cell migration and the invasion of the poorly aggressive prostate cancer cell line LNCaP. Finally, we also showed that SIRT7 overexpression induced the resistance to docetaxel. Our results demonstrate that SIRT7 promotes prostate cancer cell aggressiveness and chemoresistance and suggest that SIRT7 is a good predictive biomarker of PCa aggressiveness.

The energy disruptor metformin targets mitochondrial integrity via modification of calcium flux in cancer cells.

Mitochondrial integrity is critical for the regulation of cellular energy and apoptosis. Metformin is an energy disruptor targeting complex I of the respiratory chain. We demonstrate that metformin induces endoplasmic reticulum (ER) stress, calcium release from the ER and subsequent uptake of calcium into the mitochondria, thus leading to mitochondrial swelling. Metformin triggers the disorganization of the cristae and inner mitochondrial membrane in several cancer cells and tumors. Mechanistically, these alterations were found to be due to calcium entry into the mitochondria, because the swelling induced by metformin was reversed by the inhibition of mitochondrial calcium uniporter (MCU). We also demonstrated that metformin inhibits the opening of mPTP and induces mitochondrial biogenesis. Altogether, the inhibition of mPTP and the increase in mitochondrial biogenesis may account for the poor pro-apoptotic effect of metformin in cancer cells.

Impact of Expert Pathologic Review of Lymphoma Diagnosis: Study of Patients From the French Lymphopath Network.

Purpose To prospectively assess the clinical impact of expert review of lymphoma diagnosis in France. Materials and Methods From January 2010 to December 2013, 42,145 samples from patients with newly diagnosed or suspected lymphomas were reviewed, according to the 2008 WHO classification, in real time by experts through the Lymphopath Network. Changes in diagnosis between referral and expert review were classified as major or minor according to their potential impact on patient care. Results The 42,145 reviewed samples comprised 36,920 newly diagnosed mature lymphomas, 321 precursor lymphoid neoplasms, 314 myeloid disorders, and 200 nonhematopoietic neoplasms, with 4,390 benign lesions. There were 4,352 cutaneous and 32,568 noncutaneous lymphomas. The most common mature noncutaneous lymphomas were diffuse large B-cell lymphomas (32.4%), follicular lymphomas (15.3%), classic Hodgkin lymphomas (13%), peripheral T-cell lymphomas (6.3%) of which angioimmunoblastic T-cell lymphomas (2.3%) were the most frequent, and mucosa-associated lymphoid tissue lymphomas (5.8%). A diagnostic change between referral and expert review occurred in 19.7% of patients, with an estimated impact on patient care for 17.4% of patients. This rate was significantly higher for patients sent with a provisional diagnosis seeking expert second opinion (37.8%) than for patients sent with a formal diagnosis (3.7%). The most frequent discrepancies were misclassifications in lymphoma subtype (41.3%), with 12.3% being misclassifications among small B-cell lymphoma entities. Fewer than 2% of changes were between benign and malignant lymphoid conditions. Minor changes (2.3%) mostly consisted of follicular lymphoma misgrading and diffuse large B-cell lymphoma subtype misclassification. Conclusion To our knowledge, this study provides the largest ever description of the distribution of lymphoma entities in a western country and highlights how expert review significantly contributes to a precise lymphoma diagnosis and optimal clinical management in a proportion of patients.

The differential spatiotemporal expression pattern of shelterin genes throughout lifespan.

Shelterin forms the core complex of telomere proteins and plays critical roles in protecting telomeres against unwanted activation of the DNA damage response and in Emaintaining telomere length homeostasis. Although shelterin expression is believed to be ubiquitous for stabilization of chromosomal ends. Evidences suggest that some shelterin subunits have tissue-specific functions. However, very little is known regarding how shelterin subunit gene expression is regulated during development and aging. Using two different animal models, the mouse and zebrafish, we reveal herein that shelterin subunits exhibit distinct spatial and temporal expression patterns that do not correlate with the proliferative status of the organ systems examined. Together, this work shows that the shelterin subunits exhibit distinct spatiotemporal expression patterns, suggesting important tissue-specific functions during development and aging.

Small RNA-directed epigenetic programming of embryonic stem cell cardiac differentiation.

Microinjection of small noncoding RNAs in one-cell embryos was reported in several instances to result in transcriptional activation of target genes. To determine the molecular mechanisms involved and to explore whether such epigenetic regulations could play a role in early development, we used a cell culture system as close as possible to the embryonic state. We report efficient cardiac differentiation of embryonic stem (ES) cells induced by small non-coding RNAs with sequences of Cdk9, a key player in cardiomyocyte differentiation. Transfer of oligoribonucleotides representing parts of the Cdk9 mRNA into ES and mouse embryo fibroblast cultures resulted in upregulation of transcription. Dependency on Argonaute proteins and endogenous antisense transcripts indicated that the inducer oligoribonucleotides were processed by the RNAi machinery. Upregulation of Cdk9 expression resulted in increased efficiency of cardiac differentiation suggesting a potential tool for stem cell-based regenerative medicine.

Adequately defining tumor cell proportion in tissue samples for molecular testing improves interobserver reproducibility of its assessment.

Gene mutation status assessment of tumors has become standard practice in diagnostic pathology. This is done using samples comprising tumor cells but also non-tumor cells, which may dramatically dilute the proportion of tumor DNA and induce false negative results. Increasing sensitivity of molecular tests presently allows detection of a targeted mutation in a sample with a small percentage of tumor cells, but assessment of tumor cellularity remains essential to adequately interpret the results of molecular tests. Comprehensive tumor cell counting would provide the most reliable approach but is time consuming, and therefore rough global estimations are used, the reliability of which has been questioned in view of their potential clinical impact. The French association for quality assurance in pathology (AFAQAP) conducted two external quality assurance schemes, partly in partnership with the French group of oncology cytogenomics (GFCO). The purpose of the schemes was to (1) evaluate how tumor cellularity is assessed on tissue samples, (2) identify reasons for discrepancies, and (3) provide recommendations for standardization and improvement. Tumor cell percentages in tissue samples of lung and colon cancer were estimated by 40-50 participants, on 10 H&E virtual slides and 20 H&E conventional slides. The average difference between lowest and highest estimated percentage was 66. This was largely due to inadequate definition of cellularity, reflecting confusion between the percentage of tumor cells and the percentage of the area occupied by tumor in the assessed region. The widest range of interobserver variation was observed for samples with dense or scattered lymphocytic infiltrates or with mucinous stroma. Estimations were more accurate in cases with a low percentage of tumor cells. Macrodissection of the most homogeneous area in the tissue reduced inter-laboratory variation. We developed a rating system indicating potential clinical impact of a discrepancy. Fewer discrepancies were clinically relevant since the study was conducted. Although semi-quantitative estimations remain somewhat subjective, their reliability improves when tumor cellularity is adequately defined and heterogeneous tissue samples are macrodissected for molecular analysis.