The highly conserved let-7 microRNA (miRNA) regulates developmental pathways across animal phyla. Mis-expression of let-7 causes lethality in Caenorhabditis elegans and has been associated with several human diseases. We show that timing of let-7 expression in developing worms is under complex transcriptional and post-transcriptional control. Expression of let-7 primary transcripts oscillates during each larval stage but precursor and mature let-7 miRNAs do not accumulate until later in development after lin-28 activity has diminished. We demonstrate that LIN-28 binds endogenous primary let-7 transcripts co-transcriptionally. We further show that LIN-28 binds endogenous primary let-7 transcripts in the nuclear compartment of human ES cells, suggesting that this LIN-28 activity is conserved across species. We conclude that co-transcriptional interaction of LIN-28 with let-7 primary transcripts blocks Drosha processing and, thus, precocious expression of mature let-7 during early development.

Background Germline genetic variants in PLCE1 (10q23) have demonstrated consistent associations with risk of esophageal squamous cell carcinoma (ESCC) and gastric cancer among Chinese. We evaluated PLCE1 mRNA and protein expression in paired tumor-normal tissues, and their relationship with survival. Methods PLCE1 mRNA was profiled using three probes in the Affymetrix GeneChip U133 for paired tumor-normal tissues of ESCC (n=132), gastric cardia adenocarcinoma (GCA, n=62) and gastric noncardia adenocarcinoma (GNCA, n=72). We used immunohistochemistry to detect PLCE1 protein on slides from tissue microarrays in paired tumor-normal tissues of ESCC (n=303), and tumors of GCA (n=298) and GNCA (n=124). Results Compared with normal tissues, PLCE1 mRNA expression was significantly reduced in ESCC tumors (P=0.03, probe_205112_at), as well as in GCA and GNCA tumors (P<0.0001, each probe). Protein expression was non-significantly reduced in ESCC tumors (P=0.51). Increased tumor-normal mRNA fold change (probe_205112_at) was associated with longer survival in ESCC (9.6 months for highest vs lowest quartile; P-trend=0.02). Increased mRNA tumor-normal fold change (probe_205111_at) was associated with longer survival for GCA (10.7 months for highest quartile; P-trend=0.04), but not for GNCA cases (P=0.72). Similar to mRNA, elevated tumor-normal fold change for protein in ESCC was also associated with improved survival (8.1 months for highest quartile; P-trend=0.04). Conclusions Dysregulated PLCE1 mRNA expression was observed for both ESCC (one probe only) and GCA tumors, and the altered PLCE1 expression appears to be associated with cancer prognosis. Impact A potential role for PLCE1 in the early detection and/or therapy of ESCC and GCA warrants further investigation.

Effective therapies are needed to enhance the long-term survival of patients with pancreatic ductal adenocarcinoma (PDAC), which is the fourth leading cause of cancer-related deaths in the United States and eighth worldwide. The mere 5-year survival rate of 5% is the lowest of all cancers and is due in part to late diagnosis and resistance to conventional therapy. Therefore, it is critical to identify molecular vulnerabilities in PDAC for developing targeted therapies. Activating point mutations in the small GTPase, K-Ras are present in 90% of PDAC cases. However, successful strategies that exploit the reliance of PDAC on mutant K-Ras have not been developed. Recent studies have shown that PDAC cell lines demonstrate a pronounced dependence on autophagy and that oncogenic Ras activates autophagy to maintain tumorigenesis. However, the mechanism by which oncogenic Ras induces autophagy is poorly understood. Notably, Tank Binding Kinase 1 (TBK1) operates downstream of the Ras effector, RalB to directly activate AKT pro-survival signaling, independent of mTOR and PI3K. In addition to supporting oncogenic transformation in cancer cells, TBK1 is a crucial component in antibacterial autophagy. This selective form of autophagy requires TBK1 to activate autophagic cargo receptors, p62 and Optineurin for enhanced autophagic clearance. Furthermore, mouse embryonic fibroblasts (MEFs) harvested from mice expressing mutant TBK1 show a block in autophagy, implicating TBK1 as a mediator of non-selective autophagy in addition to xenophagy. Therefore, we hypothesize that TBK1 is the Ras effector driving autophagy in pancreatic cancers to support tumorigenic growth. We assessed the effect of pharmacological inhibition of TBK1 with a derivative of 6-aminopyrazolopyrimidine (Compound II) in human pancreatic cancer cell lines and in a pre-clinical model of PDAC. Inhibition of TBK1 with Compound II substantially reduced cell viability in cancer cells with diverse oncogenotypes. Initial studies in a genetic mouse model of PDAC (p48-Cre; LSL-KrasG12D; Cdkn2alox/lox, KIC) treated with Compound II, show a reduction in tumor burden. Moreover, Compound II reduced the activity of AKT in tumor tissues and decreased disease progression as determined by histology and immunohistochemistry for amylase, a marker of normal acinar tissue. Further studies will be performed to examine the relative level of active autophagy in animals treated with Compound II. Additionally, the contribution of TBK1 to the autophagic pathway and development and progression of PDAC is being assessed by crossing TBK1 mutant animals with KIC mice. These results will further our understanding of Ras signaling in pancreatic cancer and are critical for exploring a new avenue of targeted therapy. Citation Format: Victoria H. Burton, Yi-Hung Ou, Jason E. Toombs, Michael A. White, Rolf A. Brekken. TBK1 as a novel mediator of K-Ras driven pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A04. doi: 10.1158/1557-3125.RASONC14-A04

<p>PDF - 194K, Supplementary Figure S1. Immunohistochemistry for PLCE1 protein expression with areas of annotation selected for image quantification (20x.)Supplementary Table S1. PLCE1 mRNA expression in GCA, GNCA and ESCC tumor tissues and tumor-normal expression fold change by characteristics of the subjects. Supplementary Table S2. PLCE1 protein expression in GCA, GNCA and ESCC tumor tissues and tumor-normal expression fold change in ESCC by characteristics of the participants. Supplementary Table S3. The probe-specific association between PLCE1 mRNA expression in tumors and tumor-normal fold change and mortality of ESCC, GCA, and GNCAs. Supplementary Table S4. Spearman correlation coefficients (P values) between PLCE1 mRNA expression in normal tissues of ESCC, GCA, and GNCA and selected PLCE1 SNPs.</p>

<div>Abstract<p><b>Background:</b> Germline genetic variants in <i>PLCE1</i> (10q23) have demonstrated consistent associations with risk of esophageal squamous cell carcinoma (ESCC) and gastric cancer among Chinese. We evaluated PLCE1 mRNA and protein expression in paired tumor-normal tissues, and their relationship with survival.</p><p><b>Methods:</b><i>PLCE1</i> mRNA was profiled using three probes in the Affymetrix GeneChip U133 for paired tumor-normal tissues of ESCC (<i>n</i> = 132), gastric cardia adenocarcinoma (GCA, <i>n</i> = 62), and gastric noncardia adenocarcinoma (GNCA, <i>n</i> = 72). We used immunohistochemistry to detect PLCE1 protein on slides from tissue microarrays in paired tumor-normal tissues of ESCC (<i>n</i> = 303), and tumors of GCA (<i>n</i> = 298) and GNCA (<i>n</i> = 124).</p><p><b>Results:</b> Compared with normal tissues, <i>PLCE1</i> mRNA expression was significantly reduced in ESCC tumors (<i>P</i> = 0.03, probe_205112_at), as well as in GCA and GNCA tumors (<i>P</i> < 0.0001, each probe). Protein expression was nonsignificantly reduced in ESCC tumors (<i>P</i> = 0.51). Increased tumor-normal mRNA fold change (probe_205112_at) was associated with longer survival in ESCC (9.6 months for highest vs. lowest quartile; <i>P</i>_trend = 0.02). Increased mRNA tumor-normal fold change (probe_205111_at) was associated with longer survival for GCA (10.7 months for highest quartile; <i>P</i>_trend = 0.04), but not for GNCA cases (<i>P</i> = 0.72). Similar to mRNA, elevated tumor-normal fold change for protein in ESCC was also associated with improved survival (8.1 months for highest quartile; <i>P</i>_trend = 0.04).</p><p><b>Conclusions:</b> Dysregulated PLCE1 mRNA expression was observed for both ESCC (one probe only) and GCA tumors, and the altered PLCE1 expression seems to be associated with cancer prognosis.</p><p><b>Impact:</b> A potential role for PLCE1 in the early detection and/or therapy of ESCC and GCA warrants further investigation. <i>Cancer Epidemiol Biomarkers Prev; 23(8); 1579–88. ©2014 AACR</i>.</p></div>