1、Part 1,Amplification Of MIR548K Contributes To Aggressiveness And Lymphangiogenesis Of Esophageal Squamous Cell Carcinoma (ESCC) 2、Part 2,Role Of PCLO In The Aggressiveness Of Esophageal Squamous Cell Carcinoma (ESCC) 3、Part 3, Screening For P53-d

Esophageal cancer is the eighth most common cancer and the sixth most common cause of cancer relative deaths worldwide. According to histopathologic types, there are two main types of esophageal carcinomas (EC), the esophageal adenocarcinoma (EAC) and the esophageal squamous cell carcinoma (ESCC). There are 90% of EC have been diagnosed as ESCC of which about 70% occur in China. The latest three decades have witnessed the great improvement of diagnosis, staging and treatment of patients with ESCC. However, the long-term outcome of this cancer is still dismal, with 5-year survival rate ranging from 15% to 25%. Further studies are warranted to reveal the underlying mechanisms of carcinogenesis and figure out more effective molecular biomarkers and drug targets, which would be of great value for clinical practice.In the present study, basing on the whole genome sequencing (WGS) and comparative genomic hybridization (aCGH), we found that the region 11q13.3-11q13.4 was significantly amplified in patients with ESCC and associated with regional lymph node involvement (p<0.0001). In this region, we identified one miRNA, MIR548K, that has never been associated with human cancer. Additionally, we performed RNA in situ hybrization in 93 ESCC tissues and matched adjacent normal epithelial tissues and found that miR-548k was overexpressed in tumor tissues, which was significantly correlated with poor overall survival and lymph node metastasis.Furthermore, comprehensive study in vitro and in vivo demonstrated that miR-548k play oncogenic roles in promoting cell proliferation, cell colony formation, cell cycle progression, cell motility and invasiveness, xenograft tumor formation and regional invasiveness and lung metastasis.Lymphangiogenesis is essential for cancer cell disseminating to the regional lymph nodes In the present study, we found that the conditional medium of miR-548k overexpression cells could enhance the motility and matrigel tube formation of human dermal lymphatic endothelial cell.The effect of miR-548k on lymphangiogenesis and lymphatic metastasis in ESCC was investigated in vivo using a popliteal lymph node metastasis model. We found that the lymph nodes in tumors formed from miR-548k overexpression cells had larger volumes and displayed higher numbers of GFP-positive tumor cells than tumors formed from control cells. Additionally, the tumors formed by miR-548k overexpression cells displayed increased levels of microlymphatic vessel density (MLD) compared with the control tumors, within the tumors and peri-tumoral tissues.Conversely, MLD was markedly increased in the peripheral of popliteal lymph nodes from tumors formed by miR-548k overexpression cells.Further studies unmasked the underlying mechanism that miR-548k promoted cell cycle progression by targeting the G2 phase checkpoint kinase Weel. Additionally, the transcriptional repressor of EGFR, KLF10, was another target of miR-548k. By downregulated KLF10, miR-548k activated the EGFR downstream pathways Akt and Erk indirectly, which promoted the cell proliferation, motility and invasiveness of ESCC as a result. Furthermore, miR-548k may target ADAMTS1 to promote lymphangiogesis.In summary, here we provide evidences that miR-548k is overexpressed in ESCC and significantly associated with overall survival and lymph node metastasis. Functional studies reveal oncogenic characteristics of miR-548k in ESCC aggressiveness. Mechanistic studies demonstrate that miR-548k target Weel, KLF10 and ADAMTS1 to promote cell cycle progression, proliferation, cell motility and lymphangiogesis.Esophageal squamous cell carcinoma (ESCC) is one of the most common aggressive and lethal malignancies in the world and especially in China, where ESCC is the fourth leading cause of cancer-related mortality44. Recently, basing on the whole genome sequencing (WGS) and whole-exome sequencing (WES) conducted by our previous work, we represent a landscape of genomic alteration in ESCC, which indeed provide insights into the genetic mechanisms of ESCC malignancy.In the present study, PCLO displayed 15.9% mutations and 11.4% CNV gains the ESCC sequencing cohort, which was one of the most altered genes. We further studied the association between PCLO protein (Piccolo) level and different clinicopathological features of ESCC. Piccolo expression was examined in 469 pair paraffin-embedded, archived ESCC tissues and matched adjacent normal epithelial tissues using IHC. Consistently, we further confirmed that Piccolo was overexpression in ESCC tumor tissues (p=0.0028). Additionally, Piccolo was often co-overexpression in tumor tissues and the metastatic lymph nodes (p<0.001, rs=0.502), indicating that patients with Piccolo overexpression were vulnerable to lymph node metastasis. Furthermore, statistical analyses revealed that expression of Piccolo was significantly correlated with clinical stage (p=0.001), patient overall survival (p=0.008), patient disease free survival (p=0.004) and tumor embolus (p=0.013), while weakly positive correlated with the lymph node metastasis (p=0.099) and pathological grade (p=0.095).Notably, Piccolo overexpression strongly associated with poor survival (both overall survival and disease free survival) of patients with ESCC (p=0.001, Kaplan-Meier survival analysis and log-rank test), and the 5-year survival rate in the Piccolo-positive group (47.8%) was substantially lower than that of the Piccolo-negative group (68.9%).The median survival in the Piccolo negative group was 150 months (95%CI 68.136 to 231.864), while 47 months (95% CI 34.345 to 59.655) in the Piccolo positive group. The median progression free survival in the Piccolo negative group was 141 months (95% CI 82.992 to 199.008), while 37 months (95% CI 27.235 to 46.765) in the Piccolo positive group. Univariate Cox regression survival analysis indicated that patients with positive Piccolo staining were at a higher risk of poor outcome than those Piccolo were negative (HR=1.665,95% CI 1.227 to 2.259 for overall survival and HR=1.618,95% CI 1.208 to 2.168 for disease free survival).Interestingly, Kaplan-Meier survival analysis and log-rank test showed that Piccolo overexpression was correlated with shorter overall survival (p=0.004) and disease free survival (p=0.004). Additionally, patients with Piccolo overexpression were at a high risk of poor outcome (HR=2.393,95% CI 1.295 to 4.424 and HR=2.398,95% CI 1.324 to 4.341 for overall survival and disease free survival, respectively). Multivariate Cox regression survival analysis adjusting for lymph node metastasis (LNM), Piccolo level, tumor embolus, gender, age and family history of patients consistently reported strong correlation between Piccolo overexpression and shorter survival (p<0.001, HR=4.77,95% CI 2.129 to 10.689 for overall survival and p<0.001, HR=4.003,95% CI 1.871 to 8.564 for disease free survival), indicating that Piccolo expression was an independent prognostic factor for outcome in ESCC.Functional studies demonstrated that knockdown of PCLO remarkably attenuated the malignances of ESCC cells in vitro and in vivo, while elevated expression of EGFR compromised the phenotypes of Piccolo loss. Knockdown of Piccolo promoted ubiquitination and degradation of EGFR which was dependent on the negative regulation effect of Piccolo on E3 ligase Siahl. Meanwhile, Piccolo may play a role in mediating EGFR endosomal sorting. Therapeutic data in mouse model implied that targeting Piccolo by monoclonal antibody may be a promising precision medicine strategy.Our results comprehensively demonstrate that Piccolo plays critical roles in tumor aggressiveness of ESCC via mediating the EGFR malignant signaling pathway, and may represent a novel prognostic biomarker and therapeutic target for patients with ESCC.To maintain genomic integrity, cells have evolved a sophisticated cellular network to deal with DNA lesions, which is collectively termed as the DNA damage response (DDR). When cells suffer DNA damage, pathways of DDR are activated to sense the DNA lesions, transmit these signals and promote their repair. The tumor suppressor gene p53 is one of the most important genes that can maintain the genomic integrity by regulating the cell cycle, promoting DNA repair and inducing apoptosis.In response to environmental and cellular stress,p53 could exert its functions by directly transcribing several downstream miRNA genes, affecting miRNAs biogenesis.In the present study, miRNA microarrays were performed to characterize the miRNAs expression pattern at varying time points (0h、4h、8h and 12h) after UV-C irradiation in both HCT116 p53+/+and HCT116 p53-/-cell lines. Interestingly, compared to non UV-C irradiation (0h), the number of the differentially expressed miRNAs at 4h in HCT116 p53+/+ cell line was similar to HCT116 p53-/- cell line and most of which were downregulated. These results suggested that a few miRNAs were downregulated in the early stage of DDR. Moreover, UV-C irradiation induced differential expression of miRNAs were partially relied on the p53 status, for the miRNAs expression pat terns were distinct between these two cell linesIntriguingly, a large number of downregulated miRNAs in HCT116 p53+/+ cell line were observed after irradiation, while the differentially expressed miRNAs in HCT116 p53-/-cell line were mainly upregulated. Additionally, more and more miRNAs were differentially expressed in a time-dependent course in p53 null cell line, however, most miRNAs were altered 8h after radiation in the p53 wild type cell line. To further identify miRNAs regulated by p53, miRNAs expression patterns were analyzed by comparing HCT116 p53-/- cell line to HCT116 p53+/+ cell line at different time points. We noted that many miRNAs were upregulated at 4 h after irradiation in HCT116 p53+/+ cell line relative to its null counterpart, but downregulated at 8h and 12h subsequently. This analysis indicated that some p53-medaited miRNAs are involved in the early stage of DDR and then downregulated gradually during the progression of DDR. Furthermore, many miRNAs were dynamic regulated in response to DNA damage. On the other hand, conserved p53-DNA binding sites were searching within the 2kb region upstream of the pre-miRNA start sites. About half (59/120) of the differential expressed miRNAs were harbored a high score of conserved p53-DNA binding sites.Hypergeometric distribution model was applied to analyze the chromosomal location priority, and found that miRNAs located in chromosomes 13,17 and X might play important roles in DDR. We used an online integrated miRNAs analysis system, miRSystem, to characterize the functions and pathways of these differerentially expressed miRNAs. The results demonstrated that MAPK pathway was one of the most perturbed pathways after UV-C irradiation. Additionally, Wnt signaling pathway, focal adhesion pathway and p53 pathway were also enriched here.At last, we focus on miR-320a, which was involved in the UV-C induced DDR, and found that it could promote the survival of colon cancer cells under modest doses of UV-C irradiation.In conclusion, our results identified a number of DNA damage responsive miRNAs that would be worthwhile for future study. The comprehensive UV-C induced miRNAs reported here represent an important contribution to our understanding of the significance of miRNAs involvement in the DNA damage response in colon cancer.