ATOH8 binds SMAD3 to induce cellular senescence and prevent Ras-driven malignant transformation.

The process of oncogene-induced senescence (OIS) and the conversion between OIS and malignant transformation during carcinogenesis is poorly understood. Here, we show that following overactivation of oncogene Ras in lung epithelial cells, high-level transforming growth factor ß1 (TGF-ß1)-activated SMAD3, but not SMAD2 or SMAD4, plays a determinant role in inducing cellular senescence independent of the p53/p16/p15 senescence pathways. Importantly, SMAD3 binds a potential tumor suppressor ATOH8 to form a transcriptional complex that directly represses a series of cell cycle-promoting genes and consequently causes senescence in lung epithelial cells. Interestingly, the prosenescent SMAD3 converts to being oncogenic and essentially facilitates oncogenic Ras-driven malignant transformation. Furthermore, depleting Atoh8 rapidly accelerates oncogenic Ras-driven lung tumorigenesis, and lung cancers driven by mutant Ras and Atoh8 loss, but not by mutant Ras only, are sensitive to treatment of a specific SMAD3 inhibitor. Moreover, hypermethylation of the ATOH8 gene can be found in approximately 12% of clinical lung cancer cases. Together, our findings demonstrate not only epithelial cellular senescence directed by a potential tumor suppressor-controlled transcriptional program but also an important interplay between the prosenescent and transforming effects of TGF-ß/SMAD3, potentially laying a foundation for developing early detection and anticancer strategies.

Dual specific phosphatase 4 suppresses ferroptosis and enhances sorafenib resistance in hepatocellular carcinoma.

AIMS: This investigation aims to elucidate the mechanism underlying sorafenib-induced ferroptosis in hepatocellular carcinoma (HCC). METHODS: The role of dual specificity phosphatase 4 (DUSP4) in sorafenib-treated HCC was investigated using comprehensive assessments both in vitro and in vivo, including Western blotting, qRT-PCR, cell viability assay, lipid reactive oxygen species (ROS) assay, immunohistochemistry, and xenograft tumor mouse model. Additionally, label-free quantitative proteomics was employed to identify potential proteins associated with DUSP4. RESULTS: Our study revealed that suppression of DUSP4 expression heightens the susceptibility of HCC cells to ferroptosis inducers, specifically sorafenib and erastin, in both in vitro and in vivo settings. Furthermore, we identified DUSP4-mediated regulation of key ferroptosis-related markers, such as ferritin light chain (FTL) and ferritin heavy chain 1 (FTH1). Notably, label-free quantitative proteomics unveiled the phosphorylation of threonine residue T148 on YTH Domain Containing 1 (YTHDC1) by DUSP4. Further investigations unraveled that YTHDC1, functioning as an mRNA nuclear export regulator, is a direct target of DUSP4, orchestrating the subcellular localization of FTL and FTH1 mRNAs. Significantly, our study highlights a strong correlation between elevated DUSP4 expression and sorafenib resistance in HCC. CONCLUSIONS: Our findings introduce DUSP4 as a negative regulator of sorafenib-induced ferroptosis. This discovery opens new avenues for the development of ferroptosis-based therapeutic strategies tailored for HCC treatment.

Mining the interpretable prognostic features from pathological image of intrahepatic cholangiocarcinoma using multi-modal deep learning.

BACKGROUND: The advances in deep learning-based pathological image analysis have invoked tremendous insights into cancer prognostication. Still, lack of interpretability remains a significant barrier to clinical application. METHODS: We established an integrative prognostic neural network for intrahepatic cholangiocarcinoma (iCCA), towards a comprehensive evaluation of both architectural and fine-grained information from whole-slide images. Then, leveraging on multi-modal data, we conducted extensive interrogative approaches to the models, to extract and visualize the morphological features that most correlated with clinical outcome and underlying molecular alterations. RESULTS: The models were developed and optimized on 373 iCCA patients from our center and demonstrated consistent accuracy and robustness on both internal (n = 213) and external (n = 168) cohorts. The occlusion sensitivity map revealed that the distribution of tertiary lymphoid structures, the geometric traits of the invasive margin, the relative composition of tumor parenchyma and stroma, the extent of necrosis, the presence of the disseminated foci, and the tumor-adjacent micro-vessels were the determining architectural features that impacted on prognosis. Quantifiable morphological vector extracted by CellProfiler demonstrated that tumor nuclei from high-risk patients exhibited significant larger size, more distorted shape, with less prominent nuclear envelope and textural contrast. The multi-omics data (n = 187) further revealed key molecular alterations left morphological imprints that could be attended by the network, including glycolysis, hypoxia, apical junction, mTORC1 signaling, and immune infiltration. CONCLUSIONS: We proposed an interpretable deep-learning framework to gain insights into the biological behavior of iCCA. Most of the significant morphological prognosticators perceived by the network are comprehensible to human minds.

Correction: Systemic Delivery of MicroRNA-101 Potently Inhibits Hepatocellular Carcinoma In Vivo by Repressing Multiple Targets.

[This corrects the article DOI: 10.1371/journal.pgen.1004873.].

Intensive cycles of neoadjuvant camrelizumab combined with chemotherapy in locally advanced esophageal squamous cell carcinoma: a single-arm, phase II trial.

BACKGROUND: Two cycles of neoadjuvant PD-1 blockade plus chemotherapy induced favorable pathological response and tolerant toxicity in patients with locally advanced esophageal squamous cell carcinoma (ESCC). However, approximately 25% of patients relapsed within 1 year after surgery, indicating that a short course of treatment may not be sufficient. Therefore, exploring the effects of intensive treatment is needed for optimal clinical outcomes. METHODS: Locally advanced ESCC patients were administered three cycles of camrelizumab plus nab-paclitaxel and capecitabine, followed by thoracoscopic esophagectomy. The primary endpoint was pathologic response. Secondary endpoints included safety, feasibility, radiologic response, survival outcomes, and immunologic/genomic correlates of efficacy. RESULTS: Forty-seven patients were enrolled in the study. Forty-two patients received surgery, and R0 resection was achieved in all cases. The complete and major pathological response rates were 33.3% and 64.3%, respectively, and the objective response rate was 80.0%. Three cycles of treatment significantly improved T down-staging compared to two cycles (P = 0.03). The most common treatment-related adverse events were grades 1-2, and no surgical delay was reported. With a median follow-up of 24.3 months, the 1-year disease-free survival and overall survival rates were both 97.6%, and the 2-year disease-free survival and overall survival rates were 92.3% and 97.6%, respectively. Three patients experienced disease recurrence or metastasis ranging from 12.5 to 25.8 months after surgery, and one patient died 6 months after surgery due to cardiovascular disease. Neither programmed death-ligand 1 expression nor tumor mutational burden was associated with pathological response. An increased infiltration of CD56dim natural killer cells in the pretreatment tumor was correlated with better pathological response in the primary tumor. CONCLUSIONS: It seems probable that intensive cycles of neoadjuvant camrelizumab plus nab-paclitaxel and capecitabine increased tumor regression and improved survival outcomes. Randomized controlled trials with larger sample sizes and longer follow-up periods are needed to validate these findings. Trial registration Chinese Clinical Trial Registry, ChiCTR2000029807, Registered February 14, 2020, https://www.chictr.org.cn/showproj.aspx?proj=49459 .

A novel peptide encoded by N6-methyladenosine modified circMAP3K4 prevents apoptosis in hepatocellular carcinoma.

BACKGROUND: Circular RNAs (circRNAs) regulate various biological activities and have been shown to play crucial roles in hepatocellular carcinoma (HCC) progression. However, only a few coding circRNAs have been identified in cancers, and their roles in HCC remain elusive. This study aimed to identify coding circRNAs and explore their function in HCC. METHODS: CircMAP3K4 was selected from the CIRCpedia database. We performed a series of experiments to determine the characteristics and coding capacity of circMAP3K4. We then used in vivo and in vitro assays to investigate the biological function and mechanism of circMAP3K4 and its protein product, circMAP3K4-455aa, in HCC. RESULTS: We found circMAP3K4 to be an upregulated circRNA with coding potential in HCC. IGF2BP1 recognized the circMAP3K4 N6-methyladenosine modification and promoted its translation into circMAP3K4-455aa. Functionally, circMAP3K4-455aa prevented cisplatin-induced apoptosis in HCC cells by interacting with AIF, thus protecting AIF from cleavage and decreasing its nuclear distribution. Moreover, circMAP3K4-455aa was degraded through the ubiquitin-proteasome E3 ligase MIB1 pathway. Clinically, a high level of circMAP3K4 is an independent prognostic factor for adverse overall survival and adverse disease-free survival of HCC patients. CONCLUSIONS: CircMAP3K4 is a highly expressed circRNA in HCC. Driven by m6A modification, circMAP3K4 encoded circMAP3K4-455aa, protected HCC cells from cisplatin exposure, and predicted worse prognosis of HCC patients. Targeting circMAP3K4-455aa may provide a new therapeutic strategy for HCC patients, especially for those with chemoresistance. CircMAP3K4 is a highly expressed circRNA in HCC. Driven by m6A modification, IGF2BP1 facilitates circMAP3K4 peptide translation, then the circMAP3K4 peptide inhibits AIF cleavage and nuclear distribution, preventing HCC cells from cell death under stress and promoting HCC progression.

Distribution and density of tertiary lymphoid structures predict clinical outcome in intrahepatic cholangiocarcinoma.

BACKGROUND & AIMS: The prognostic value and clinical relevance of tertiary lymphoid structures (TLSs) in intrahepatic cholangiocarcinoma (iCCA) remain unclear. Thus, we aimed to investigate the prognostic value and functional involvement of TLSs in iCCA. METHODS: We retrospectively included 962 patients from 3 cancer centers across China. The TLSs at different anatomic subregions were quantified and correlated with overall survival (OS) by Cox regression and Kaplan-Meier analyses. Multiplex immunohistochemistry (mIHC) was applied to characterize the composition of TLSs in 39 iCCA samples. RESULTS: A quaternary TLS scoring system was established for the intra-tumor region (T score) and peri-tumor region (P score) respectively. T scores positively correlated with favorable prognosis (p <0.001), whereas a high P score signified worse survival (p <0.001). mIHC demonstrated that both T follicular helper and regulatory T cells were significantly increased in intra-tumoral TLSs compared to peri-tumoral counterparts (p <0.05), and regulatory T cell frequencies within intra-tumoral TLSs were positively associated with P score (p <0.05) rather than T score. Collectively, the combination of T and P scores stratified iCCAs into 4 immune classes with distinct prognoses (p <0.001) that differed in the abundance and distribution pattern of TLSs. Patients displaying an immune-active pattern had the lowest risk, with 5-year OS rates of 68.8%, whereas only 3.4% of patients with an immune-excluded pattern survived at 5 years (p <0.001). The C-index of the immune class was statistically higher than the TNM staging system (0.73 vs. 0.63, p <0.001). These results were validated in an internal and 2 external cohorts. CONCLUSIONS: The spatial distribution and abundance of TLSs significantly correlated with prognosis and provided a useful immune classification for iCCA. T follicular helper and regulatory T cells may play a critical role in determining the functional orientation of spatially different TLSs. LAY SUMMARY: Tertiary lymphoid structures (TLSs) are associated with favorable prognosis in a number of cancers. However, their role in intrahepatic cholangiocarcinoma (iCCA) remains unclear. Herein, we comprehensively evaluated the spatial distribution, abundance, and cellular composition of TLSs in iCCA, and revealed the opposite prognostic impacts of TLSs located within or outside the tumor. This difference could be mediated by the different immune cell subsets present within the spatially distinct TLSs. Based on our analysis, we were able to stratify iCCAs into 4 immune subclasses associated with varying prognoses.

Immune-related adverse events predict responses to PD-1 blockade immunotherapy in hepatocellular carcinoma.

Immune checkpoint blockade has demonstrated remarkable efficacy in hepatocellular carcinoma (HCC) but is also commonly accompanied by immune-related adverse events (irAEs). However, the association between irAEs and antitumor efficacy in HCC patients remains unknown. All patients with HCC treated with anti-PD-1 antibodies from July 2018 to November 2019 were analyzed and divided into different groups according to their irAEs' status. In total, 101 HCC patients, including 21 (20.8%) patients who presented with irAEs (irAEs+ ), were enrolled. Among the adverse events, rash (n = 9, 8.9%) was the most frequent irAE, followed by mucositis (n = 3, 3.0%) and thyroiditis (n = 3, 3.0%). Patients in the irAEs+ group showed a higher tumor response rate than those in the irAEs- group (overall response rate: 28.6% vs 6.3%, P = .011; disease control rate: 85.7% vs 60.0%, P = .028). The median progression-free survival (PFS) times were 14.8 months in the irAEs+ group and 4.1 months in the irAEs- group (P < .001). Further analysis based on the presence or absence of rash showed that the PFS of the patients in the irAEs+ /rash+ group was better than that of those in the irAEs+ /rash- or irAEs- group (all P < .05). Multivariate analysis showed that irAEs were an independent prognostic factor for PFS (hazard ratio [HR]: 0.22, P = .002). Thus, the occurrence of irAEs, especially rash, was associated with markedly improved PFS. Awareness of irAEs may help classify the subtype of HCC patients with an unprecedented survival benefit from anti-PD-1 antibodies.

Tumor necrosis as a poor prognostic predictor on postoperative survival of patients with solitary small hepatocellular carcinoma.

BACKGROUND: Small hepatocellular carcinoma (sHCC) is a special subtype of HCC with the maximum tumor diameter ≤ 3 cm and excellent long-term outcomes. Surgical resection or radiofrequency ablation provides the greatest chance for cure; however, many patients still undergo tumor recurrence after primary treatment. To date, there is no clinical applicable method to assess biological aggressiveness in solitary sHCC. METHODS: In the current study, we retrospectively evaluated tumor necrosis of 335 patients with solitary sHCC treated with hepatectomy between December 1998 and 2010 from Sun Yat-sen University Cancer Center. RESULTS: The presence of tumor necrosis was observed in 157 of 335 (46.9%) sHCC patients. Further correlation analysis showed that tumor necrosis was significantly correlated with tumor size and vascular invasion (P = 0.026, 0.003, respectively). The presence of tumor necrosis was associated closely with poorer cancer-specific overall survival (OS) and recurrence-free survival (RFS) as evidenced by univariate (P <  0.001; hazard ratio, 2.821; 95% CI, 1.643-4.842) and multivariate analysis (P = 0.005; hazard ratio, 2.208; 95% CI, 1.272-3.833). Notably, the combined model by tumor necrosis, vascular invasion and tumor size can significantly stratify the risk for RFS and OS and improve the ability to discriminate sHCC patients' outcomes (P <  0.0001 for both). CONCLUSIONS: Our results provide evidence that tumor necrosis has the potential to be a parameter for cancer aggressiveness in solitary sHCC. The combined prognostic model may be a useful tool to identify solitary sHCC patients with worse outcomes.

Universal screening for Lynch syndrome in a large consecutive cohort of Chinese colorectal cancer patients: High prevalence and unique molecular features.

The prevalence of Lynch syndrome (LS) varies significantly in different populations, suggesting that ethnic features might play an important role. We enrolled 3330 consecutive Chinese patients who had surgical resection for newly diagnosed colorectal cancer. Universal screening for LS was implemented, including immunohistochemistry for mismatch repair (MMR) proteins, BRAFV600E mutation test and germline sequencing. Among the 3250 eligible patients, MMR protein deficiency (dMMR) was detected in 330 (10.2%) patients. Ninety-three patients (2.9%) were diagnosed with LS. Nine (9.7%) patients with LS fulfilled Amsterdam criteria II and 76 (81.7%) met the revised Bethesda guidelines. Only 15 (9.7%) patients with absence of MLH1 on IHC had BRAFV600E mutation. One third (33/99) of the MMR gene mutations have not been reported previously. The age of onset indicates risk of LS in patients with dMMR tumors. For patients older than 65 years, only 2 patients (5.7%) fulfilling revised Bethesda guidelines were diagnosed with LS. Selective sequencing of all cases with dMMR diagnosed at or below age 65 years and only of those dMMR cases older than 65 years who fulfill revised Bethesda guidelines results in 8.2% fewer cases requiring germline testing without missing any LS diagnoses. While the prevalence of LS in Chinese patients is similar to that of Western populations, the spectrum of constitutional mutations and frequency of BRAFV600E mutation is different. Patients older than 65 years who do not meet the revised Bethesda guidelines have a low risk of LS, suggesting germline sequencing might not be necessary in this population.

Eukaryotic Initiation Factor 5A2 Contributes to the Maintenance of CD133(+) Hepatocellular Carcinoma Cells via the c-Myc/microRNA-29b Axis.

Cancer stem cells (CSCs)/cancer-initiating cells (CICs) are suggested responsible for driving cancer resistance to conventional therapies and for cancer recurrence and/or metastasis. CD133 is served as a key biomarker to identify and characterize this subpopulation of cells in hepatocellular carcinoma (HCC). Our previous study indicated that overexpression of eukaryotic initiation factor 5A2 (EIF5A2) promotes HCC cell metastasis and angiogenesis. In this study, we demonstrated that EIF5A2 might play a crucial role in CSCs regulation and investigated its potential molecular mechanisms. Using quantitative real-time polymerase chain reaction assay, we observed that the expression of EIF5A2 positively correlated with CD133 levels in a cohort of cancerous and noncancerous liver tissues and cells. Next, HCC cells with high expression of EIF5A2 have a strong capacity to form undifferentiated tumor spheres in vitro and show elevated levels of stem cell-related genes, leading to an increased ability to develop tumors when subcutaneously injected into nude mice. Furthermore, differential microRNA expression was profiling between two EIF5A2-depleted HCC cell lines and their control one identified a decreased expression of miR-29b in EIF5A2-depleted cell lines. Further functional studies illustrated that downregulated miR-29b level is responsible for EIF5A2-maintained HCC cell stemness either in vitro or in vivo. Moreover, enforced expression of EIF5A2 in HCC cells largely enhanced the binding of c-Myc on the promoter of miR-29b and downregulation of miR-29b by EIF5A2 was dependent on c-Myc. Our findings, collectively, reveal that EIF5A2 contributes to the maintenance of CD133+ HCC cells via the c-Myc/miR-29b axis. Stem Cells 2018;36:180-191.

Systemic delivery of microRNA-101 potently inhibits hepatocellular carcinoma in vivo by repressing multiple targets.

Targeted therapy based on adjustment of microRNA (miRNA)s activity takes great promise due to the ability of these small RNAs to modulate cellular behavior. However, the efficacy of miR-101 replacement therapy to hepatocellular carcinoma (HCC) remains unclear. In the current study, we first observed that plasma levels of miR-101 were significantly lower in distant metastatic HCC patients than in HCCs without distant metastasis, and down-regulation of plasma miR-101 predicted a worse disease-free survival (DFS, P<0.05). In an animal model of HCC, we demonstrated that systemic delivery of lentivirus-mediated miR-101 abrogated HCC growth in the liver, intrahepatic metastasis and distant metastasis to the lung and to the mediastinum, resulting in a dramatic suppression of HCC development and metastasis in mice without toxicity and extending life expectancy. Furthermore, enforced overexpression of miR-101 in HCC cells not only decreased EZH2, COX2 and STMN1, but also directly down-regulated a novel target ROCK2, inhibited Rho/Rac GTPase activation, and blocked HCC cells epithelial-mesenchymal transition (EMT) and angiogenesis, inducing a strong abrogation of HCC tumorigenesis and aggressiveness both in vitro and in vivo. These results provide proof-of-concept support for systemic delivery of lentivirus-mediated miR-101 as a powerful anti-HCC therapeutic modality by repressing multiple molecular targets.

Nomogram individually predicts the overall survival of patients with gastroenteropancreatic neuroendocrine neoplasms.

BACKGROUND: The current study aimed to establish a novel nomogram to predict the overall survival of individual Chinese patients with gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs). Furthermore, this study sought to externally validate this nomogram using the Surveillance, Epidemiology, and End Results (SEER) database. METHODS: The records of 1183 patients with GEP-NENs treated at five high-capacity institutions in China between 2005 and 2015 were retrospectively analysed. In addition, 10 236 GEP-NEN cases from the SEER database were included as an external validation set. RESULTS: A multivariate analysis using Cox proportional hazards (PHs) regression was performed, and a nomogram was constructed. Discrimination, calibration, and external validation were performed using the SEER data set. The multivariate Cox model indicated that age, tumour size, differentiation, lymph node metastases, and distant metastases were independent covariates associated with survival. With respect to the training set, the nomogram exhibited better discrimination power than TNM classification (Harrell's concordance index (C-index): 0.837 vs 0.784, P=0.006). Discrimination was also excellent and superior to that of TNM classification for the SEER-based validation set (C-index: 0.808 vs 0.717, P<0.001). The calibrated nomogram predicted a survival rate that closely corresponded to the actual survival rate. CONCLUSIONS: We developed a nomogram that predicted the 3- and 5-year overall survival rates of patients with GEP-NENs. Validation revealed excellent discrimination and calibration for this nomogram, suggesting that it exhibits satisfactory clinical utility that might improve individualised predictions of survival risks and lead to the creation of additional clinical therapies.

Prognostic Factors for Locoregional Recurrence in Patients with Thoracic Esophageal Squamous Cell Carcinoma Treated with Radical Two-Field Lymph Node Dissection: Results from Long-Term Follow-Up.

OBJECTIVE: To aim of this study was to determine the clinical and biological prognostic factors for locoregional recurrence (LRR) in patients with thoracic esophageal squamous cell carcinoma (ESCC) undergoing radical two-field lymph node dissection (2FLD). METHODS: A total of 462 patients diagnosed with thoracic ESCC underwent radical esophagectomy between March 2001 and May 2010 at Sun Yat-Sen University Cancer Center. Clinical characteristics, CD44 expression, and tumor-infiltrating lymphocyte (TIL) levels were evaluated in 198 patients who underwent R0 dissection with long-term follow-up. Partial Cox regression analysis with leave-one-out cross-validation was performed to validate the selected risk factors. RESULTS: With a median follow-up of 54 months, the 5-year local failure-free survival (LFFS) rate of 198 patients was 62.5%. Multivariate analysis revealed that T stage (p = 0.043), pathological positive tumor above the carina (p = 0.000), CD44 expression level (p = 0.045) and TIL level (p = 0.007) were prognostic factors for LFFS, while the Cox model with risk scores had an area under the curve value of 83.6% for the prediction of 5-year LFFS. The best cut-off value (sum score = 11.19) was used to determine the high- and low-risk groups, with patients at high risk having a significantly shorter 5-year LFFS than patients at low risk (p = 0.000). The LRR pattern revealed significantly high incidences of recurrent disease at the supraclavicular and cervical sites, mediastinum (above the carina), and anastomosis. CONCLUSIONS: Our predictive model was able to distinguish between patients at high risk for LRR and patients at low risk for LRR. LRR primarily involved the upper thorax and this area must be considered in future study designs for radical trimodality treatment.

p53R2 as a novel prognostic biomarker in nasopharyngeal carcinoma.

BACKGROUND: p53R2 is a target of p53 gene, which is essential for DNA repair, mitochondrial DNA synthesis, protection against oxidative stress, chromosomal instability, chronic inflammation and tumorigenesis. This study is aimed to investigate the expression of ribonucleotide reductase (RR) subunit p53R2 in nasopharyngeal carcinoma and its significance in the prognosis. METHODS: The expression levels of p53R2 in 201 patients with NPC were examined by immunohistochemical assay. The correlations of p53R2 expression and clinicopathological features of nasopharyngeal carcinoma patient were analysed by chi-square test. The Kaplan-Meier survival analysis and Cox multivariate regression model were used to analyze the prognostic significance of the patients with NPC. RESULTS: Immunohistochemical results showed that p53R2 was positively expressed in 92.5% (186/201) of nasopharyngeal carcinoma and the high expression rate was 38.3% (77/201). Further analysis observed that the negative correlation between expression of p53R2 and pT status had statistical significance (P < 0.05). Kaplan-Meier survival analysis found that the mean survival time of patients with high expression of p53R2 was 143.32 months, while the patients with low expression level of p53R2 was 121.63 months (P < 0.05). Cox regression analysis suggested that p53R2 protein expression could be used as an independent prognostic factor for nasopharyngeal carcinoma (P < 0.05). CONCLUSIONS: This study drew a conclusion that p53R2 could be used as a prognostic biomarker indicative of the favorable outcome for patients with nasopharyngeal carcinoma.

Increased expression of Solute carrier family 12 member 5 via gene amplification contributes to tumour progression and metastasis and associates with poor survival in colorectal cancer.

OBJECTIVE: Using whole genome sequencing, we identified gene amplification of solute carrier family 12 member 5 (SLC12A5) located at 20q13.12 in colorectal cancer (CRC). We analysed its amplification, overexpression, biological effects and prognostic significance in CRC. DESIGN: SLC12A5 amplification status was evaluated by fluorescence in situ hybridisation (FISH). The effects of SLC12A5 re-expression or knockdown were determined in proliferation, apoptosis, invasion and metastasis assays. SLC12A5 target genes and related pathways were identified by reporter activity and cDNA microarray analyses. Clinical impact of SLC12A5 overexpression was assessed in 195 patients with CRC. RESULTS: Amplification of SLC12A5 was verified in 78 out of 191 (40.8%) patients with primary CRC by FISH, which was positively correlated with its protein overexpression (p<0.001). Biofunctional investigation of SLC12A5 revealed that SLC12A5 significantly increased cell proliferation, G1-S cell cycle transition, invasion/migration abilities, but suppressed apoptosis in vitro and promoted xenograft tumour growth as well as lung metastasis in vivo. The antiapoptosis effect by SLC12A5 was mediated through inhibiting apoptosis-inducing factor and endonuclease G-dependent apoptotic signalling pathway; and the pro-metastasis role was by regulating key elements of the matrix architecture, including matrix metallopeptidase and fibronectin. After a median follow-up of 50.16 months, multivariate analysis revealed that patients with SLC12A5 protein overexpression had a significant decrease in overall survival. Kaplan-Meier survival curves showed that SLC12A5 overexpression was significantly associated with shortened survival in patients with CRC. CONCLUSIONS: SLC12A5 plays a pivotal oncogenic role in colorectal carcinogenesis; its overexpression is an independent prognostic factor of patients with CRC.

CLDN14 is epigenetically silenced by EZH2-mediated H3K27ME3 and is a novel prognostic biomarker in hepatocellular carcinoma.

Trimethylation of lysine 27 on histone H3 (H3K27ME3) is a transcription-suppressive histone mark mediated by enhancer of zeste homolog 2 (EZH2). We have previously suggested that EZH2-mediated H3K27ME3 plays a critical oncogenic role in human hepatocellular carcinoma (HCC) aggressiveness. However, the direct downstream targets of EZH2-H3K27ME3 and the molecular mechanisms by which regulates HCC pathogenesis remain unclear. In this study, we used chromatin immunoprecipitation together with high-throughput sequencing (ChIP-seq) and gene expression profiling by microarray analysis to assess genome-wide chromatin occupancy of H3K27ME3 in HCC cells. We identified that claudin14 (CLDN14) is a potentially direct target for EZH2-mediated H3K27ME3 in HCC. In a large cohort of clinical HCC tissues, we found that low expression of CLDN14 was significantly associated with advanced tumor stage and determined to be an independent predictor of shortened survival of HCC patients. Next, functional experiment demonstrated that depletion of CLDN14 substantially restored EZH2-silenced HCC cells motility and invasive capacities and supported cell epithelial-mesenchymal transition (EMT). Furthermore, downregulation of CLDN14 dramatically re-enhanced the wnt/ß-catenin signaling activity in EZH2-silenced HCC cells by increasing the levels of active ß-catenin and promoting the nuclear localization of ß-catenin. These results, collectively, uncover that CLDN14 is a novel direct target of EZH2-mediated H3K27ME3, and provide an explanation for the aggressive nature of HCC with downregulation of CLDN14 and the underling mechanism that links the tumor suppressor CLDN14 to the wnt/ß-catenin signaling pathway.

Erratum to: Low expression of ULK1 is associated with operable breast cancer progression and is an adverse prognostic marker of survival for patients.

Erratum to: Breast Cancer Res Treat (2012),134:549­560,DOI 10.1007/s10549-012-2080-y. In the original publication of the article, Fig. 5c was published incorrectly. The authors apologize for this error and the correct Fig. 5c is given below.

Overexpression of Hexokinase 1 as a poor prognosticator in human colorectal cancer.

It has been suggested that hexokinase 1 (HK1) is involved in tumorigenesis. However, the expression dynamics of HK1 and its prognostic significance in human colorectal cancer (CRC) still remain unclear. The aim of the present study was to investigate the expression of HK1 and its prognostic significance in CRC. In this study, immunohistochemical analysis was used to examine the expression dynamics of HK1 in CRC tissues from two independent cohorts. Receiver operating characteristic curve analysis, Kaplan-Meier curves, and Cox regression analysis were utilized to investigate the prognostic significance. Results showed that a high expression of HK1 was observed in 106 of 393 (27.0 %) and 69 of 229 (30.1 %) of CRC in the training cohort and validation cohort, respectively. Further correlation analyses indicated that the increased HK1 expression was strongly correlated with the pN classification and TNM stage. Both cohorts showed a close association between the overexpression of HK1 and poorer overall survival. Importantly, multivariate analysis identified HK1 expression in CRC as an independent prognostic factor. Overexpression of HK1 may act as a significant biomarker of poor prognosis for patients with CRC.

Acylglycerol kinase is over-expressed in early-stage cervical squamous cell cancer and predicts poor prognosis.

Acylglycerol kinase (AGK) had been shown to contribute to cancer progression and unfavorable clinical outcomes of patients. Our study aimed to investigate the expression pattern and clinical significance of AGK in patients with early-stage cervical squamous cell cancer (CSCC). The protein and messenger RNA (mRNA) expression of AGK was analyzed in six cervical cancer cell lines and four paired early-stage CSCC specimens and normal cervical tissues (NCT), using Western blotting and real-time PCR (RT-PCR). And we investigated the AGK protein expression in paraffin-embedded specimens from 140 patients with early-stage CSCC and 30 cases of NCT by immunohistochemistry (IHC). Statistical analyses were performed to evaluate the clinicopathological significance of AGK expression. The expressions of AGK protein and mRNA were significantly up-regulated in cervical cancer cell lines and cancer tissues. IHC analyses revealed that AGK was highly expressed in 93 (66.4 %) of 140 early-stage CSCC specimens, but in none of the NCT. Moreover, AGK expression in early-stage CSCC was significantly correlated with tumor stage (P < 0.001), tumor size (P < 0.001), and tumor type (P < 0.001). Early-stage CSCC patients with high AGK expression level had shorter progress-free survival (PFS) and overall survival (OS) time compared with patients with low AGK expression levels. Univariate and multivariate analyses identified AGK expression level as an independent prognostic factor for survival of early-stage CSCC patients. We showed that AGK was over-expressed in cervical cancer cell lines and clinical tissues, and over-expression of AGK was associated with poor survival outcomes of early-stage CSCC patients. AGK can be used as an independent prognostic marker for early-stage CSCC.