Gainullin, Vladimir G.

Dent disease 1, an X‐linked inherited kidney disease caused by mutations in the CLCN5 gene, is characterized by low molecular weight proteinuria (LMWP), hypercalciuria, nephrocalcinosis, nephrolithiasis, rickets, and progressive renal failure. CLCN5 encodes the CLC‐5 protein found at the apical membrane in the kidney proximal tubule as well as endosome membranes of other tubule segments. Approximately 80 human mutations of the CLCN5 gene have been identified consisting of nonsense, missense, splice site, insertion and deletion mutations. However, the genotype and phenotype correlation are poor, and the mechanisms of CLC‐5 dysfunction that cause disease are still unclear. Moreover, there has been a lack of systematic study of CLC‐5 mutational effects on Cl − /H + exchanger activity. To address this need we used ion selective microelectrodes to monitor intracellular pH (pH i ) and intracellular Cl − concentration ([Cl i ]) of Xenopus oocytes expressing WT and mutant CLC‐5 while clamping at +40 mV. Common and novel mutations were identified in Dent patients by collaborators in the Rare Kidney Stone Consortium. The apparent H + :Cl − coupling ratio through the CLC‐5 protein was calculated using the Gibbs‐free energy equation. Cellular localization and protein trafficking of WT and mutant CLC‐5 in renal epithelial cells were also examined by immunofluorescence microscopy using eGFP/HA double‐tagged CLC‐5 and organelle‐specific markers and quantified by flow cytometry. In the presence of 104 mM Cl − (ND96), the pH i and [Cl − ] i of the CLC‐5 WT increased 340 ± 59 ×10 −4 pH unit/min and 2.8 ± 0.5 mM/min, respectively. T657S increased at a similar rate as WT, while there was a significant decrease in the rate of pH i and [Cl − ] i change for S244L, R345W, and Q629X compared to WT. Decreasing extracellular chloride to 5 mM decreased WT ClC‐5 pH i and [Cl − ] i by reversing transport: −7 ± 65 ×10 −4 pH unit/min and −0.2 ± 0.2 mM/min, respectively. Using these measurements, the stoichiometry of WT CLC‐5 was calculated to be 1.80 ± 0.02 (nCl − :H + ) in ND96, similar to the stoichiometry estimated for T657S. However, the stoichiometry was significantly altered for S244L, R345W, and Q629X. WT ClC‐5 acts as Cl − /H + exchanger at +40mV with 104 mM Cl − in the extracellular solution. Lowering extracellular Cl − reverses the transport direction, and the ClC‐5 exchanger functions at a significantly slower rate. Although S244L has normal surface expression, S244L and R345W mutations have significantly decreased pH and Cl − exchange rates in response to solution manipulations, suggesting these mutations have an altered ion exchange coupling ratio (stoichiometry). Interestingly, while R345W was retained in the ER suggesting defective protein maturation, R345W surface localization was rescued by lowering the incubation temperature from 37 to 30°C. Q629X was retained in the ER, not localized in early endosomes, and also has abolished Cl − /H + exchange activity, suggesting that Q629X affects protein maturation and trafficking while T657S is a benign, non‐disease causing mutation. These data suggest studying CLC‐5 mutational effects on both transport function and cellular localization is essential to better understand divergent phenotypes in Dent disease. Support or Funding Information NIH DK101405, U54KD083908, U54DK100227, Mayo O'Brien Urology Research Center, Mayo Clinic Rare Kidney Stone Consortium

Aberrant activation of the Hedgehog (Hh) signaling pathway, through which the GLI family of transcription factors (TF) is stimulated, is commonly observed in cancer cells. One well-established mechanism of this increased activity is through the inactivation of Suppressor of Fused (SUFU), a negative regulator of the Hh pathway. Relief from negative regulation by SUFU facilitates GLI activity and induction of target gene expression. Here, we demonstrate a novel role for SUFU as a promoter of GLI activity in pancreatic ductal adenocarcinoma (PDAC). In non-ciliated PDAC cells unresponsive to Smoothened agonism, SUFU overexpression increases GLI transcriptional activity. Conversely, knockdown (KD) of SUFU reduces the activity of GLI in PDAC cells. Through array PCR analysis of GLI target genes, we identified B-cell lymphoma 2 (BCL2) among the top candidates down-regulated by SUFU KD. We demonstrate that SUFU KD results in reduced PDAC cell viability, and overexpression of BCL2 partially rescues the effect of reduced cell viability by SUFU KD. Further analysis using as a model GLI1, a major TF activator of the GLI family in PDAC cells, shows the interaction of SUFU and GLI1 in the nucleus through previously characterized domains. Chromatin immunoprecipitation (ChIP) assay shows the binding of both SUFU and GLI1 at the promoter of BCL2 in PDAC cells. Finally, we demonstrate that SUFU promotes GLI1 activity without affecting its protein stability. Through our findings, we propose a novel role of SUFU as a positive regulator of GLI1 in PDAC, adding a new mechanism of Hh/GLI signaling pathway regulation in cancer cells.

10526 Background: Cancer screening provides a straightforward diagnostic path compared to non-screening, which often requires complex evaluation for patients with non-specific symptoms. The time to diagnosis (from clinical presentation or screening event to definitive diagnosis) captures the clinical efficiency of the journey. The study characterizes time to diagnosis for cancers with and without USPSTF Grade A/B screening recommendations. This could inform the value of screening in diagnostic management and care efficiency improvements. Methods: The Geisinger Health System Phenomics Initiative Database provides deidentified EHR and tumor registry data. Patients aged 50-74 with encounters at least biennially for six years prior to a cancer diagnosis were frequency-matched to a non-cancer control group by diagnosis year. From 18 months prior to diagnosis, equally-sized random samples were drawn (with replacement) from a time-bounded sliding window at monthly intervals. Aggregated procedure and diagnostic code counts for cancer and non-cancer samples were used to calculate the Bray-Curtis dissimilarity (BCd) statistic. The start of the diagnostic path was defined as the earliest date with a monotonically-increasing statistically-significant difference in BCd (95% CI), and was stratified into cancers with and without screening and by stage. Results: 8188 new cancer cases were identified between 2010-2019; 40% had an established screening modality (breast, lung, colorectal, cervical), 55% of which were screened prior to diagnosis. The time to diagnosis for non-screenable cancers was 6 months versus 3 months for screenable cancers. The BCd for procedures differed by 0.029 (95% CI: 0.021-0.037) between the two groups at 6 months prior to diagnosis. Stages I and IV had a time to diagnosis of 3 months across all cancers versus 7 and 6 months for stages II and III, respectively. Conclusions: We report the first known quantification of time to diagnosis across multiple cancers. Screenable cancers were associated with shorter time to diagnosis than non-screenable cancers. Stage II and III cancers took longer to diagnose than stage I and IV, likely related to early detection by screening in the former and severe symptom presentation in the latter. These novel data highlight improved time to diagnosis as another achievable benefit from expanded, multi-cancer screening.

ABSTRACT Approximately 15% of autosomal dominant polycystic kidney disease (ADPKD) is caused by variants in PKD2. PKD2 encodes polycystin-2, which forms an ion channel in primary cilia and endoplasmic reticulum (ER) membranes of renal collecting duct cells. Elevated internal Ca2+ modulates polycystin-2 voltage-dependent gating and subsequent desensitization – two biophysical regulatory mechanisms that control its function at physiological membrane potentials. Here, we refute the hypothesis that Ca2+ occupancy of the polycystin-2 intracellular EF hand is responsible for these forms of channel regulation, and, if disrupted, results in ADPKD. We identify and introduce mutations that attenuate Ca2+-EF hand affinity but find channel function is unaltered in the primary cilia and ER membranes. We generated two new mouse strains that harbor distinct mutations that abolish Ca2+-EF hand association but do not result in a PKD phenotype. Our findings suggest that additional Ca2+-binding sites within polycystin-2 or Ca2+-dependent modifiers are responsible for regulating channel activity.

Abstract Reflux-induced injury promotes esophageal adenocarcinoma, one of the most rapidly increasing, highly lethal cancers in Western countries. Here, we investigate the efficacy of a combinatorial chemoprevention strategy for esophageal adenocarcinoma and characterize the underlying molecular mechanisms. Specifically, our approach involves the use of ursodeoxycholic acid (Urso) due to its ability to decrease injury-inducing bile salts in combination with Aspirin to mitigate the consequences of injury. We find that Urso-Aspirin combination reduces the risk of adenocarcinoma in vivo in animals with reflux, decreases the proliferation of esophageal adenocarcinoma cells, and downregulates a key cell cycle regulator, CDK2. Mechanistically, using cell growth, luciferase reporter, expression, and chromatin immunoprecipitation assays, we identify GLI1, a Hedgehog-regulated transcription factor, as a novel target of Urso-Aspirin combination. We show that GLI1 is upregulated during esophageal carcinogenesis, and GLI1 can bind to the CDK2 promoter and activate its expression. Although the Urso-Aspirin combination downregulates GLI1, the GLI1 overexpression not only abrogates the effect of this combination on proliferation but it also restores CDK-2 expression. These findings support that the chemopreventive effect of the Urso-Aspirin combination occurs, at least in part, through a novel GLI1-CDK2–dependent mechanism. To further understand the regulation of CDK2 by GLI1, both pharmacologic and RNAi-mediated approaches show that GLI1 is a transcriptional activator of CDK2, and this regulation occurs independent of Smoothened, the central transducer of the Hedgehog canonical pathway. Collectively, these results identify a novel GLI1-to-CDK2 pathway in esophageal carcinogenesis, which is a bona fide target for effective combinatorial chemoprevention with Urso and Aspirin. Cancer Res; 70(17); 6787–96. ©2010 AACR.