Michael G. Izban

Spontaneous preterm labor is associated with an increase in the proinflammatory signal transducer TLR4 receptor on maternal blood monocytes

BackgroundLocalized inflammation and increased expression of TLR4 receptors within the uterus has been implicated in the pathogenesis of preterm labor. It remains unclear whether intrauterine inflammatory responses activate the maternal peripheral circulatory system. Therefore we determined whether increased TLR4 expression is present in the peripheral maternal white blood cells of women with spontaneous preterm labor.MethodsThis is a cross-sectional study of 41 preterm labor cases and 41 non-preterm controls. For each case and control sample, RNA was purified from white blood cells and TLR4 mRNA pool size was evaluated by quantitative PCR. Protein expression levels were determined by flow cytometry. Statistical evaluation using multiple linear regressions was used to determine any significant differences between the cases and controls. The purpose was to determine association prevalence of TLR4 levels and preterm labor.ResultsAdjusted mean TLR4 mRNA levels of 0.788 ± 0.037 (standard error) for preterm labor and 0.348 ± 0.038 for the corresponding pregnant control women were statistically significantly different (P = 0.002). Using the lower 95% confidence interval of the mean expression level in PTL subjects (0.7) as a cutoff value for elevated TLR4 mRNA levels, 25/41 (60.9%) of PTL patients expressed elevated TLR4 mRNA as compared to 0/41 (0%) in control subjects. The TLR4 receptor levels in the granulocyte fraction of white blood cells from preterm labor and pregnant controls were similar. However, TLR4+/CD14+monocytes were 2.3 times more frequent (70% vs. 30%) and TLR4 also had a 2.6-fold higher density (750 vs. 280 molecules per cell) in preterm labor women compared with pregnant controls. There was no difference in the levels of TLR4 in patients at term.ConclusionsPatients with preterm labor exhibited elevated levels of CD14+ maternal blood monocytes each bearing enhanced expression of TLR4, indicating that the peripheral circulatory system is activated in patients with preterm labor. Elevated leukocyte TLR4 levels may be a useful biomarker associated with preterm labor.

SKP2 loss destabilizes EZH2 by promoting TRAF6-mediated ubiquitination to suppress prostate cancer

EZH2 is crucial for the progression of prostate cancer (PCa) and castration-resistant prostate cancer (CRPC) through upregulation and activation of progenitor genes, as well as androgen receptor (AR)-target genes. However, the mechanisms by which EZH2 is regulated in PCa and CRPC remain elusive. Here we report that EZH2 is post-transcriptionally regulated by SKP2 in vitro in cultured cells and in vivo in mouse models. We observed aberrant upregulation of Skp2, Ezh2 and histone H3 lysine 27 trimethylation (H3K27me3) in both Pten null mouse embryonic fibroblasts (MEFs) and Pten null mouse prostate tissues. Loss of Skp2 resulted in a striking decrease of Ezh2 levels in Pten/Trp53 double-null MEFs and in prostate tumors of Pten/Trp53 double-null mutant mice. SKP2 knockdown decreased EZH2 levels in human PCa cells through upregulation of TRAF6-mediated and lysine(K) 63-linked ubiquitination of EZH2 for degradation. Ectopic expression of TRAF6 promoted the K63-linked ubiquitination of EZH2 to decrease EZH2 and H3K27me3 levels in PCa cells. In contrast, TRAF6 knockdown resulted in a reduced EZH2 ubiquitination with an increase of EZH2 and H3K27me3 levels in PCa cells. Furthermore, the catalytically dead mutant TRAF6 C70A abolished the TRAF6-mediated polyubiquitination of recombinant human EZH2 in vitro. Most importantly, a concurrent elevation of Skp2 and Ezh2 was found in CRPC tumors of Pten/Trp53 mutant mice, and expression levels of SKP2 and EZH2 were positively correlated in human PCa specimens. Taken together, our findings revealed a novel mechanism on EZH2 ubiquitination and an important signaling network of SKP2-TRAF6-EZH2/H3K27me3, and targeting SKP2-EZH2 pathway may be a promising therapeutic strategy for CRPC treatment.

Preterm labor: CD55 in maternal blood leukocytes.

Problem  Intrauterine inflammation is a frequent and significant factor associated with the pathogenesis of preterm labor/birth (PTL/PTB). However, it remains unclear whether the intrauterine inflammatory responses activate the maternal peripheral circulation. We explored the association between PTL/PTB and the ‘activation’ of the peripheral circulatory system by determining whether CD55 mRNA expression within peripheral WBCs differed between PTL and control patients not in labor.

1,25-Dihydroxyvitamin D3 promotes a sustained LPS-induced NF-κB-dependent expression of CD55 in human monocytic THP-1 cells.

The vitamin D3 system imposes immunosuppressive effects on monocytic cells, in part, by inhibiting NF-κB-dependent expression of proinflammatory mediators. CD55, a cell surface complement regulatory protein that promotes protective and anti-inflammatory properties, is reportedly an NF-κB target gene transiently induced in monocytic cells by the bacterial endotoxin LPS. CD55 is elevated on white cells in women experiencing preterm labor (a pathophysiology commonly associated with bacterial infection) and failure to maintain CD55 was associated with subsequent preterm delivery. We examined the influence of vitamin D3 signaling on LPS-induced expression of CD55 in human monocytic THP-1 cells using quantitative PCR, immunoblot, immunohistochemistry, and NF-κB activation pathway inhibitors. Non-NF-κB targets CD14 and CD11b, which modulate bacterial surveillance and eradication, respectively, were also examined. LPS produced a rapid transient 1.6-fold increase in CD55 mRNA. 1,25-D3 alone did not affect CD55 mRNA expression within the first 48 h. However, in 1,25-D3 pretreated cells, LPS produced a >4-fold immediate and sustained increase in CD55 mRNA and protein expression, which was blocked by NF-κB inhibitors. Our results unexpectedly suggest that vitamin D3 signaling may promote an anti-inflammatory response through an NF-κB-dependent increase in CD55 expression. As expected, LPS or 1,25-D3 alone led to sustained increases in CD14 and CD11b expression. In 1,25-D3 pretreated cells, LPS differentially regulated protein expression - CD14 (21-fold increase) and CD11b (a transient 2-fold decrease) - principally at the posttranscriptional level. The coordinated temporal expression of CD55, CD14 and CD11b would contribute to an anti-inflammatory response by providing protection against complement-mediated cell lysis during pathogen recognition and eradication. Overall, the vitamin D3 system may play a role coordinating an anti-inflammatory response pattern of the host complement immune system. This may be particularly important when considering the high rates of preterm births in blacks, a population that exhibits reduced circulating vitamin D3 levels.

ORIGINAL ARTICLE: Preterm Labor: CD55 in Maternal Blood Leukocytes

Problem  Intrauterine inflammation is a frequent and significant factor associated with the pathogenesis of preterm labor/birth (PTL/PTB). However, it remains unclear whether the intrauterine inflammatory responses activate the maternal peripheral circulation. We explored the association between PTL/PTB and the ‘activation’ of the peripheral circulatory system by determining whether CD55 mRNA expression within peripheral WBCs differed between PTL and control patients not in labor.

Human alpha defensin 5 is a candidate biomarker to delineate inflammatory bowel disease

Inability to distinguish Crohns colitis from ulcerative colitis leads to the diagnosis of indeterminate colitis. This greatly effects medical and surgical care of the patient because treatments for the two diseases vary. Approximately 30 percent of inflammatory bowel disease patients cannot be accurately diagnosed, increasing their risk of inappropriate treatment. We sought to determine whether transcriptomic patterns could be used to develop diagnostic biomarker(s) to delineate inflammatory bowel disease more accurately. Four patients groups were assessed via whole-transcriptome microarray, qPCR, Western blot, and immunohistochemistry for differential expression of Human α-Defensin-5. In addition, immunohistochemistry for Paneth cells and Lysozyme, a Paneth cell marker, was also performed. Aberrant expression of Human α-Defensin-5 levels using transcript, Western blot, and immunohistochemistry staining levels was significantly upregulated in Crohns colitis, p< 0.0001. Among patients with indeterminate colitis, Human α-Defensin-5 is a reliable differentiator with a positive predictive value of 96 percent. We also observed abundant ectopic crypt Paneth cells in all colectomy tissue samples of Crohns colitis patients. In a retrospective study, we show that Human α-Defensin-5 could be used in indeterminate colitis patients to determine if they have either ulcerative colitis (low levels of Human α-Defensin-5) or Crohns colitis (high levels of Human α-Defensin-5). Twenty of 67 patients (30 percent) who underwent restorative proctocolectomy for definitive ulcerative colitis were clinically changed to de novo Crohns disease. These patients were profiled by Human α-Defensin-5 immunohistochemistry. All patients tested strongly positive. In addition, we observed by both hematoxylin and eosin and Lysozyme staining, a large number of ectopic Paneth cells in the colonic crypt of Crohns colitis patient samples. Our experiments are the first to show that Human α-Defensin-5 is a potential candidate biomarker to molecularly differentiate Crohns colitis from ulcerative colitis, to our knowledge. These data give us both a potential diagnostic marker in Human α-Defensin-5 and insight to develop future mechanistic studies to better understand crypt biology in Crohns colitis.

Fetuin-A (alpha 2HS glycoprotein) modulates growth, motility, invasion, and senescence in high-grade astrocytomas

Glioblastomas (high‐grade astrocytomas) are highly aggressive brain tumors with poor prognosis and limited treatment options. In the present studies, we have defined the role of fetuin‐A, a liver‐derived multifunctional serum protein, in the growth of an established glioblastoma cell line, LN229. We hereby demonstrate that these cells synthesize ectopic fetuin‐A which supports their growth in culture in the absence of serum. We have demonstrated that a panel of tissue microarray (TMA) of glioblastomas also express ectopic fetuin‐A. Knocking down fetuin‐A using shRNA approach in LN229, significantly reduced their in vitro growth as well as growth and invasion in vivo. The fetuin‐A knockdown subclones of LN229 (A and D) also had reduced motility and invasive capacity. Treatment of LN229 cells with asialofetuin (ASF), attenuated their uptake of labeled fetuin‐A, and induced senescence in them. Interestingly, the D subclone that had ~90% reduction in ectopic fetuin‐A, underwent senescence in serum‐free medium which was blunted in the presence of purified fetuin‐A. Uptake of labeled exosomes was attenuated in fetuin‐A knockdown subclones A and D. Taken together, the studies demonstrate the impact of fetuin‐A as significant node of growth, motility, and invasion signaling in glioblastomas that can be targeted for therapy.

A rapidly growing human papillomavirus‑positive oral tongue squamous cell carcinoma in a 21‑year old female: A case report

Oral tongue squamous cell carcinoma (OTSCC) has a median age at diagnosis of 62 years. The incidence of OTSCC in young adults has been increasing, and the reason is unclear. The present study describes a case, and molecular analysis, of OTSCC in a 21-year-old female. Clinical and pathological information were collected from medical records. Formalin-fixed paraffin-embedded biopsy tissue from the patient was reassessed using standard hematoxylin & eosin staining, and immunohistochemistry was used to assess the expression of cellular p16, MutL homolog (MLH)1, MLH2, MutS homolog 6 (MSH6) and PMS1 homolog 2 (PMS2). The human papilloma virus (HPV) genome was detected by PCR analysis of the extracted DNA. The young age of the patient with OTSCC was unusual. The original pathology report indicated koilocytotic atypia, a cellular abnormality associated with HPV. Although HPV-positive oral cancer tends to occur in ‘younger’ individuals, 21 years is unusual. The confirmation of biologically active HPV in the tumor was obtained via the observation of strong positive staining for cellular p16. The patient described a maternal family cluster of rare cancer types, thus the possibility that this rapidly growing cancer resulted from HPV infection combined with an underlying genetic mutation causing decreased DNA-mismatch repair was explored. However, MSH1, MSH2, MSH6 and PSM2, proteins that are associated with Lynch Syndrome, were expressed at normal levels. A rapidly growing OTSCC of a 21-year-old female was determined to be HPV-positive. The patient underwent combination chemotherapy and radiation and has experienced long-term survival without recurrence. The reason this tumor grew so quickly in such a young individual remains unknown. These types of cases warrant additional genomic and proteomic studies to improve understanding of this phenomenon.

Correction: Human alpha defensin 5 is a candidate biomarker to delineate inflammatory bowel disease

[This corrects the article DOI: 10.1371/journal.pone.0179710.].

Abstract 5326: EZH2 regulation through lysine 63 linked ubiquitination in prostate cancer

Enhancer of zeste homolog 2 (EZH2) plays crucial roles on the development of cells and tissues as well as the progression of prostate cancer (PCa) and castration resistant prostate cancer (CRPC). EZH2 can upregulate and activate progenitor genes including androgen receptor (AR) and the target genes. However, the mechanisms by which EZH2 is regulated in PCa remain elusive. Literature reports that tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6) is essential for both normal tissue development and regulation of oncogenic signaling pathways in cancers. In this study, we reported that EZH2 is regulated by PTEN and SKP2 network in PCa. Specifically, we showed that the aberrant upregulation of EZH2 and histone H3 lysine 27 trimethylation (H3K27me3) in both Pten null mouse embryonic fibroblasts (MEFs) in vitro and prostate tumors of Pten null mice in vivo, as compared to the controls. EZH2 levels were negatively correlated with TRAF6 in human PCa cells upon SKP2 dysregulation. Immunofluorescence (IF) staining results showed a co-localization of EZH2 and TRAF6 in nucleus of PC3 cells, and co-immunoprecipitation (co-IP) analysis further confirmed a physical binding of EZH2 and TRAF6 in PC3 cells. Ectopic expression of TRAF6 promoted the K63-linked polyubiquitination of EZH2 to decrease EZH2 and H3K27me3 levels in PCa cells. Conversely, TRAF6 knockdown resulted in a reduction of EZH2 polyubiquitination with an increase of EZH2 and H3K27me3 levels in PCa cells. Furthermore, the catalytically dead mutant TRAF6 C70A abolished the TRAF6-mediated polyubiquitination of human EZH2 in vivo in 293T cells and in vitro in the recombinant human EZH2 protein, as compared with TRAF6 WT. Together, we report for the first time a novel mechanism on EZH2 ubiquitination and an important signaling network of SKP2-TRAF6-EZH2-H3K27me3 in PCa cells. Our findings provide valuable clues on the mechanism and efficiency of targeting EZH2 in PCa and CRPC . Note: This abstract was not presented at the meeting. Citation Format: Wenfu Lu, Shenji Lu, Bo Li, Yingqiu Xie, Michael G. Izban, Billy R. Ballard, Sandeep A. Sathyanarayana, Samuel E. Adunyah, Robert J. Matusik, Zhenbang Chen. EZH2 regulation through lysine 63 linked ubiquitination in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5326. doi:10.1158/1538-7445.AM2017-5326