Leptin Promotes Greater Ki67 Expression in CD4+ T Cells From Obese Compared to Lean Persons Living With HIV

While antiretroviral therapy (ART) has proven effective in suppressing viremia and disease progression among people living with human immunodeficiency virus (HIV; PLWH), suboptimal CD4+ T cell reconstitution remains a major obstacle in nearly 30% of ART-treated individuals. Epidemiological studies demonstrate that obesity, or a body mass index (BMI) ≥ 30 kg/m2, is positively correlated with greater CD4+ T cell recovery in PLWH on ART. Leptin is a known immunomodulator that is produced in proportion to fat mass and is increased in obese individuals, including PLWH. We hypothesized that CD4+ T cells from obese PLWH have increased cell proliferation and cytokine production compared to cells from lean PLWH, potentially modulated by differential effects of leptin signaling. To test this hypothesis, peripheral blood mononuclear cells from obese and lean PLWH with long-term virologic suppression on the same ART regimen were pretreated with recombinant leptin and then stimulated with anti-CD3/CD28 or PMA/ionomycin to measure Ki67 expression, leptin receptor (LepR) surface expression and cytokine production. In the absence of leptin, Ki67 expression and IL-17A production were significantly higher in CD4+ T cells from obese compared to lean PLWH. However, LepR expression was significantly lower on CD4+ T cells from obese compared to lean PLWH. After leptin treatment, Ki67 expression was significantly increased in CD4+ T cells from obese PLWH compared to the lean participants. Leptin also increased IL-17A production in CD4+ T cells from obese healthy controls. In contrast, leptin decreased IL-17A production in CD4+ T cells from both obese and lean PLWH. Combined, these results demonstrate that obesity is associated with greater CD4+ T cell proliferation among PLWH, and that higher circulating leptin levels in obesity may contribute to improved CD4+ T reconstitution in PLWH initiating ART.

Iodine-125 seed represses the growth and facilitates the apoptosis of colorectal cancer cells by suppressing the methylation of miR-615 promoter

Background Colorectal cancer (CRC) represents a common malignancy in gastrointestinal tract. Iodine-125 (125I) seed implantation is an emerging treatment technology for unresectable tumors. This study investigated the mechanism of 125I seed in the function of CRC cells. Methods The CRC cells were irradiated with different doses of 125I seed (0.4, 0.6 and 0.8 mCi). miR-615 expression in CRC tissues and adjacent tissues was detected by RT-qPCR. miR-615 expression was intervened with miR-615 mimic or miR-615 inhibitor, and then the CRC cells were treated with 5-AZA (methylation inhibitor). The CRC cell growth, invasion and apoptosis were measured. The methylation level of miR-615 promoter region was detected. The xenograft tumor model irradiated by 125I seed was established in nude mice. The methylation of miR-615, Ki67 expression and CRC cell apoptosis were detected. Results 125I seed irradiation repressed the growth and facilitated apoptosis of CRC cells in a dose-dependent manner. Compared with adjacent tissues, miR-615 expression in CRC tissues was downregulated and miR-615 was poorly expressed in CRC cells. Overexpression of miR-615 suppressed the growth of CRC cells. 125I seed-irradiated CRC cells showed increased miR-615 expression, reduced growth rate and enhanced apoptosis. The methylation level of miR-615 promoter region in CRC cells was decreased after 125I seed treatment. In vivo experiments confirmed that 125I seed-irradiated xenograft tumors showed reduced methylation of the miR-615 promoter and increased miR-615 expression, as well as decreased Ki67 expression and enhanced apoptosis. The target genes of miR-615 and its regulatory downstream pathway were further predicted by bioinformatics analysis. Conclusions 125I seed repressed the growth and facilitated the apoptosis of CRC cells by suppressing the methylation of the miR-615 promoter and thus activating miR-615 expression. The possible mechanism was that miR-615-5p targeted MAPK13, thus affecting the MAPK pathway and the progression of CRC.

Endometrial Metaplastic/Reactive Changes Coexistent with Endometrial Hyperplasia and Carcinoma: A Morphological and Immunohistochemical Study

The aim of this study was to assess the relationship between endometrial metaplastic/reactive changes (EMRCs) and endometrial neoplastic lesions. Twenty cases of “simple” (without architecture complexity) EMRCs coexistent with endometrial malignant/premalignant lesions, twenty cases of neoplasia-unassociated EMRCs, and eight cases of complex metaplastic lesions were assessed by immunohistochemistry. EMRCs coexisted with endometrioid carcinoma (n = 12), atypical endometrial hyperplasia (n = 3), serous carcinoma (n = 2), and clear cell carcinoma (n = 3). Neoplasia-associated EMRCs showed a mean Ki67 labeling index of 12.6% (range 0–30%); with nuclear atypia in 16/20 (80%) cases; diffuse p16 expression in 15/20 (75%) cases; and heterogeneous ER, PR, and vimentin expression. Compared to the associated neoplasia, EMRCs showed a lower Ki67 expression (p < 0.001) and higher p16 expression (p < 0.001). No EMRC case showed mitotic activity, PTEN loss, MMR deficiency, nuclear β-catenin, p53-mutant pattern, Napsin A, or AMACR expression. No significant differences were found between neoplasia-associated and neoplasia-unassociated EMRCs. Complex metaplastic lesions showed a lower Ki67 expression than EMRCs (p = 0.044) and PTEN loss in 5/8 cases, even in the absence of nuclear atypia. In conclusion, neoplasia-associated simple EMRCs may show evident atypia and a worrisome immunophenotype, but no data support their involvement in endometrial carcinogenesis. Architectural complexity appears as a crucial factor to identify precancerous lesions.

O-L09 Determining the Prognostic Utility of Immuno-histochemically detected Trans-membranous Human Equilibriative Nucleoside Transporter 1 (hENT1) in Patients Undergoing Hilar Cholangiocarcinoma Resection

Background Human equilibriative nucleoside transporter protein 1 (hENT1) is a trans-membranous protein which facilitates nucleoside transport in to the cell. Immunohistochemically-detected hENT1 abundance is increased in cholangiocarcinoma tumour cells compared to matched non-tumour cells and increased in highly metabolising cells. The privately-held Mackey 10D7G2 hybridoma has demonstrated prognostic utility in Pancreatic Ductal Adenocarcinoma patients. The commercially available Proteintech Polyclonal hENT1 antibody’s prognostic utility has not been previously assessed. Cellular Ki67 expression has been linked to mitotic indices of tumour proliferation. This proof-of-concept study aims to assess the antibodies prognostic utility for hilar cholangiocarcinoma patients undergoing surgical resection. Methods Between February 2009 and February 2016 54 patients underwent resection for peri-hilar cholangiocarcinoma. Formalin-Fixed Paraffin Embedded (FFPE) blocks from a sub-set of 44 resected specimens were retrieved. Appropriate areas of tumour were sampled from the blocks and a Tissue-Matched Array (TMA) was constructed. The TMA underwent staining for each antibody. H-scores were utilised to determine intensity of expression. Correlation of expression between antibodies was determined by Pearson correlation co-efficient and Chi-squared where appropriate. Silencing RNA transfected HepG2 cell-lines was used to determine hENT1 staining by the Proteintech antibody. Demographic and survival characteristics for the patients were acquired from a prospectively held database linked to Hospital Episode Statistics. Survival characteristics were calculated with global log-rank calculations. Results There was significant correlation between the Mackey 10D7G2 and the Proteintech antibodies (p &lt; 0.001). There was significant correlation between the Proteintech hENT1 antibody expression and Ki67 expression (p = 0.02). Knockdown of hENT1 with silencing RNA transfected HepG2 cells was confirmed by Western blot in a time-dependent fashion over 72 hours. The antibodies (Mackey; Proteintech; Ki67) did not achieve significance for predicting OS (p = 0.75; 0.63; 0.22 respectively). Nodal stage (p = 0.03) and grade of tumour differentiation (p = 0.02) were the univariate tumour variables with prognostic utility. Conclusions While the Proteintech antibody demonstrates concordance with the 10D7G2 antibody in determining hENT1 expression the antibodies did not demonstrate significant prognostic ability in this proof-of-concept study. Standard histopathological co-variates retain prognostic utility within the cohort.

Magnetic resonance imaging-based radiomics signature for preoperative prediction of Ki67 expression in bladder cancer

Purpose The Ki67 expression is associated with the advanced clinicopathological features and poor prognosis in bladder cancer (BCa). We aimed to develop and validate magnetic resonance imaging (MRI)-based radiomics signatures to preoperatively predict the Ki67 expression status in BCa. Methods and materials We retrospectively collected 179 BCa patients with Ki67 expression and preoperative MRI. Radiomics features were extracted from T2-weighted (T2WI) and dynamic contrast-enhancement (DCE) images. The synthetic minority over-sampling technique (SMOTE) was used to balance the minority group (low Ki67 expression group) in the training set. Minimum redundancy maximum relevance was used to identify the best features associated with Ki67 expression. Support vector machine and Least Absolute Shrinkage and Selection Operator algorithms (LASSO) were used to construct radiomics signatures in training and SMOTE-training sets, and diagnostic performance was assessed by the area under the curve (AUC) and accuracy. The decision curve analyses (DCA) and calibration curve and were used to investigate the clinical usefulness and calibration of radiomics signatures, respectively. The Kaplan-Meier test was performed to investigate the prognostic value of radiomics-predicted Ki67 expression status. Results 1218 radiomics features were extracted from T2WI and DCE images, respectively. The SMOTE-LASSO model based on nine features achieved the best predictive performance in the SMOTE-training (AUC, 0.859; accuracy, 80.3%) and validation sets (AUC, 0.819; accuracy, 81.5%) with a good calibration performance and clinical usefulness. Immunohistochemistry-based high Ki67 expression and radiomics-predicted high Ki67 expression based on the SMOTE-LASSO model were significantly associated with poor disease-free survival in training and validation sets (all P < 0.05). Conclusions The SMOTE-LASSO model could predict the Ki67 expression status and was associated with survival outcomes of the BCa patients, thereby may aid in clinical decision-making.

When Left Does Not Seem Right: Epigenetic and Bioelectric Differences Between Left- and Right-Sided Breast Cancer

Background: During embryogenesis, lateral symmetry is broken giving rise to Left/Right (L/R) breast tissues with distinct identity. L/R-sided breast tumors exhibit consistently-biased incidence, gene expression, and DNA methylation. We postulate that a differential L/R tumor-microenvironment crosstalk generates different tumorigenesis mechanisms. Methods: We performed in-silico analyses on breast tumors of public datasets, developed xenografted tumors, and conditioned MDA-MB-231 cells with L/R mammary extracts. Results: We found L/R differential DNA methylation involved in embryogenic and neuron-like functions. Focusing on ion-channels, we discovered significant L/R epigenetic and bioelectric differences. Specifically, L-sided cells presented increased methylation of hyperpolarizing ion channel genes and increased Ca2+ concentration and depolarized membrane potential, compared to R-ones. Functional consequences were associated with increased proliferation in left tumors, assessed by KI67 expression and mitotic count. Conclusions: Our findings reveal considerable L/R asymmetry in cancer processes, and suggest specific L/R epigenetic and bioelectric differences as future targets for cancer therapeutic approaches in the breast and many other paired organs.

MKL1 regulates hepatocellular carcinoma cell proliferation, migration and apoptosis via the COMPASS complex and NF-κB signaling

Background Histone modification plays essential roles in hepatocellular carcinoma (HCC) pathogenesis, but the regulatory mechanisms remain poorly understood. In this study, we aimed to analyze the roles of Megakaryoblastic leukemia 1 (MKL1) and its regulation of COMPASS (complex of proteins associated with Set1) in HCC cells. Methods MKL1 expression in clinical tissues and cell lines were detected by bioinformatics, qRT-PCR and western blot. MKL1 expression in HCC cells were silenced with siRNA, followed by cell proliferation evaluation via Edu staining and colony formation, migration and invasion using the Transwell system, and apoptosis by Hoechst staining. HCC cell tumorigenesis was assessed by cancer cell line-based xenograft model, combined with H&E staining and IHC assays. Results MKL1 expression was elevated in HCC cells and clinical tissues which was correlated with poor prognosis. MKL1 silencing significantly repressed proliferation, migration, invasion and colony formation but enhanced apoptosis in HepG2 and Huh-7 cells. MKL1 silencing also inhibited COMPASS components and p65 protein expression in HepG2 and Huh-7 cells. HepG2 cell tumorigenesis in nude mice was severely impaired by MKL1 knockdown, resulted into suppressed Ki67 expression and cell proliferation. Conclusion MKL1 promotes HCC pathogenesis by regulating hepatic cell proliferation, migration and apoptosis via the COMPASS complex and NF-κB signaling.