Fas/APO-1 is a cell surface protein known to trigger apoptosis upon specific antibody engagement. Because wild-type p53 can activate transcription as well as induce apoptosis, we queried whether p53 might upregulate Fas/APO-1. To explore this possibility, we examined human p53-null (H358 non-small-cell lung adenocarcinoma and K562 erythroleukemia) and wild-type p53-containing (H460 non-small-cell lung adenocarcinoma) cell lines. When H358 or H460 cells were transduced with a replication-deficient adenovirus expression construct containing the human wild-type p53 gene but not with vector alone, a marked upregulation (approximately a three-to fourfold increase) of cell surface Fas/APO-1 was observed by flow cytometry. Similarly, K562, cells stably transfected with a plasmid vector containing the temperature-sensitive human p53 mutant Ala-143 demonstrated a four- to sixfold upregulation of Fas/APO-1 by flow-cytometric analysis at the permissive temperature of 32.5 degrees C. Temperature-sensitive upregulation of Fas/APO-1 in K562 Ala-143 cells was verified by immunoprecipitation and demonstrated to result from enhanced mRNA production by nuclear run-on and Northern (RNA) analyses. Stably transfected K562 cells expressing temperature-insensitive, transcriptionally inactive p53 mutants (His-175, Trp-248, His-273, or Gly-281) failed to upregulate Fas/APO-1 at either 32.5 degrees or 37.5 degrees C. The temperature-sensitive transcription of Fas/APO-1 occurred in the presence of cycloheximide, indicating that de novo protein synthesis was not required and suggested a direct involvement of p53. Collectively, these observations argue that Fas/APO-1 is a target gene for transcriptional activation by p53.

Purpose Colorectal cancer (CRC) is one of the most common malignancies worldwide. Recently, a novel circular RNA, ciRS-7, was proposed to be a potential miR-7 sponge. Since miR-7 regulates the expression of several important drivers of CRC, we analyzed the clinical significance of ciRS-7 in CRC patients. Experimental Design Initially, we evaluated the expression levels of ciRS-7 in a training cohort comprising of 153 primary CRC tissues and 44 matched normal mucosae. We subsequently confirmed its clinical relevance in an independent validation cohort (n=165), and evaluated the effect of ciRS-7 on miR-7, and its targets genes EGFR and RAF1. Functional analysis were performed in cell lines and an animal model to support clinical findings. Results Our data revealed that ciRS-7 was significantly up-regulated in CRC tissues compared with matched normal mucosae (P=0.0018), and its overexpression was associated with poor patient survival (P=0.0224 and 0.0061 in the training and validation cohorts, respectively). Multivariate survival analysis revealed that ciRS-7 emerged as an independent risk factor for overall survival (P=0.0656 and 0.0324 in the training and validation cohorts, respectively). Overexpression of ciRS-7 in HCT116 and HT29 cells led to blocking of tumor suppressive effects of miR-7 and resulted in a more aggressive oncogenic phenotype, and ciRS-7 overexpression permitted inhibition of miR-7 and subsequent activation of EGFR and RAF1 oncogenes. Conclusions CiRS-7 is a promising prognostic biomarker in CRC patients and may serve as a therapeutic target for reducingEGFR-RAF1 activity in CRC patients.

Telomerase activation is a critical step for human carcinogenesis through the maintenance of telomeres, but the activation mechanism during carcinogenesis remains unclear. Transcriptional regulation of the human telomerase reverse transcriptase (hTERT) gene is the major mechanism for cancer-specific activation of telomerase, and a number of

Cancer-associated fibroblasts (CAFs) in the tumor microenvironment (TME) play a central role in tumor progression. We investigated whether CAFs can regulate tumor-infiltrating lymphocytes (TILs) and their role in tumor immunosuppression. A total of 140 cases of esophageal cancer were analyzed for CAFs and CD8 or forkhead box protein 3 (FoxP3) TILs by IHC. We analyzed cytokines using murine or human fibroblasts and cancer cells. Murine-derived fibroblasts and cancer cells were also inoculated into BALB/c or BALB/c- mice and the tumors treated with recombinant IL6 or anti-IL6 antibody. CD8 TILs and CAFs were negatively correlated in intratumoral tissues ( < 0.001), whereas FoxP3 TILs were positively correlated ( < 0.001) in esophageal cancers. Cocultured Colon26 cancer cells and fibroblasts resulted in accelerated tumor growth in BALB/c mice, along with decreased CD8 and increased FoxP3 TILs, compared with cancer cells alone. , IL6 was highly secreted in both murine and human cancer cell/fibroblast cocultures. IL6 significantly increased Colon26 tumor growth in immune-competent BALB/c ( < 0.001) with fewer CD8 TILs than untreated tumors ( < 0.001), whereas no difference in BALB/c- mice. In contrast, FoxP3 TILs increased in IL6-treated tumors ( < 0.001). IL6 antibody blockade of tumors cocultured with fibroblasts resulted not only in regression of tumor growth but also in the accumulation of CD8 TILs in intratumoral tissues. CAFs regulate immunosuppressive TIL populations in the TME via IL6. IL6 blockade, or targeting CAFs, may improve preexisting tumor immunity and enhance the efficacy of conventional immunotherapies. .

Purpose: Replication-selective tumor-specific viruses present a novel approach for treating neoplastic disease. These vectors are designed to induce virus-mediated lysis of tumor cells after selective viral propagation within the tumor. Telomerase activation is considered to be a critical step in carcinogenesis, and its activity is closely correlated with human telomerase reverse transcriptase (hTERT) expression. We investigated the antitumor effect of the hTERTspecific replication-competent adenovirus on human cancer cells.Experimental Design: We constructed an adenovirus 5 vector [tumor-or telomerase-specific replication-competent adenovirus (TRAD)], in which the hTERT promoter element drives expression of E1A and E1B genes linked with an internal ribosome entry site, and we examined the selective replication and antitumor effect in human cancer cells in vitro and in vivo.Results: TRAD induced selective E1A and E1B expression in human cancer cells, but not in normal cells such as human fibroblasts. TRAD replicated efficiently and induced marked cell killing in a panel of human cancer cell lines, whereas replication as well as cytotoxicity was highly attenuated in normal human fibroblasts lacking telomerase activity. In nu/nu mice carrying s.c. human lung tumor xenografts, intratumoral injection of TRAD resulted in a significant inhibition of tumor growth. No evidence of TRAD was identified in tissues outside of the tumors, despite the presence of TRAD in the circulation. Moreover, TRAD replication in the distant, noninjected tumors was demonstrated.Conclusions: Our results suggest that the hTERT promoter confers competence for selective replication of TRAD in human cancer cells, an outcome that has important implications for the treatment of human cancers.