A MiR181/Sirtuin1 regulatory circuit modulates drug response in biliary cancers.

Despite recent advances, biliary tract cancer (BTC) remains one of the most lethal tumor worldwide due to late diagnosis, limited therapeutic strategies and resistance to conventional therapies. In recent years, high-throughput technologies have enabled extensive genome, and transcriptome sequencing unveiling, among others, the regulatory potential of microRNAs (miRNAs). Compelling evidence shown that miRNA are attractive therapeutic targets and promising candidates as biomarkers for various therapy-resistant tumors. The analysis of miRNA profile successfully identified miR-181c and -181d as significantly downregulated in BTC patients. Low miR-181c and -181d expression levels were correlated with worse prognosis and poor treatment efficacy. In fact, progression-free survival analysis indicated poor survival rates in miR-181c and -181d low expressing patients. The expression profile of miR-181c and -181d in BTC cell lines revealed that both miRNAs were dysregulated. Functional in vitro experiments in BTC cell lines showed that overexpression of miR-181c and -181d affected cell viability and increased sensitivity to chemotherapy compared to controls. In addition, by using bioinformatic tools we showed that the miR-181c/d functional role is determined by binding to their target SIRT1 (Sirtuin 1). Moreover, BTC patients expressing high levels of miR-181 and low SIRT1 shown an improved survival and treatment response. An integrative network analysis demonstrated that, miR-181/SIRT1 circuit had a regulatory effect on several important metabolic tumor-related processes. Our study demonstrated that miR-181c and -181d act as tumor suppressor miRNA in BTC, suggesting the potential use as therapeutic strategy in resistant cancers and as predictive biomarker in the precision medicine of BTC.

Combining ß-Carotene with 5-FU via Polymeric Nanoparticles as a Novel Therapeutic Strategy to Overcome uL3-Mediated Chemoresistance in p53-Deleted Colorectal Cancer Cells.

Colorectal cancer (CRC) is one of the leading causes of cancer-related death worldwide. Despite recent therapeutic advancements, resistance to 5-fluorouracil (5-FU) remains a major obstacle to the successful treatment of this disease. We have previously identified the ribosomal protein uL3 as a key player in the cell response to 5-FU, and loss of uL3 is associated with 5-FU chemoresistance. Natural products, like carotenoids, have shown the ability to enhance cancer cell response to drugs and may provide a safer choice to defeat chemoresistance in cancer. Transcriptome analysis of a cohort of 594 colorectal patients revealed a correlation between uL3 expression and both progression-free survival and response to treatment. RNA-Seq data from uL3-silenced CRC cells demonstrated that a low uL3 transcriptional state was associated with an increased expression of specific ATP-binding cassette (ABC) genes. Using two-dimensional (2D) and three-dimensional (3D) models of 5-FU-resistant CRC cells stably silenced for uL3, we investigated the effect of a novel therapeutic strategy by combining ß-carotene and 5-FU using nanoparticles (NPs) as a drug delivery system. Our results indicated that the combined treatment might overcome 5-FU chemoresistance, inducing cell cycle arrest in the G2/M phase and apoptosis. Furthermore, the combined treatment significantly reduced the expression levels of analyzed ABC genes. In conclusion, our findings suggest that ß-carotene combined with 5-FU may be a more effective therapeutic approach for treating CRC cells with low levels of uL3.

Targeting the MITF/APAF-1 axis as salvage therapy for MAPK inhibitors in resistant melanoma.

Melanoma is a deadly form of cancer characterized by remarkable therapy resistance. Analyzing the transcriptome of MAPK inhibitor sensitive- and resistant-melanoma, we discovered that APAF-1 is negatively regulated by MITF in resistant tumors. This study identifies the MITF/APAF-1 axis as a molecular driver of MAPK inhibitor resistance. A drug-repositioning screen identified quinacrine and methylbenzethonium as potent activators of apoptosis in a context that mimics drug resistance mediated by APAF-1 inactivation. The compounds showed anti-tumor activity in in vitro and in vivo models, linked to suppression of MITF function. Both drugs profoundly sensitize melanoma cells to MAPK inhibitors, regulating key signaling networks in melanoma, including the MITF/APAF-1 axis. Significant activity of the two compounds in inhibiting specific epigenetic modulators of MITF/APAF-1 expression, such as histone deacetylases, was observed. In summary, we demonstrate that targeting the MITF/APAF-1 axis may overcome resistance and could be exploited as a potential therapeutic approach to treat resistant melanoma.

Generation and Characterization of a Tumor Stromal Microenvironment and Analysis of Its Interplay with Breast Cancer Cells: An In Vitro Model to Study Breast Cancer-Associated Fibroblast Inactivation.

Breast cancer-associated fibroblasts (BCAFs), the most abundant non-cancer stromal cells of the breast tumor microenvironment (TME), dramatically sustain breast cancer (BC) progression by interacting with BC cells. BCAFs, as well as myofibroblasts, display an up regulation of activation and inflammation markers represented by α-smooth muscle actin (α-SMA) and cyclooxygenase 2 (COX-2). BCAF aggregates have been identified in the peripheral blood of metastatic BC patients. We generated an in vitro stromal model consisting of human primary BCAFs grown as monolayers or 3D cell aggregates, namely spheroids and reverted BCAFs, obtained from BCAF spheroids reverted to 2D cell adhesion growth after 216 h of 3D culture. We firstly evaluated the state of activation and inflammation and the mesenchymal status of the BCAF monolayers, BCAF spheroids and reverted BCAFs. Then, we analyzed the MCF-7 cell viability and migration following treatment with conditioned media from the different BCAF cultures. After 216 h of 3D culture, the BCAFs acquired an inactivated phenotype, associated with a significant reduction in α-SMA and COX-2 protein expression. The deactivation of the BCAF spheroids at 216 h was further confirmed by the cytostatic effect exerted by their conditioned medium on MCF-7 cells. Interestingly, the reverted BCAFs also retained a less activated phenotype as indicated by α-SMA protein expression reduction. Furthermore, the reverted BCAFs exhibited a reduced pro-tumor phenotype as indicated by the anti-migratory effect exerted by their conditioned medium on MCF-7 cells. The deactivation of BCAFs without drug treatment is possible and leads to a reduced capability of BCAFs to sustain BC progression in vitro. Consequently, this study could be a starting point to develop new therapeutic strategies targeting BCAFs and their interactions with cancer cells.

Integrated Genomics Identifies miR-181/TFAM Pathway as a Critical Driver of Drug Resistance in Melanoma.

MicroRNAs (miRNAs) are attractive therapeutic targets and promising candidates as molecular biomarkers for various therapy-resistant tumors. However, the association between miRNAs and drug resistance in melanoma remains to be elucidated. We used an integrative genomic analysis to comprehensively study the miRNA expression profiles of drug-resistant melanoma patients and cell lines. MicroRNA-181a and -181b (miR181a/b) were identified as the most significantly down-regulated miRNAs in resistant melanoma patients and cell lines. Re-establishment of miR-181a/b expression reverses the resistance of melanoma cells to the BRAF inhibitor dabrafenib. Introduction of miR-181 mimics markedly decreases the expression of TFAM in A375 melanoma cells resistant to BRAF inhibitors. Furthermore, melanoma growth was inhibited in A375 and M14 resistant melanoma cells transfected with miR-181a/b mimics, while miR-181a/b depletion enhanced resistance in sensitive cell lines. Collectively, our study demonstrated that miR-181a/b could reverse the resistance to BRAF inhibitors in dabrafenib resistant melanoma cell lines. In addition, miR-181a and -181b are strongly down-regulated in tumor samples from patients before and after the development of resistance to targeted therapies. Finally, melanoma tissues with high miR-181a and -181b expression presented favorable outcomes in terms of Progression Free Survival, suggesting that miR-181 is a clinically relevant candidate for therapeutic development or biomarker-based therapy selection.

Up-regulation of miR-34b/c by JNK and FOXO3 protects from liver fibrosis.

α1-Antitrypsin (AAT) deficiency is a common genetic disease presenting with lung and liver diseases. AAT deficiency results from pathogenic variants in the SERPINA1 gene encoding AAT and the common mutant Z allele of SERPINA1 encodes for Z α1-antitrypsin (ATZ), a protein forming hepatotoxic polymers retained in the endoplasmic reticulum of hepatocytes. PiZ mice express the human ATZ and are a valuable model to investigate the human liver disease of AAT deficiency. In this study, we investigated differential expression of microRNAs (miRNAs) between PiZ and control mice and found that miR-34b/c was up-regulated and its levels correlated with intrahepatic ATZ. Furthermore, in PiZ mouse livers, we found that Forkhead Box O3 (FOXO3) driving microRNA-34b/c (miR-34b/c) expression was activated and miR-34b/c expression was dependent upon c-Jun N-terminal kinase (JNK) phosphorylation on Ser574 Deletion of miR-34b/c in PiZ mice resulted in early development of liver fibrosis and increased signaling of platelet-derived growth factor (PDGF), a target of miR-34b/c. Activation of FOXO3 and increased miR-34c were confirmed in livers of humans with AAT deficiency. In addition, JNK-activated FOXO3 and miR-34b/c up-regulation were detected in several mouse models of liver fibrosis. This study reveals a pathway involved in liver fibrosis and potentially implicated in both genetic and acquired causes of hepatic fibrosis.

Down-regulation of hepatocyte nuclear factor-4α and defective zonation in livers expressing mutant Z α1-antitrypsin.

α1 -Antitrypsin (AAT) deficiency is one of the most common genetic disorders and the liver disease due to the Z mutant of AAT (ATZ) is a prototype of conformational disorder due to protein misfolding with consequent aberrant intermolecular protein aggregation. In the present study, we found that livers of PiZ transgenic mice expressing human ATZ have altered expression of a network of hepatocyte transcriptional factors, including hepatocyte nuclear factor-4α, that is early down-regulated and induces a transcriptional repression of ATZ expression. Reduced hepatocyte nuclear factor-4α was associated with activation of ß-catenin, which regulates liver zonation. Livers of PiZ mice and human patients with AAT deficiency were both found to have a severe perturbation of liver zonation. Functionally, PiZ mice showed a severe defect of ureagenesis, as shown by increased baseline ammonia, and reduced urea production and survival after an ammonia challenge. Down-regulation of hepatocyte nuclear factor-4α expression and defective zonation in livers have not been recognized so far as features of the liver disease caused by ATZ and are likely involved in metabolic disturbances and in the increased risk of hepatocellular carcinoma in patients with AAT deficiency. CONCLUSION: The findings of this study are consistent with the concept that abnormal AAT protein conformation and intrahepatic accumulation have broad effects on metabolic liver functions. (Hepatology 2017;66:124-135).

Recruitment of rural healthcare professionals for live continuing education.

INTRODUCTION: The availability of rural healthcare is a growing concern in the United States as fewer healthcare providers choose to work in rural areas. Accessing quality continuing education (CE) for rural healthcare practitioners (HCPs) remains a challenge and may pose a barrier to quality care. METHODS: To maximize attendance at a live, in-person, free CE program focusing on geriatric medication and issues specifically targeted to HCPs in rural areas, two methods were implemented sequentially. The first method used formal advertising implemented by a professional marketing service to promote CE events. The second method enlisted local healthcare organizations and physician groups to promote the CE event to their employees. Cost per attendee was calculated for comparison. RESULTS: Professional marketing services recruited 31 HCPs (March 2011) and resulted in a per-participant recruitment cost of US$428.62. Local healthcare organizations and physician groups' marketing recruited 48 HCPs (July-August 2011) and resulted in a per-participant recruitment cost of US$55.19. DISCUSSION: Providing free CE coordinated through local healthcare organizations and physician groups was the most cost-effective method of recruiting rural HCPs for CE. Formal advertising added cost without increasing the number of participants per event. Although this is the first study of the cost-effectiveness of recruitment methods targeting HCPs in rural areas, results are consistent with research on cost-effectiveness of outreach to rural lay community members.

Cost-effective strategies for rural community outreach, Hawaii, 2010-2011.

Three strategies designed to maximize attendance at educational sessions on chronic disease medication safety in older adults in rural areas were implemented sequentially and compared for cost-effectiveness: 1) existing community groups and events, 2) formal advertisement, and 3) employer-based outreach. Cost-effectiveness was measured by comparing overall cost per attendee recruited and number of attendees per event. The overall cost per attendee was substantially higher for the formal advertising strategy, which produced the lowest number of attendees per event. Leveraging existing community events and employers in rural areas was more cost-effective than formal advertisement for recruiting rural community members.

Preliminary findings concerning the use of prazosin for the treatment of posttraumatic nightmares in a refugee population.

Prazosin, a centrally active alpha-1 adrenergic receptor antagonist, has reduced nightmares and sleep disturbances in placebo-controlled studies involving patients with combat and civilian related posttraumatic stress disorder (PTSD). In this retrospective chart review, we analyzed data from 23 refugees diagnosed with chronic PTSD who were treated with prazosin. The recurrent distressing dreams item of the Clinician Administered PTSD Scale (CAPS) was used to quantify nightmare severity. A Clinical Global Impressions-Change (CGI-C) score assessed change in overall PTSD severity exclusive of nightmares. Using a paired-samples t-test, we found that CAPS scores decreased significantly (p <0.0005) from baseline after 8 weeks of treatment with a stable dose of prazosin. Overall PTSD severity was "markedly improved" in 6 patients, "moderately improved" in 11 patients, and "minimally improved" in 6 patients. These data provide preliminary support for the use of prazosin in targeting reduction of trauma-related nightmares and promoting improvement of global clinical status within an international sample of severely traumatized refugee patients.

Renal tract ultrasonography and calcium homeostasis in Williams-Beuren syndrome.

Renal ultrasound scan, circulating creatinine and calcium, and the urinary calcium excretion rate were investigated in 57 patients with clinically and genetically typical Williams-Beuren syndrome (25 female and 32 male subjects, aged from 1.0 year to 23 years, median 8.5 years) on regular follow up at our institution. Twenty-three unilateral abnormalities were detected in 20 patients: pelvic dilatation ( n=6), renal hypoplasia ( n=5), isolated renal cyst ( n=3), kidney surface irregularity ( n=3), kidney duplication ( n=2), renal agenesis ( n=1), megaureter ( n=1), pelvic kidney dystopia ( n=1), and renal stone ( n=1). Both infantile hypercalcemia and nephrocalcinosis was absent in the 57 patients. Mild hypercalcemia was noted in 1 and mild hypercalciuria in 2 patients after the 1st year of life. In conclusion, the study indicates the frequent occurrence of intrinsic renal tract abnormalities detected by ultrasonography in Williams-Beuren syndrome. However, the study does not confirm the importance given in the past to the occurrence of hypercalcemia and hypercalciuria.