Yes-Associated Protein (YAP) Modulates Oncogenic Features and Radiation Sensitivity in Endometrial Cancer

<div><p>Background</p><p>Yes-associated protein (YAP) is a transcriptional co-activator and regulates cell proliferation and apoptosis. We investigated the clinical and biological significance of YAP in endometrial cancer (EMCA).</p><p>Methods</p><p>YAP expression in 150 primary tumor tissues from patients with EMCA was evaluated by immunohistochemistry and its association with clinicopathological data was assessed. The biological functions of YAP were determined in EMCA cell lines through knockdown/overexpression of YAP. The role of YAP in modulating radiation sensitivity was also investigated in EMCA cells.</p><p>Results</p><p>Increased nuclear YAP expression was significantly associated with higher grade, stage, lympho-vascular space invasion, postoperative recurrence/metastasis and overall survival in estrogen mediated EMCA, called type 1 cancer (p = 0.019,  = 0.028,  = 0.0008,  = 0.046 and  = 0.015, respectively). In multivariate analysis, nuclear YAP expression was confirmed as an independent prognostic factor for overall survival in type 1 EMCA. YAP knockdown by siRNA resulted in a significant decrease in cell proliferation (p<0.05), anchorage-dependent growth (p = 0.015) and migration/invasion (p<0.05), and a significant increase in the number of cells in G0/G1 phase (p = 0.002). Conversely, YAP overexpression promoted cell proliferation. Clonogenic assay demonstrated enhanced radiosensitivity by approximately 36% in YAP inhibited cells.</p><p>Conclusions</p><p>Since YAP functions as a transcriptional co-activator, its differential localization in the nucleus of cancer cells and subsequent impact on cell proliferation could have important consequences with respect to its role as an oncogene in EMCA. Nuclear YAP expression could be useful as a prognostic indicator or therapeutic target and predict radiation sensitivity in patients with EMCA.</p></div

Yes-Associated Protein (YAP) Modulates Oncogenic Features and Radiation Sensitivity in Endometrial Cancer - Figure 2

<p>(A) Expression of YAP and phospho-YAP (Ser127) in three EMCA cell lines by western blotting. (B) Immunofluorescent cytochemical staining of endogenous YAP using anti-YAP antibody (YAP: green, DAPI: blue).</p

Clonogenic assay in HEC-1-B cells after radiation exposure.

<p>Knockdown of YAP expression by siRNA reduced clonogenic survival in HEC-1-B cells, resulting in an increase in radiation sensitivity with a dose enhancement factor at 10% survival (DEF 0.1) of 1.36. Results are shown in means ±standard deviations (bars) in triplicate experiments. Similar trends were obtained in other three independent experiments.</p

Comparison of demographic data and YAP expression between type 1 and type 2 EMCA.

<p>BMI; Body mass index, LVSI; Lymphovascular space involvement, N.S.; not significant.</p><p>(a): Student's t-test, (b): Chi square test (there is no <10 number in data)/Fisher's exact test (there is <10 number in data).</p

The Kaplan-Meier curve for overall survival rate of patients with type 1 EMCA (n = 120) according to the nuclear expression of YAP.

<p>Increased nuclear immunoreactivity of YAP was significantly associated with worse overall survival (P = 0.015, log-rank test).</p

Long-Term Weight-Loss in Gastric Bypass Patients Carrying Melanocortin 4 Receptor Variants

<div><p>Background</p><p>The melanocortin 4 receptor (MC4R) critically regulates feeding and satiety. Rare variants in <i>MC4R</i> are predominantly found in obese individuals. Though some rare variants in <i>MC4R</i> discovered in patients have defects in localization, ligand binding and signaling to cAMP, many have no recognized defects.</p><p>Subjects/Methods</p><p>In our cohort of 1433 obese subjects that underwent Roux-en-Y Gastric Bypass (RYGB) surgery, we found fifteen variants of <i>MC4R</i>. We matched rare variant carriers to patients with the <i>MC4R</i> reference alleles for gender, age, starting BMI and T2D to determine the variant effect on weight-loss post-RYGB. <i>In vitro</i>, we determined expression of mutant receptors by ELISA and western blot, and cAMP production by microscopy.</p><p>Results</p><p>While carrying a rare <i>MC4R</i> allele is associated with obesity, carriers of rare variants exhibited comparable weight-loss after RYGB to non-carriers. However, subjects carrying three of these variants, <i>V95I</i>, <i>I137T</i> or <i>L250Q</i>, lost less weight after surgery. <i>In vitro</i>, the R305Q mutation caused a defect in cell surface expression while only the I137T and C326R mutations showed impaired cAMP signaling. Despite these apparent differences, there was no correlation between <i>in vitro</i> signaling and pre- or post-surgery clinical phenotype.</p><p>Conclusions</p><p>These data suggest that subtle differences in receptor signaling conferred by rare <i>MC4R</i> variants combined with additional factors predispose carriers to obesity. In the absence of complete <i>MC4R</i> deficiency, these differences can be overcome by the powerful weight-reducing effects of bariatric surgery. In a complex disorder such as obesity, genetic variants that cause subtle defects that have cumulative effects can be overcome after appropriate clinical intervention.</p></div

BMIs of patients with rare variants only found in obese populations.

<p>The pre-surgery BMIs of patients with rare variants were matched with non-carrier patients of the same gender, T2D status, insulin medication status and similar age (within 5 years) (black symbols). The variant carrier's age, sex and T2D status is also listed (○). *The starting BMI range for the matched patient was extended to ±2.</p

Expression of MC4R mutants.

<p>A) ELISA of HEK293 cells expressing wild-type MC4R or mutations. The cell surface expression was normalized to total expression for each mutant and then to wild-type receptor for that batch (* denotes p<0.05 compared to wild-type by a one way ANOVA with Dunnet's post-hoc test). □ MC4R mutant D90N was not found in our cohort. B) Cell surface localization of HEK-293 cells expressing mutant or wild-type (BBS-MC4R) constructs labeled with Bungarotoxin-Texas Red. C) Normalized GFP loading reveals no differences in HA-MC4R mutant lifespan. HA-MC4R expression was normalized to GFP expression and plotted as a percentage of wild-type for each blot (n≥3).</p

Clinical variables for Roux-en Y gastric bypass patients with rare <i>MC4R</i> variants.

<p>Non-carrier patients are separated into non-diabetics, type 2 diabetics (T2D) and T2Ds taking insulin. Pre-surgery BMIs and blood pressure are reported as three month averages. No data were collected after surgery for the S4F (denoted ¶).</p><p>*The total number of patients differ in the six month remission data because those patients that did not have enough follow-up data collected were excluded from analysis (T2D-25 patients, T2D insulin dependent-9 patients).</p

cAMP Assay of MC4R mutants.

<p>cAMP production of MC4R mutants after stimulation with 10 nmol/L melanotan II (MTII) and then 100 µmol/L forskolin to activate maximum receptor-independent cAMP response. MTII stimulation was normalized to baseline cAMP production and plotted as a percentage of forskolin in the same cell. Mutants similar to wild-type (Black ○) are designated with the symbol (Gray ○). MC4R novel mutant G34A (Red ○) and those that have different statistically altered cAMP signaling (p<0.01 compared to wild-type by a one way ANOVA with Dunnet's post hoc test) (I137T (Green ○), D90N (Blue ○) and C326R (Purple ○)) are highlighted with different colored symbols. The D90N variant was not found in this cohort, but included as a control.</p