Driving effect of P16 methylation on telomerase reverse transcriptase-mediated immortalization and transformation of normal human fibroblasts.

BACKGROUND: P16 inactivation is frequently accompanied by telomerase reverse transcriptase (TERT) amplification in human cancer genomes. P16 inactivation by DNA methylation often occurs automatically during immortalization of normal cells by TERT. However, direct evidence remains to be obtained to support the causal effect of epigenetic changes, such as P16 methylation, on cancer development. This study aimed to provide experimental evidence that P16 methylation directly drives cancer development. METHODS: A zinc finger protein-based P16-specific DNA methyltransferase (P16-Dnmt) vector containing a "Tet-On" switch was used to induce extensive methylation of P16 CpG islands in normal human fibroblast CCD-18Co cells. Battery assays were used to evaluate cell immortalization and transformation throughout their lifespan. Cell subcloning and DNA barcoding were used to track the diversity of cell evolution. RESULTS: Leaking P16-Dnmt expression (without doxycycline-induction) could specifically inactivate P16 expression by DNA methylation. P16 methylation only promoted proliferation and prolonged lifespan but did not induce immortalization of CCD-18Co cells. Notably, cell immortalization, loss of contact inhibition, and anchorage-independent growth were always prevalent in P16-Dnmt&TERT cells, indicating cell transformation. In contrast, almost all TERT cells died in the replicative crisis. Only a few TERT cells recovered from the crisis, in which spontaneous P16 inactivation by DNA methylation occurred. Furthermore, the subclone formation capacity of P16-Dnmt&TERT cells was two-fold that of TERT cells. DNA barcoding analysis showed that the diversity of the P16-Dnmt&TERT cell population was much greater than that of the TERT cell population. CONCLUSION: P16 methylation drives TERT-mediated immortalization and transformation of normal human cells that may contribute to cancer development.

Angiopoietin 2 stimulates trophoblast invasion via a mechanism associated with JNK signaling.

Extravillous trophoblast cell (EVT) invasion is tightly controlled, and its dysregulation can lead to altered spiral artery remodeling and contribute to a number of different pregnancy complications. Angiopoietin-2 (Ang-2) is expressed by trophoblast cells and various cells in the decidua, and trophoblast cells express its receptor, Tie2. Ang-2 has been shown to play roles in tumor progression and metastasis but it is not known if it also regulates EVT invasion. Here, we show that both the HTR-8/SVneo cell line and primary isolates of human EVT expressed various integrins and the Tie2 receptor, and Ang-2 stimulated their migration and/or invasion. Ang-2 increased expression of matrix metalloproteinase (MMP)2 and MMP9, altered the cytoskeleton of HTR-8/SVneo cells and also induced phosphorylation of Tie2, JNK and c-Jun. Inhibition of p-JNK (using SP600125) blocked the Ang-2 induced invasion of HTR-8/SVneo cells. In addition, inhibition of Tie2 (pexmetinib) and integrin signaling (RGDS and ATN-161) also blocked Ang-2-induced invasion. In conclusion, we demonstrate that Ang-2 can stimulate EVT invasion via a mechanism associated with activation of both the Tie2 receptor and integrins, which appear to work through different pathways; Tie2 through the JNK/c-JUN pathway and integrins through an as yet unidentified pathway(s). We therefore propose that any alterations in Ang-2 expression in the decidua would lead to an imbalance in pro- and anti-invasive factors, disrupting regulation of EVT invasion and spiral artery remodeling and thereby contribute to the etiology of several complications of pregnancy.

Garcinol inhibits the proliferation of endometrial cancer cells by inducing cell cycle arrest.

Endometrial cancer (EC) is the most common gynecological cancer, and one of the most important causes of cancer­related deaths in women worldwide. The long­term survival rate is lower in advanced­stage and recurrent EC, therefore it is important to identify new anticancer drugs. Garcinol, a polyisoprenylated benzophenone, is a promising anticancer drug for various cancer types but its effects on EC remain unclear. To investigate the anticancer effects of garcinol on EC, cell proliferation and cell cycle were assessed by real­time cell proliferation, cell counting, and colony formation assays, flow cytometric analysis, and 5­ethynyl­2'­deoxyuridine (EdU) incorporation assay, in EC Ishikawa (ISH) and HEC­1B cell lines. Western blotting was used to evaluate the expression of cell cycle­related protein cyclins, cyclin­dependent kinase and tumor suppression proteins. Garcinol inhibited ISH and HEC­1B cell proliferation in a dose­dependent manner, and induced ISH and HEC­1B cell cycle arrest at the G1 phase and G2/M phase, respectively, and decreased the S phase and DNA synthesis in these two cell lines. Following garcinol treatment the expression levels of p53 and p21 were increased, while the expression levels of CDK2, CDK4, cyclin D1 and cyclin B1 were gradually decreased in a dose­dependent manner in both ISH and HEC­1B cells. In addition, the expression levels of phosphorylated c­JUN N­terminal kinase (JNK) and p­c­JUN were significantly increased in both types of cells. Collectively, garcinol can induce EC cell cycle arrest and may be a promising candidate for EC chemotherapy.

To what extent should the intestinal be resected proximally after radiotherapy: hint from a pathological view.

BACKGROUND: Neoadjuvant chemoradiotherapy (nCRT) is associated with post-operative anastomotic complications in rectal-cancer patients. Anastomosis involving at least one non-irradiated margin reportedly significantly reduces the risk of post-operative anastomotic complications in radiation enteritis. However, the exact scope of radiotherapy on the remaining sigmoid colon remains unknown. METHODS: We evaluated the radiation damage of proximally resected colorectal segments in 44 patients with rectal cancer, who received nCRT followed by conventional resection (nCRT-C, n = 21) or proximally extended resection (nCRT-E, n = 23). The segments from another 13 patients undergoing neoadjuvant chemotherapy (nCT) were used as control. We dissected these samples at a distance of 2 cm between the two adjacent sections. Radiation damage in proximally resected colorectal segments was evaluated using the radiation injury score (RIS) and the concentration and distribution patterns of angiostatin. RESULTS: Compared to those in the nCT group, the nCRT group showed higher RIS, levels of angiostatin, and proportion of diffuse pattern of angiostatin. With increasing distance from the tumor site, these parameters all gradually decreased; and the differences came to be not significant at the site that is over 20 cm from the tumor. The nCRT-E group showed lower RIS (median: 2 vs 4, P = 0.002) and a greater proportion of non-diffuse angiostatin (87% vs 55%, P = 0.039) at the proximal margins compared with the nCRT-C group. CONCLUSIONS: The severity of the radiation damage of the proximal colon is inversely proportional to the proximal-resection margin length. Little damage was left on the proximal margin that was over 20 cm from the tumor. Removal of an initial length of ≥20 cm from the tumor may be beneficial for rectal-cancer patients after nCRT.

Vitamin D Promotes Trophoblast Cell Induced Separation of Vascular Smooth Muscle Cells in Vascular Remodeling via Induction of G-CSF.

Vitamin D deficiency is associated with complications of pregnancy such as pre-eclampsia, fetal growth restriction, and miscarriage, all of which are also associated with incomplete spiral artery (SpA) remodeling. We have previously shown that both uterine natural killer (uNK) cells and extravillous trophoblast cells (EVT) are required for successful SpA remodeling, but whether their activity in this process is modulated by vitamin D is not known. In the current study, we use a previously described chorionic plate artery (CPA) ex vivo model of vascular remodeling to determine the effects of 1,25(OH)2D treated uNK cell, placental explant (PEx), and uNK/PEx conditioned medium (CM) on vascular smooth muscle cell (VSMC) disorganization and phenotypic switching. Significant results were followed up in VSMCs in vitro. We demonstrate that 1,25(OH)2D can enhance the ability of PEx to induce SpA remodeling, via a mechanism associated with increased secretion of granulocyte-colony stimulating factor (G-CSF). G-CSF appears able to increase VSMC disorganization and phenotypic switching in both an ex vivo vascular model and in vitro VSMC cultures. The clinical relevance of these findings are still to be determined. G-CSF may have differential effects depending on dose and vascular bed, and vitamin D may play a role in potentiating these actions. G-CSF may be an interesting potential therapeutic target for facilitating physiological vascular remodeling for the prevention of adverse obstetric outcomes.

Impact of Cold Ischemic Time and Freeze-Thaw Cycles on RNA, DNA and Protein Quality in Colorectal Cancer Tissues Biobanking.

Tissue-derived RNA, DNA and protein samples become more and more crucial for molecular detection in clinical research, personalized and targeted cancer therapy. This study evaluated how to biobanking colorectal tissues through examining the influences of cold ischemic time and freeze-thaw cycles on RNA, DNA and protein integrity. Here, 144 pairs of tumor and normal colorectal tissues were used to investigate the impact of cold ischemic times (0-48h) on RNA, DNA and protein integrity at on ice or room temperature conditions. Additionally, 45 pairs of tissues experienced 0-9 freeze-thaw cycles, and then the RNA, DNA and protein quality were analyzed. On ice, RNA, DNA and protein from colorectal tumor and normal tissues were all stable up to 48h after surgery. At room temperature, RNA in colorectal tumor and normal tissues began to degrade at 8h and 24h, respectively. Meanwhile, the tumor tissues DNA degradation occurred at 24h after surgery at room temperature. Similarly, the protein expression level of tumor and normal tissues began to change at 24h after the surgery at room temperature. Interestingly, tissue RNA and DNA remained stable even after 9 freeze-thaw cycles, whereas the proteins levels were remarkably changed after 7 freeze-thaw cycles. This study provided a useful evidence on how to store human colorectal tissues for biobanking. Preserving the surgical colorectal tissue on ice was an effective way to prevent RNA, DNA and protein degradation. Importantly, more than 7 repeated freeze-thaw cycles were not recommended for colorectal tissues.

Molecular Decision Tree Algorithms Predict Individual Recurrence Pattern for Locally Advanced Nasopharyngeal Carcinoma.

Background: Recurrence remains one of the key reasons of relapse after the radical radiation for locally advanced nasopharyngeal carcinoma (NPC). Here, the multiple molecular and clinical variables integrated decision tree algorithms were designed to predict individual recurrence patterns (with VS without recurrence) for locally advanced NPC. Methods: A total of 136 locally advanced NPC patients retrieved from a randomized controlled phase III trial, were included. For each patient, the expression levels of 33 clinicopathological biomarkers in tumor specimen, 3 Epstein-Barr virus related serological antibody titer and 5 clinicopathological variables, were detected and collected to construct the decision tree algorithm. The expression level of 33 clinicopathological biomarkers in tumor specimen was evaluated by immunohistochemistry staining. Results: Three algorithm classifiers, augmented by the adaptive boosting algorithm for variable selection and classification, were designed to predict individual recurrence pattern. The classifiers were trained in the training subset and further tested using a 10-fold cross-validation scheme in the validation subset. In total, 13 molecules expression level in tumor specimen, including AKT1, Aurora-A, Bax, Bcl-2, N-Cadherin, CENP-H, HIF-1α, LMP-1, C-Met, MMP-2, MMP-9, Pontin and Stathmin, and N stage were selected to construct three 10-fold cross-validation decision tree classifiers. These classifiers showed high predictive sensitivity (87.2-93.3%), specificity (69.0-100.0%), and overall accuracy (84.5-95.2%) to predict recurrence pattern individually. Multivariate analyses confirmed the decision tree classifier was an independent prognostic factor to predict individual recurrence (algorithm 1: hazard ration (HR) 0.07, 95% confidence interval (CI) 0.03-0.16, P < 0.01; algorithm 2: HR 0.13, 95% CI 0.04-0.44, P < 0.01; algorithm 3: HR 0.13, 95% CI 0.03-0.68, P = 0.02). Conclusion: Multiple molecular and clinicopathological variables integrated decision tree algorithms may individually predict the recurrence pattern for locally advanced NPC. This decision tree algorism provides a potential tool to select patients with high recurrence risk for intensive follow-up, and to diagnose recurrence at an earlier stage for salvage treatment in the NPC endemic region.

Radiation-induced injury on surgical margins: a clue to anastomotic leakage after rectal-cancer resection with neoadjuvant chemoradiotherapy?

BACKGROUND: Few studies on anastomotic condition after rectal-cancer resection and its effect on anastomotic leakage (AL) are available up to now. This study aimed to investigate potential radiation-induced injury left on surgical margins of anterior resection after neoadjuvant chemoradiotherapy (nCRT) and its association with AL. METHODS: We retrospectively identified 161 consecutive patients who underwent anterior resection with nCRT, neoadjuvant chemotherapy without radiation (nCT) or no neoadjuvant therapy between 2014 and 2015. Tissue samples of resection margins were assessed using a specific histopathological score and microvessel density in submucosa. Propensity score matching was used to balance the baseline characteristics. Association between AL and histopathological features was analysed. RESULTS: AL occurred in 13 of 54 patients undergoing nCRT, 5 of 48 patients undergoing nCT and 7 of 59 patients without neoadjuvant therapy. Comparisons after matching showed median (range) histopathological scores as follows: 3 (0-8) vs 0 (0-3) vs 0 (0-2) for the proximal margin (P < 0.001); 4 (2-9) vs 0 (0-4) vs 0 (0-3) for the distal margin (P < 0.001). Correspondingly, mean (SD) microvessel densities were as follows: 21.7 (7.9) vs 27.2 (8.6) vs 27.3 (9.4) for the proximal margin (P = 0.003); 18.1 (9.3) vs 25.2 (12.9) vs 24.9 (7.4) for the distal margin (P < 0.001). Among patients undergoing nCRT, AL was associated with increased histopathological score (P = 0.003) and decreased microvessel density (P = 0.004) on the proximal margin. CONCLUSIONS: Surgical margins of rectal-cancer resection are exposed to certain radiation-induced injury after nCRT. AL is associated with aggravated radiation damage on the proximal margin.

Demographic trends and KRAS/BRAFV600E mutations in colorectal cancer patients of South China: A single-site report.

The incidence of colorectal cancer (CRC) is increasing in China. Here, we aimed to evaluate the latest demographic trends and KRAS/BRAF mutations status of Chinese CRC. Five thousand five hundred and forty-six CRC patients diagnosed from 2010 to 2017 were involved. KRAS exon 2 and BRAFV600E mutations were detected by Sanger sequencing and high-resolution melting analysis or allelic-specific probe method. Gene mutation profiles and clinicopathologic characteristics of 5495 patients were analyzed. The joinpoint regression model was used to predict the demographic data in 2018. We found KRAS exon 2 and BRAFV600E mutation rates were 37.7 and 2.8% in CRC patients. Tumors with KRAS exon 2 mutations were more likely to be present in female and patients aged older than 75 years, right colon and have well-differentiated histology. Tumors with BRAFV600E mutations were more likely to be present in the female, right colon and have poorly differentiated histology. From 2010 to 2017, the percentage of colon cancer and tubular adenocarcinoma in CRC increased substantially (from 39.3 to 51.8%, from 78.6 to 93.4%, respectively). The percentage of right colon cancer increased from 18.3 to 20.5%, which predictively may keep at 22.6% in 2018. The rise trends for patients with moderate differentiation tumor or KRAS exon 2 mutated tumor were apparent (from 50.3 to 78.6%, from 32.8 to 39.7%, respectively). In conclusion, in recent 8 years, there is a shift to the colon, especially right colon in the incidence of Chinese CRC. Moreover tubular adenocarcinoma is becoming the primary histology type.

Role of Angiogenesis in Chronic Radiation Proctitis: New Evidence Favoring Inhibition of Angiogenesis Ex Vivo.

BACKGROUND: Chronic radiation proctitis (CRP), a common complication after radiotherapy for pelvic malignancies, compromises patient quality of life. Vascular damage and aberrant angiogenesis in the mucosal layer are essential histological features, but changes to the submucosal layer are unclear. Thus, we evaluated the histological characteristics and distribution changes of key angiogenic factors in full-layered human CRP samples. METHODS: Thirty paraffin-embedded CRP and twenty-nine non-CRP tissues were used to evaluate histopathological changes. Immunohistochemistry with anti-CD34 antibody was performed to calculate microvascular density (MVD). Frozen tissues from eight CRP patients and five non-CRP controls were collected and analyzed by antibody array, which contained sixty human angiogenesis-related factors. Quality controls with positive and negative controls were performed during antibody array analysis. Two differentially expressed factors were confirmed by ELISA. RESULTS: CRP lesions showed vasculopathy, fibrosis, mucosal ulceration, edema, and inflammatory cell infiltration. Human angiogenesis antibody array and ELISA confirmed the increased angiostatin in CRP lesions. Immunohistochemical staining showed dispersed distribution of angiostatin throughout the mucosal and submucosal layers in CRP lesions, while angiostatin accumulated within the vessel lumens in non-CRP tissues. MVD significantly decreased in the submucosal layer of CRP, suggesting a potential association with increased angiostatin. CONCLUSIONS: Angiostatin increased and had a distinct distribution in CRP lesions. Compensatory telangiectasia in the mucosa, vessel stenosis, and reduced MVD might attenuate blood flow in the submucosa and contribute to CRP progression. Restoration of vascular functionality by promoting angiogenesis in the submucosal layer may help alleviate CRP in clinical practice.

Increased urothelial cancer associated 1 is associated with tumor proliferation and metastasis and predicts poor prognosis in colorectal cancer.

Long non-coding RNA, urothelial cancer associated 1 (UCA1), is reported to play a critical role in progression of carcinogenesis. In the present study, we identified differential expression of UCA1 in colorectal cancer (CRC) and paired peritumoral tissues using gene expression microarray analyses. qPCR analysis confirmed that UCA1 was upregulated in CRC (p<0.001) and the expression of UCA1 was statistically correlated with lymph node metastasis (P=0.040), distant metastasis (P=0.043) and tumor stage (P=0.010). Kaplan-Meier analysis indicated that patients with high UCA1 expression had a poor prognosis. Moreover, multivariate analysis identified UCA1 overexpression as an independent predictor for CRC. We also found that knockdown of UCA1 significantly suppressed cell proliferation and metastasis in CRC cells. Flow cytometry assays showed UCA1 silencing induced G0/G1 growth arrest and apoptosis of CRC cells. To further investigate the regulatory mechanisms of UCA1, we identified that Ets-2 bound to the UCA1 core promoter using luciferase assays. Collectively, our findings suggested that UCA1 might be an important prognostic indicator in CRC and may be a potential target for diagnosis and gene therapy.

High expression of Beclin-1 predicts favorable prognosis for patients with colorectal cancer.

PURPOSE: Beclin-1 is an autophagy gene. It promotes the formation of the autophagic vesicle as well as plays an essential role in guarding the cells against chromosomal instability. Overexpression of Beclin-1 has been reported to predict a favorable survival in various cancers. However, little is known about its prognostic significance in colorectal cancer. METHODS AND MATERIALS: A total of three hundred and sixty-three (363) colorectal tissues from colorectal cancer (CRC) patients were collected. Tissue micro-arrays and immunohistochemistry were used to investigate the expression and prognostic significance of Beclin-1 in CRC. The associations among Beclin-1 expression, clinicopathological parameters and prognosis were evaluated. RESULTS: Beclin-1 had a higher expression in CRC tissues than in normal tissues. A high expression of Beclin-1 was positively correlated with gender (P=0.027), histological grade (P=0.003), pM status (P=0.003) and clinical stage (P=0.024). Patients with a high Beclin-1 expression, when compared to those with a lower expression had both a better overall survival (OS, P=0.006) and disease-free survival (DFS, P=0.008). In the pT3 subgroup, Beclin-1 was also found to be a good prognostic indicator (P<0.05). Multivariate analysis showed a high expression of Beclin-1 was indeed a positive independent prognostic factor of OS and DFS for CRC patients (P<0.05). CONCLUSION: Our results demonstrated that a high expression of Beclin-1 correlated with a better overall survival and disease-free survival, thus serving as a favorable independent prognostic marker in CRC.

Phosphorylated p38, a negative prognostic biomarker, complements TNM staging prognostication in colorectal cancer.

Phosphorylated p38 (p-p38) played a pivotal role in the regulation of disease progression and correlated with tumor prognosis. Here, we characterized the prognostic effect of p-p38 in colorectal cancer (CRC). Three hundred and sixteen CRC patients in stages I-III were recruited in this study. P-p38 expression was semi-quantitatively evaluated using tissue microarrays and immunohistochemistry staining. Overall survival (OS), disease-free survival (DFS), local failure-free survival (LFFS), and distant metastasis-free survival (DMFS) of patient subgroups, segregated by p-p38 expression level and clinical stage, were compared using Kaplan-Meier analysis. We found that p-p38 was overexpressed in 48.1 % (152/316) CRC tissues, whereas low or deficiently expressed in normal adjacent epithelia. Overexpression of p-p38 predicted poor OS (P < 0.001), DFS (P = 0.002), LFFS (P = 0.016), and DMFS (P = 0.025) in CRC. Importantly, patient subgroups in the early stage (stages I + II) and with low p-p38 had similar OS, PFS, LFFS, and DMFS probabilities to that of stage I, whereas those with high p-p38 were similar to stage III disease. In addition, for stage III disease, the subgroup with low p-p38 had a similar survival probability to that of stage I, whereas the subgroup with high p-p38 had the worst survival. Multivariate Cox analysis confirmed that p-p38 was indeed a significantly independent factor for death, recurrence, and distant metastases in CRC. Our results demonstrated that p-p38 was a negative independent prognostic factor for CRC. Complementing TNM staging with p-p38 might refine the risk definition more accurately for a subset of patients.