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Studies of molecular and cellular functions of small-molecule inhibitors in cancer treatment, eliciting effects by targeting genome and epigenome associated proteins, requires measurement of drug-target engagement in single-cell resolution. Here we present EpiChem for in situ single-cell joint mapping of small molecules and multimodal epigenomic landscape. We demonstrate single-cell co-assays of three small molecules together with histone modifications, chromatin accessibility or target proteins in human colorectal cancer (CRC) organoids. Integrated multimodal analysis reveals diverse drug interactions in the context of chromatin states within heterogeneous CRC organoids. We further reveal drug genomic binding dynamics and adaptive epigenome across cell types after small-molecule drug treatment in CRC organoids. This method provides a unique tool to exploit the mechanisms of cell type-specific drug actions., (© 2024. The Author(s).)

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., (Copyright © 2024 The Chinese Medical Association, produced by Wolters Kluwer, Inc. under the CC-BY-NC-ND license.)

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., (© The Author(s) 2021. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Endometrial cancer (EC) is the most common gynecological cancer, and one of the most important causes of cancer‑related deaths in women worldwide. The long‑term survival rate is lower in advanced‑stage 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 real‑time cell proliferation, cell counting, and colony formation assays, flow cytometric analysis, and 5‑ethynyl‑2'‑deoxyuridine (EdU) incorporation assay, in EC Ishikawa (ISH) and HEC‑1B cell lines. Western blotting was used to evaluate the expression of cell cycle‑related protein cyclins, cyclin‑dependent kinase and tumor suppression proteins. Garcinol inhibited ISH and HEC‑1B cell proliferation in a dose‑dependent manner, and induced ISH and HEC‑1B 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 dose‑dependent manner in both ISH and HEC‑1B cells. In addition, the expression levels of phosphorylated c‑JUN N‑terminal kinase (JNK) and p‑c‑JUN 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.

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) 2 D 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) 2 D 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Zhang, Wu, Chen, Ning, Lu, Qiu, Hewison, Tamblyn, Kilby and Lash.)

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., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

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 BRAF V600E 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 BRAF V600E 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 BRAF V600E 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., (© 2018 UICC.)

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.

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.

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.

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., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)