<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>
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.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).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 nonsignificantly 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; Ptrend = 0.02). Increased mRNA tumor-normal fold change (probe_205111_at) was associated with longer survival for GCA (10.7 months for highest quartile; Ptrend = 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; Ptrend = 0.04).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.A potential role for PLCE1 in the early detection and/or therapy of ESCC and GCA warrants further investigation.
Abstract 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
In a recent study, a unique gene expression signature was observed when comparing esophageal squamous cell carcinoma (ESCC) epithelial cells to normal esophageal epithelial cells using laser capture microdissection (LCM) and cDNA microarray technology. To validate the expression of several intriguing genes from that study (KRT17, cornulin, CD44, and EpCAM), we employed two new technologies, expression microdissection (xMD) for high-throughput microdissection facilitating protein analysis and RNAscope for the evaluation of low abundant transcripts in situ. For protein measurements, xMD technology was utilized to specifically procure sufficient tumor and normal epithelium from frozen human tissue for immunoblot analysis of KRT17 (CK17) and cornulin. A novel in situ hybridization method (RNAscope) was used to determine the transcript level of two relatively low expressed genes, CD44 and EpCAM in both individual formalin-fixed paraffin-embedded (FFPE) tissue sections and in an ESCC tissue microarray (TMA). The results successfully confirmed the initial expression pattern observed for all four genes, potentially implicating them in the pathogenesis of ESCC. Additionally, the study provides important methodological information on the overall process of candidate gene validation.
Abstract Initial stages of human pancreatic ductal adenocarcinoma (PDA) are commonly characterized by an activating mutation in K-RAS along with extensive immune cell infiltration. Direct inhibition of K-RAS through pharmacological means remains a challenge; however targeted inhibition of TANK Binding Kinase 1 (TBK1), a critical downstream effector of mutant active K-RAS is an attractive alternative. High levels of active TBK1 are associated with inflammatory disease and cancer progression. TBK1 and homolog, IKKe activate the immune response transcription factor NF-κB. In metabolically challenged mice, IKKe regulates energy balance by sustaining chronic, low-grade inflammation. We hypothesize that TBK1 signaling is also critical in metabolic regulation and is required for progression of inflammation-induced diseases such as PDA. A kinase dead, Tbk1Δ/Δ mouse was used to determine the contribution of TBK1 to metabolic disease and PDA progression. Metabolic phenotyping experiments were performed with Tbk1Δ/Δ and Tbk1+/+ mice including a high fat diet weight study, body composition and metabolic chamber analyses. Tbk1+/+ and Tbk1Δ/Δ mice were crossed into a genetically engineered mouse model of PDA to determine the consequences of genetically removing Tbk1 on tumor development and overall survival. Here we report that Tbk1Δ/Δ mice are significantly smaller, leaner and have less fat than Tbk1+/+ mice on high fat chow diets (HFD) for 14 weeks. Tbk1Δ/Δ mice are more active and have smaller and more abundant adipocytes relative to Tbk1+/+ mice. Additionally, Tbk1Δ/Δ mice are protected from HFD induced hypercholesterolemia and liver steatosis. White adipose tissue (WAT) from HFD fed Tbk1Δ/Δ mice exhibit an induction of brown fat gene expression suggesting that thermogenesis is a contributor to their healthier and more active phenotype. Macrophage infiltration is fairly low in WAT from Tbk1+/+ and Tbk1Δ/Δ mice on normal chow. However on HFD, WAT from Tbk1+/+ mice display a significant increase in macrophage marker expression compared to Tbk1Δ/Δ mice. These findings indicate that HFD fed Tbk1Δ/Δ mice are protected from classic phenotypes of metabolic syndrome. In PDA, TBK1 is expressed and more active in human PDA cell lines relative to immortalized pancreatic epithelial lines and fibroblasts. Human PDA cell lines are sensitive to a small molecule inhibitor of TBK1 (compound II) in the low micromolar range. In a K-Ras driven genetic mouse model of PDA, TBK1 supports spontaneous pancreatic tumor growth as evidenced by smaller tumors in Tbk1Δ/Δ: PDA mice relative to Tbk1+/+: PDA mice. Our results suggest that TBK1 contributes to metabolic regulation and demonstrate the therapeutic potential of targeting TBK1 in pancreatic malignancies. Current work is focused on delineating the inflammatory and metabolic dysregulation in these animals and determining the precise mechanism by which TBK1 supports the progression of PDA. Citation Format: Victoria H. Burton, Rolf A. Brekken. Investigating the functional contribution of TANK binding kinase 1 to inflammation induced disease progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 22.
Abstract Effective therapies are needed to enhance the long-term survival of patients with pancreatic ductal adenocarcinoma (PDA), which is the fourth leading cause of cancer-related deaths in the United States and eighth worldwide. Initial stages of PDA are commonly characterized by an activating mutation in K-RAS, yet direct inhibition of K-RAS through pharmacological means remains a challenge. Higher levels of TANK Binding Kinase 1 (TBK1) mRNA, a critical downstream effector of mutant active K-RAS in lung cancer, are associated with poorer overall survival in a cohort of human PDA patients. We hypothesize that TBK1 is also an effective mediator of K-RAS driven pancreatic cancer. Here we report that TBK1 is expressed and more active in human PDA cell lines relative to immortalized pancreatic epithelial lines (HPNE) and fibroblasts. We found that human PDA cell lines are sensitive to a small molecule inhibitor of TBK1 in the low micromolar range. Further mice engineered to express a mutant kinase dead form of TBK1 (Tbk1Δ/Δ) are viable and fertile yet display smaller tumors at early time points in a genetically engineered mouse model of PDA. Interestingly, tumors from Tbk1Δ/Δ: PDA mice are more epithelial in gene expression and show significantly less collagen deposition compared to Tbk1+/+: PDA tumors. Additionally, cell lines isolated from Tbk1Δ/Δ: PDA tumors are more epithelial in morphology and less migratory and invasive relative to Tbk1+/+: PDA cell lines. Current work is focused on delineating the functional contribution of TBK1 to tumor cell motility in vivo as well as mechanistic understanding of the epithelial phenotype in Tbk1Δ/Δ: PDA tumors. 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 Haley Burton, Rolf A. Brekken, Melissa Gross, Alberto Bremauntz. Tbk1 loss in pancreatic cancer leads to changes in epithelial plasticity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1985. doi:10.1158/1538-7445.AM2017-1985
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