The role of the proline rich homeodomain transcription factor (PRH/Hhex) in cholangiocarcinoma

Cholangiocarcinoma (CCA) is cancer of the bile duct that only has a few treatment options, typically with poor prognostic outcomes. The Proline-Rich Homeodomain protein (PRH), also known as haematopoietically expressed homeobox (Hhex), is a transcription factor that regulates liver and bile duct development. PRH is not expressed in normal bile duct cells but this protein is highly expressed in CCA cells. Using a combination of overexpression and knockdown experiments, PRH was shown to increase cell proliferation, migration and invasion in tumour cell lines. Similar results were obtained in immortalized AKN-1 cells and also in primary biliary epithelial cells. In these cells PRH overexpression also led to the initiation of epithelial to mesenchymal transition (EMT) and anoikis resistance as well as increased cell proliferation and invasion. Gene Ontology and Gene Set Enrichment Analysis of RNA-sequencing data from cells with altered PRH levels reveal that in all cell types PRH regulates multiple signaling pathways and that key genes involved in the Notch signaling pathway, the cell cycle and EMT are regulated by PRH. CCA cells with elevated PRH expression were found to be more sensitive to the CDK4/6 inhibitor Palbociclib as PRH controls the cell cycle through regulation of multiple cell cycle genes including CCND2. Drug screening with a drug repurposing library was performed to identify novel drug treatments for CCA and several repurposed drugs that show a stronger cytotoxic effect when combined with Palbociclib were identified using cell viability assays. This thesis therefore reveals that PRH protein is an important oncoprotein in CCA and that this protein may play a role in initiating CCA and in driving cancer progression and identifies new drug treatment options

Cerebral Radionecrosis with Cystic Degeneration Following Radiotherapy for Nasal Cavity Squamous Cell Carcinoma: A Case Report

A 31-year-old man with nasal cavity squamous cell carcinoma was treated in our hospital with two courses of radiotherapy (120 Gy total dose) followed by surgical tumor resection. Three years after the last irradiation, he developed seizures as well as changes in behavior and consciousness. Medical therapy with diphenylhydantoin (Dilantin(r)) terminated the seizures. Dysphagia, unsteady gait, and right-side limb weakness developed 37 months after the onset of seizures. Magnetic resonance imaging showed a large, cystic mass in the left temporal lobe with left to right midline shift. Following craniotomy with decompression of the cystic mass, the patient improved clinically. No malignant cells were found in the specimen. No further progression of neurologic symptoms was noted after a 1-year follow-up. Cerebral radionecrosis is an uncommon late complication of radiotherapy and needs to be differentiated from tumor recurrence or metastasis if the irradiation field covers the cerebral region in patients with head and neck malignancies

CX-4945 Induces Methuosis in Cholangiocarcinoma Cell Lines by a CK2-Independent Mechanism

Cholangiocarcinoma is a disease with a poor prognosis and increasing incidence and hence there is a pressing unmet clinical need for new adjuvant treatments. Protein kinase CK2 (previously casein kinase II) is a ubiquitously expressed protein kinase that is up-regulated in multiple cancer cell types. The inhibition of CK2 activity using CX-4945 (Silmitasertib) has been proposed as a novel treatment in multiple disease settings including cholangiocarcinoma. Here, we show that CX-4945 inhibited the proliferation of cholangiocarcinoma cell lines in vitro. Moreover, CX-4945 treatment induced the formation of cytosolic vacuoles in cholangiocarcinoma cell lines and other cancer cell lines. The vacuoles contained extracellular fluid and had neutral pH, features characteristic of methuosis. In contrast, simultaneous knockdown of both the α and α′ catalytic subunits of protein kinase CK2 using small interfering RNA (siRNA) had little or no effect on the proliferation of cholangiocarcinoma cell lines and failed to induce the vacuole formation. Surprisingly, low doses of CX-4945 increased the invasive properties of cholangiocarcinoma cells due to an upregulation of matrix metallopeptidase 7 (MMP-7), while the knockdown of CK2 inhibited cell invasion. Our data suggest that CX-4945 inhibits cell proliferation and induces cell death via CK2-independent pathways. Moreover, the increase in cell invasion brought about by CX-4945 treatment suggests that this drug might increase tumor invasion in clinical settings

Incident heart failure and myocardial infarction in sodium-glucose cotransporter-2 vs. dipeptidyl peptidase-4 inhibitor users

Aims This study aimed to compare the rates of major cardiovascular adverse events in sodium-glucose cotransporter-2 inhibitors (SGLT2I) and dipeptidyl peptidase-4 inhibitors (DPP4I) users in a Chinese population. SGLT2I and DPP4I are increasingly prescribed for type 2 diabetes mellitus patients. However, few population-based studies are comparing their effects on incident heart failure or myocardial infarction. Methods and results This was a population-based retrospective cohort study using the electronic health record database in Hong Kong, including type 2 diabetes mellitus patients receiving either SGLT2I or DPP4I from 1 January 2015 to 31 December 2020. Propensity score matching was performed in a 1:1 ratio based on demographics, past comorbidities, and non-SGLT2I/DPP4I medications with nearest neighbour matching (caliper = 0.1). Univariable and multivariable Cox models were used to identify significant predictors for new-onset heart failure, new-onset myocardial infarction, cardiovascular mortality, and all-cause mortality. Sensitivity analyses with competing risk models and multiple propensity score matching approaches were conducted. A total of 41 994 patients (58.89% males, median admission age at 58 years old, interquartile range [IQR]: 51.2–65.3) were included with a median follow-up of 5.6 years (IQR: 5.32–5.82). In the matched cohort, SGLT2I use was significantly associated with lower risks of new-onset heart failure (hazard ratio [HR]: 0.73, 95% confidence interval [CI]: [0.66, 0.81], P < 0.0001), myocardial infarction (HR: 0.81, 95% CI: [0.73, 0.90], P < 0.0001), cardiovascular mortality (HR: 0.67, 95% CI: [0.53, 0.84], P < 0.001), and all-cause mortality (HR: 0.26, 95% CI: [0.24, 0.29], P < 0.0001) after adjusting for significant demographics, past comorbidities, and non-SGLT2I/DPP4I medications. Conclusions SGLT2 inhibitors are protective against adverse cardiovascular events including new-onset heart failure, myocardial infarction, cardiovascular mortality, and all-cause mortality. The prescription of SGLT2I is preferred when taken into consideration individual cardiovascular and metabolic risk profiles in addition to drug–drug interactions

High visit-to-visit cholesterol variability predicts heart failure and adverse cardiovascular events: a population-based cohort study

Dyslipidaemia is associated with elevated cardiovascular risks, with the INTERHEART study observing a tripling of myocardial infarction (MI) risk in patients with dyslipidaemia.1 Most studies focused on mean levels or point estimates of low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C), despite well-known visit-to-visit variability.2 Visit-to-visit cholesterol variability, reflecting fluctuations in cholesterol levels between visits, is prognostic for some adverse cardiovascular outcomes such as cardiac arrhythmias and mortality.3,4 Nonetheless, associations between cholesterol variability and heart failure (HF) remain unclear. This study therefore investigated the associations between LDL-C and HDL-C variabilities and the risk of new-onset HF and major adverse cardiovascular outcomes

Primordial Germ Cell Specification from Embryonic Stem Cells

Background: Primordial germ cell (PGC) specification is the first crucial step in germ line development. However, owing to significant challenges regarding the in vivo system, such as the complex cellular environment and potential problems with embryo manipulation, it is desirable to generate embryonic stem (ES) cells that are capable of overcoming these aforementioned limitations in order to provide a potential in vitro model to recapitulate the developmental processes in vivo. Methodology and Principal Findings: Here, we studied the detailed process of PGC specification from stella-GFP ES cells. We first observed the heterogeneous expression of stella in ES cells. However, neither Stella-positive ES cells nor Stellanegative ES cells shared a similar gene expression pattern with either PGCs or PGC precursors. Second, we derived PGCs from ES cells using two differentiation methods, namely the attachment culture technique and the embryoid body (EB) method. Compared with PGCs derived via the attachment culture technique, PGCs derived via the EB method that had undergone the sequential erasure of Peg3 followed by Igf2r resulted in a cell line in which the expression dynamics of T, Fgf8 and Sox17, in addition to the expression of the epiblast markers, were more similar to the in vivo expression, thus demonstrating that the process of PGC derivation was more faithfully recapitulated using the EB method. Furthermore, we developed an in vitro model of PGC specification in a completely chemically defined medium (CDM) that indicated that BMP4 and Wnt3a promoted PGC derivation, whereas BMP8b and activinA had no observable effect on PGC derivation

A runaway PRH/HHEX-Notch3 positive feedback loop drives cholangiocarcinoma and determines response to CDK4/6 inhibition

Aberrant Notch and Wnt signalling are known drivers of cholangiocarcinoma (CCA) but the underlying factors that initiate and maintain these pathways are not known. Here we show that the PRH/HHEX transcription factor forms a positive transcriptional feedback loop with Notch3 that is critical in CCA. PRH/HHEX expression was elevated in CCA and depletion of PRH reduced CCA tumour growth in a xenograft model. Overexpression of PRH in primary human biliary epithelial cells was sufficient to increase cell proliferation and produce an invasive phenotype. Interrogation of the gene networks regulated by PRH and Notch3 revealed that unlike Notch3, PRH directly activated canonical Wnt signalling. These data indicate that hyperactivation of Notch and Wnt signalling is independent of the underlying mutational landscape and has a common origin in dysregulation of PRH. Moreover, they suggest new therapeutic options based on the dependence of specific Wnt, Notch, and CDK4/6 inhibitors on PRH activity

The Structure of Tumor Endothelial Marker 8 (TEM8) Extracellular Domain and Implications for Its Receptor Function for Recognizing Anthrax Toxin

Anthrax toxin, which is released from the Gram-positive bacterium Bacillus anthracis, is composed of three proteins: protective antigen (PA), lethal factor (LF), and edema factor (EF). PA binds a receptor on the surface of the target cell and further assembles into a homo-heptameric pore through which EF and LF translocate into the cytosol. Two distinct cellular receptors for anthrax toxin, TEM8/ANTXR1 and CMG2/ANTXR2, have been identified, and it is known that their extracellular domains bind PA with low and high affinities, respectively. Here, we report the crystal structure of the TEM8 extracellular vWA domain at 1.7 Å resolution. The overall structure has a typical integrin fold and is similar to that of the previously published CMG2 structure. In addition, using structure-based mutagenesis, we demonstrate that the putative interface region of TEM8 with PA (consisting of residues 56, 57, and 154–160) is responsible for the PA-binding affinity differences between the two receptors. In particular, Leu56 was shown to be a key factor for the lower affinity of TEM8 towards PA compared with CMG2. Because of its high affinity for PA and low expression in normal tissues, an isolated extracellular vWA domain of the L56A TEM8 variant may serve as a potent antitoxin and a potential therapeutic treatment for anthrax infection. Moreover, as TEM8 is often over-expressed in tumor cells, our TEM8 crystal structure may provide new insights into how to design PA mutants that preferentially target tumor cells

Whole genome sequencing in paediatric channelopathy and cardiomyopathy

BackgroundPrecision medicine in paediatric cardiac channelopathy and cardiomyopathy has a rapid advancement over the past years. Compared to conventional gene panel and exome-based testing, whole genome sequencing (WGS) offers additional coverage at the promoter, intronic regions and the mitochondrial genome. However, the data on use of WGS to evaluate the genetic cause of these cardiovascular conditions in children and adolescents are limited.MethodsIn a tertiary paediatric cardiology center, we recruited all patients diagnosed with cardiac channelopathy and cardiomyopathy between the ages of 0 and 18 years old, who had negative genetic findings with prior gene panel or exome-based testing. After genetic counselling, blood samples were collected from the subjects and both their parents for WGS analysis.ResultsA total of 31 patients (11 cardiac channelopathy and 20 cardiomyopathy) were recruited. Four intronic splice-site variants were identified in three cardiomyopathy patients, which were not identified in previous whole exome sequencing. These included a pathogenic variant in TAFAZZIN:c.284+5G&gt;A (Barth syndrome), a variant of unknown significance (VUS) in MYBPC3:c.1224-80G&gt;A and 2 compound heterozygous LP variants in LZTR1 (LZTR1:c.1943-256C&gt;T and LZTR1:c1261-3C&gt;G) in a patient with clinical features of RASopathy. There was an additional diagnostic yield of 1.94% using WGS for identification of intronic variants, on top of conventional gene testing.ConclusionWGS plays a role in identifying additional intronic splice-site variants in paediatric patients with isolated cardiomyopathy. With the demonstrated low extra yield of WGS albeit its ability to provide potential clinically important information, WGS should be considered in selected paediatric cases of cardiac channelopathy and cardiomyopathy in a cost-effective manner