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Colorectal cancer (CRC) is a leading nonfamilial cause of cancer mortality among men and women. Although various genetic and epigenetic mechanisms have been identified, the full molecular mechanisms deriving CRC tumorigenesis are not fully understood. This study demonstrates that cell adhesion molecule transmembrane and immunoglobulin domain containing 1 (TMIGD1) are highly expressed in mouse and human normal intestinal epithelial cells. TMIGD1 knockout mice were developed, and the loss of TMIGD1 in mice was shown to result in the development of adenomas in small intestine and colon. In addition, the loss of TMIGD1 significantly impaired intestinal epithelium brush border membrane, junctional polarity, and maturation. Mechanistically, TMIGD1 inhibits tumor cell proliferation and cell migration, arrests cell cycle at the G2/M phase, and induces expression of p(21CIP1) (cyclin-dependent kinase inhibitor 1), and p(27KIP1) (cyclin-dependent kinase inhibitor 1B) expression, key cell cycle inhibitor proteins involved in the regulation of the cell cycle. Moreover, TMIGD1 is shown to be progressively down-regulated in sporadic human CRC, and its downregulation correlates with poor overall survival. The findings herein identify TMIGD1 as a novel tumor suppressor gene and provide new insights into the pathogenesis of colorectal cancer and a novel potential therapeutic target.

Colorectal cancer (CRC) is a leading non-familial cause of cancer mortality among men and women. Although various genetic and epigenetic mechanisms have been identified, the full molecular mechanisms deriving CRC tumorigenesis remains incompletely understood. In this study, we demonstrate that cell adhesion molecule transmembrane and immunoglobulin domain containing1 (TMIGD1) is highly expressed in mouse and human normal intestinal epithelial cells. We have developed TMIGD1 knockout mice and show that the loss of TMIGD1 in mice results in the development of adenomas in small intestine and colon. Additionally, the loss of TMIGD1 in mouse impaired intestinal epithelium brush border formation, junctional polarity and maturation. Mechanistically, TMIGD1 inhibits tumor cell proliferation, cell migration, arrests cell cycle at G2/M phase and induces expression of p21CIP1 (cyclin-dependent kinase inhibitor 1), and p27KIP1 (cyclin-dependent kinase inhibitor 1B) expression, key cell cycle inhibitor proteins involved in the regulation of the cell cycle. Moreover, we demonstrate that TMIGD1 is progressively downregulated in sporadic human CRC and correlates with poor overall survival. Our findings identify TMIGD1 as a novel tumor suppressor gene and provide insights into the pathogenesis of colorectal cancer and possibilities as a potential therapeutic target.

Emerging evidence in animal models of chronic kidney disease (CKD) implicates Aryl Hydrocarbon Receptor (AHR) signaling as a mediator of uremic toxicity. However, details about its tissue-specific and time-dependent activation in response to various renal pathologies remain poorly defined. Here, a comprehensive analysis of AHR induction was conducted in response to discrete models of kidney diseases using a transgenic mouse line expressing the AHR responsive-promoter tethered to a β-galactosidase reporter gene. Following validation using a canonical AHR ligand (a dioxin derivative), the transgenic mice were subjected to adenine-induced and ischemia/reperfusion-induced injury models representing CKD and acute kidney injury (AKI), respectively, in humans. Indoxyl sulfate was artificially increased in mice through the drinking water and by inhibiting its excretion into the urine. Adenine-fed mice showed a distinct and significant increase in β-galactosidase in the proximal and distal renal tubules, cardiac myocytes, hepatocytes, and microvasculature in the cerebral cortex. The pattern of β-galactosidase increase coincided with the changes in serum indoxyl sulfate levels. Machine-learning–based image quantification revealed positive correlations between indoxyl sulfate levels and β-galactosidase expression in various tissues. This pattern of β-galactosidase expression was recapitulated in the indoxyl sulfate–specific model. The ischemia/reperfusion injury model showed increase in β-galactosidase in renal tubules that persisted despite reduction in serum indoxyl sulfate and blood urea nitrogen levels. Thus, our results demonstrate a relationship between AHR activation in various tissues of mice with CKD or AKI and the levels of indoxyl sulfate. This study demonstrates the use of a reporter gene mouse to probe tissue-specific manifestations of uremia in translationally relevant animal models and provide hypothesis-generating insights into the mechanism of uremic toxicity that warrant further investigation.

Biomarkers are fundamental to basic and clinical research outcomes by reporting host responses and providing insight into disease pathophysiology. Measuring biomarkers with research-use ELISA kits is universal, yet lack of kit standardization and unexpected lot-to-lot variability presents analytic challenges for long-term projects. During an ongoing two-year project measuring plasma biomarkers in cancer patients, control concentrations for one biomarker (PF) decreased significantly after changes in ELISA kit lots. A comprehensive operations review pointed to standard curve shifts with the new kits, an analytic variable that jeopardized data already collected on hundreds of patient samples. After excluding other reasonable contributors to data variability, a computational solution was developed to provide a uniform platform for data analysis across multiple ELISA kit lots. The solution (ELISAtools) was developed within open-access R software in which variability between kits is treated as a batch effect. A defined best-fit Reference standard curve is modelled, a unique Shift factor “S” is calculated for every standard curve and data adjusted accordingly. The averaged S factors for PF ELISA kit lots #1-5 ranged from −0.086 to 0.735, and reduced control inter-assay variability from 62.4% to

The link between cancer and thrombosis, especially venous thromboembolism, is well established and thrombotic risk is exacerbated by cancer treatments, such as surgery and chemotherapy. This ongoing study aims to determine the impact of pre-analytic variables (PAVs) on thrombosis biomarkers in a diverse cancer patient and non-cancer subject population at an urban safety net hospital. Citrated blood from newly diagnosed, treatment-naïve patients of 11 cancer types or from non-cancer controls was processed to examine these variables: time to fractionation (2 and 4 hrs), plasma freeze-thaw cycles (2 and 3 cycles), and plasma delay to testing (24 or 72 hrs at 4oC). Regular processing (< 1 hour to centrifuge or 1 freeze-thaw cycle) served as the control sample for the variables. Current interim data presents biomarker data for D-dimer (DDE), Factor VIII activity (FVIII), soluble P-selectin (sP-Sel), prothrombin fragment 1+2 (F1+2), plasma DNA (DNA) and myeloperoxidase (MPO). Assays are performed in the hospital clinical lab (DDE, FVIII) or in our research lab following 30 detailed standard operating procedures (SOPs). Cancer patient demographics are 60% male, 40% Black/African-American, 38% Caucasian, 18% Hispanic, 3% Native American, and 1% Asian with an age range of 38-86 years. Non-cancer controls are 52% male, 59% Caucasian, 15% Black/African-American, 19% Asian, and 7% Hispanic with an age range of 23-64 years. Interim project data shows increased thrombosis biomarker levels in cancer subjects, except for sP-Sel and F1+2. Biomarker levels in cancer patients (n=9-52) were increased by approximately 300ng/ml DNA, 100ng/ml DDE, and 5ng/mL MPO with a trending increase in FVIII (~30%) when compared to non-cancer controls (n=17-22). Freeze-thaw of plasma had no effect, while a 2hr time to fractionation resulted in significantly increased MPO (~10ng/mL), FVIII (~14%) and F1+2 (~310 pg/mL). Delay to testing done for DNA and DDE showed with no apparent effect on biomarker levels after 24 or 72 hrs at 4oC. Current data show biomarker levels are impacted by presence of cancer rather than ethnicity of the patient. Donor recruitment is ongoing with shifting strategies to meet recruitment goals of non-cancer donors for greater diversity and older age to more closely reflect our cancer patient population. Rigorous control of sample handling and assay performance using SOPs that are compatible with a hospital setting contribute to identifying PAVs that matter for design of generalizable procedures. Citation Format: Morgan P. Thompson, Elizabeth R. Duffy, DJ Stearns-Kurosawa, Jasmin Bavarva, Shinichiro Kurosawa, Jiyoun Kim, Cheryl Spencer, Daniel Remick, Mark Sloan, Joel Henderson, Kerrie P. Nelson, Joseph Y. Tashjian, Yibing Wei, Rachana Agarwal, Michelle A. Berny-Lang, Chris Andry. Effect of preanalytic variables on established and emerging thrombosis-related biomarkers in an ethnically and racially diverse population of cancer patients and healthy subjects at a safety net hospital [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4529.