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

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

CK2 phosphorylation of the PRH/Hex homeodomain functions as a reversible switch for DNA binding

The proline-rich homeodomain protein (PRH/Hex) regulates transcription by binding to specific DNA sequences and regulates mRNA transport by binding to translation initiation factor eIF4E. Protein kinase CK2 plays multiple roles in the regulation of gene expression and cell proliferation. Here, we show that PRH interacts with the β subunit of CK2 in vitro and in cells and that CK2 phosphorylates PRH. Phosphorylation of PRH by CK2 inhibits the DNA binding activity of this protein and dephosphorylation restores DNA binding indicating that this modification acts as a reversible switch. We show that phosphorylation of the homeodomain is sufficient to block DNA binding and we identify two amino acids within this the domain that are phosphorylated by CK2: S163 and S177. Site-directed mutagenesis demonstrates that mutation of either of these residues to glutamic acid partially mimics phosphorylation but is insufficient to completely block DNA binding whereas an S163E/S177E double mutation severely inhibits DNA binding. Significantly, the S163E and S177E mutations and the S163E/S177E double mutation all inhibit the ability of PRH to regulate transcription in cells. Since these amino acids are conserved between many homeodomain proteins, our results suggest that CK2 may regulate the activity of several homeodomain proteins in this manner

The regulation of cell growth by the proline rich homeodomain protein and its importance in human diseases

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

PRH/Hhex Controls Cell Survival through Coordinate Transcriptional Regulation of Vascular Endothelial Growth Factor Signaling▿

The proline-rich homeodomain protein (PRH) plays multiple roles in the control of gene expression during embryonic development and in the adult. Vascular endothelial growth factor (VEGF) is a mitogen that stimulates cell proliferation and survival via cell surface receptors including VEGFR-1 and VEGFR-2. VEGF signaling is of critical importance in angiogenesis and hematopoiesis and is elevated in many tumors. Here we show that PRH binds directly to the promoter regions of the Vegf, Vegfr-1, and Vegfr-2 genes and that in each case PRH represses transcription. We demonstrate that overexpression or knockdown of PRH directly impinges on the survival of both leukemic and tumor cells and that the modulation of VEGF and VEGF receptor signaling by PRH mediates these effects. Our findings demonstrate that PRH is a key regulator of the VEGF signaling pathway and describe a mechanism whereby PRH plays an important role in tumorigenesis and leukemogenesis

In situ subcellular fractionation of adherent and non-adherent mammalian cells

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, many proteins are present as a freely diffusing population in free exchange with a sub-population that is tightly associated with a particular subcellular domain or structure. In situ subcellular fractionation allows the visualization of protein compartmentalization and can also reveal protein sub-populations that localize to specific structures. For example, removal of soluble cytoplasmic proteins and loosely held nuclear proteins can reveal the stable association of some transcription factors with chromatin. Subsequent digestion of DNA can in some cases reveal association with the network of proteins and RNAs that is collectively termed the nuclear scaffold or nuclear matrix. Here we describe the steps required during the in situ fractionation of adherent and non-adherent mammalian cells on microscope coverslips. Protein visualization can be achieved using specific antibodies or fluorescent fusion proteins and fluorescence microscopy. Antibodies and/or fluorescent dyes that act as markers for specific compartments or structures allow protein localization to be mapped in detail. In situ fractionation can also be combined with western blotting to compare the amounts of protein present in each fraction. This simple biochemical approach can reveal associations that would otherwise remain undetected

Correction: Lertsuwan et al. CX-4945 Induces Methuosis in Cholangiocarcinoma Cell Lines by a CK2-Independent Mechanism. Cancers 2018, 10, 283

Authors would like to make a correction to the previous article [...

Correction: Lertsuwan et al. CX-4945 Induces Methuosis in Cholangiocarcinoma Cell Lines by a CK2-Independent Mechanism. <i>Cancers</i> 2018, <i>10</i>, 283

Authors would like to make a correction to the previous article [...