HECT (Homologous to the E6-AP Carboxyl Terminus)-Type Ubiquitin E3 Ligase ITCH Attenuates Cardiac Hypertrophy by Suppressing the Wnt/β-Catenin Signaling Pathway

The HECT (homologous to the E6-AP carboxyl terminus)-type ubiquitin E3 ligase ITCH is an enzyme that plays an important role in ubiquitin-proteasomal protein degradation. Disheveled proteins (Dvl1 [disheveled protein 1], Dvl2, and Dvl3) are the main components of the Wnt/β-catenin signaling pathway, which is involved in cardiac hypertrophy. The aim of this study was to examine the role of ITCH during development of cardiac hypertrophy. Thoracic transverse aortic constriction (TAC) was performed in transgenic mice with cardiac-specific overexpression of ITCH (ITCH-Tg) and wild-type mice. Cardiac hypertrophy after TAC was attenuated in ITCH-Tg mice, and the survival rate was higher for ITCH-Tg mice than for wild-type mice. Protein interaction between ITCH and Dvls was confirmed with immunoprecipitation in vivo and in vitro. Expression of key molecules of the Wnt/β-catenin signaling pathway (Dvl1, Dvl2, GSK3β [glycogen synthase kinase 3β], and β-catenin) was inhibited in ITCH-Tg mice compared with wild-type mice. Notably, the ubiquitination level of Dvl proteins increased in ITCH-Tg mice. Protein and mRNA expression levels of ITCH increased in response to Wnt3a stimulation in neonatal rat cardiomyocytes. Knockdown of ITCH using small-interfering RNA increased cardiomyocyte size and augmented protein expression levels of Dvl proteins, phospho-GSK3β, and β-catenin after Wnt3a stimulation in cardiomyocytes. Conversely, overexpression of ITCH attenuated cardiomyocyte hypertrophy and decreased protein expression levels of Dvl proteins, phospho-GSK3β and β-catenin. In conclusion, ITCH targets Dvl proteins for ubiquitin-proteasome degradation in cardiomyocytes and attenuates cardiac hypertrophy by suppressing the Wnt/β-catenin signaling pathway.

MicroRNA-135a-induced formation of CD133+ subpopulation with cancer stem cell properties in cervical cancer

Cancer stem cells (CSCs) play significant roles in tumor initiation. MicroRNA-135a (miR-135a) induced the formation of a CD133+ subpopulation from a human papillomavirus-immortalized cervical epithelial cell line. Compared with the CD133− cells, the CD133+ cells expressed higher levels of miR-135a and OCT4, exhibited significantly higher tumorsphere forming capacity and the time required for tumorsphere formation was shortened in the second generation. Serum induction suppressed the expression of CD133, OCT4 and miR-135a, but increased expression of involucrin in the miR-135a-induced CD133+ cells. The miR-135a-induced CD133+ cells were tumorigenic in a limiting dilution approach in vivo. The cells expressed significantly higher level of active β-catenin and OCT4 than the CD133− counterpart. Wnt3a enhanced the expression of OCT4 and CD133 in cervical cancer cells but failed to enhance CD133 transcription in normal cervical cells. Wnt3a stimulation also increased tumorsphere size and self-renewal of miR-135a-induced CD133+ subpopulation. Wnt/β-catenin inhibition suppressed tumorsphere formation while Wnt3a partially nullified the inhibitory effect. Taken together, miR-135a induced the formation of a subpopulation of cells with CSC properties both in vitro and in vivo and the Wnt/β-catenin signaling pathway is essential to maintain its tumorigenicity.

Targeting Wnt Signaling To Overcome PARP Inhibitor Resistance

ABSTRACTEpithelial ovarian cancer (EOC) has one of the highest deaths to incidence ratios. High grade serous ovarian carcinoma (HGSOC) is the most common and deadliest EOC histotype because of the lack of secondary therapeutic options following debulking surgery and platinum/taxane-based chemotherapies. For recurrent chemosensitive HGSOC, poly(ADP)-ribose polymerase inhibitors (PARPi; olaparib, rucaparib, or niraparib) represent an emerging treatment strategy. While PARPi are most effective in homologous recombination DNA repair-deficient (HRD) HGSOCs, more recent studies have observed a significant clinical benefit in non-HRD HGSOCs. However, all HGSOC patients are likely to acquire resistance to PARPi. Therefore, there is an urgent clinical need to better understand PARPi resistance, and to introduce novel combinatorial therapies to overcome PARPi resistance and extend HGSOC disease-free intervals. Utilizing a two BRCA2-mutated and one BRCA-wildtype HGSOC cell lines that are olaparib sensitive, we established resistant cells. Transcriptome analysis of the matched olaparib-sensitive versus resistant cells did not detect BRCA2 reversion mutations, but revealed activation of Wnt/TCF signaling pathway, as TCF transcriptional activity was significantly increased in PARPi-resistant cells. In parallel, forced activation of Wnt signaling in PARPi-sensitive cells via WNT3A stimulation reduced response to PARPi. In a recurrent-HGSOC PARPi insensitive patient-derived xenograft model there was an increase in a Wnt/TCF transcriptional target. PARPi resistant cells were sensitive to inhibition of Wnt signaling using the FDA-approved compound, pyrvinium pamoate, which has been shown to inhibit Wnt signaling. We observed that combining pyrvinium pamoate with olaparib resulted in a significant decrease in tumor burden and number of tumor nodules. This study demonstrates that Wnt signaling can mediate PARPi resistance in HGSOC and provides a clinical rationale for combining PARPi and Wnt inhibitors.

Myc-interacting zinc-finger protein 1 positively regulates Wnt signalling by protecting Dishevelled from Dapper1-mediated degradation

Wnt signalling regulates embryonic development and tissue homoeostasis by modulating cell proliferation, differentiation and migration. Dapper1 (Dpr1) has been shown to be an important key negative regulator of Wnt signalling by promoting Dishevelled (Dvl) degradation. In the present study, we found that Myc-interacting zinc-finger protein 1 (MIZ1) interacts with Dpr1 and this interaction attenuates the ability of Dpr1 to induce Dvl2 degradation, thus enhancing Wnt signalling. Mechanistically, MIZ1 is translocated from the nucleus to the cytoplasm upon Wnt3a stimulation or overexpression of Dpr1 and Dvl2, disrupting the interaction between Dpr1 and Dvl2. Furthermore, MIZ1 can promote the proliferation of breast cancer MDA-MB-231 and BT-549 cells through Wnt signalling and reverse the anti-proliferative effect of Dpr1 on colorectal cancer Caco-2. Together, our findings establish a novel layer of Wnt signalling regulation via the MIZ1–Dpr1–Dvl axis.

Clathrin and AP2 are required for PtdIns(4,5)P2-mediated formation of LRP6 signalosomes

Canonical Wnt signaling is initiated by the binding of Wnt proteins to their receptors, low-density lipoprotein-related protein 5 and 6 (LRP5/6) and frizzled proteins, leading to phosphatidylinositol (4,5)bisphosphate (PtdIns(4,5)P2) production, signalosome formation, and LRP phosphorylation. However, the mechanism by which PtdIns(4,5)P2 regulates the signalosome formation remains unclear. Here we show that clathrin and adaptor protein 2 (AP2) were part of the LRP6 signalosomes. The presence of clathrin and AP2 in the LRP6 signalosomes depended on PtdIns(4,5)P2, and both clathrin and AP2 were required for the formation of LRP6 signalosomes. In addition, WNT3A-induced LRP6 signalosomes were primarily localized at cell surfaces, and WNT3A did not induce marked LRP6 internalization. However, rapid PtdIns(4,5)P2 hydrolysis induced artificially after WNT3A stimulation could lead to marked LRP6 internalization. Moreover, we observed WNT3A-induced LRP6 and clathrin clustering at cell surfaces using super-resolution fluorescence microscopy. Therefore, we conclude that PtdIns(4,5)P2 promotes the assembly of LRP6 signalosomes via the recruitment of AP2 and clathrin and that LRP6 internalization may not be a prerequisite for Wnt signaling to β-catenin stabilization.

NANOG induction of fetal liver kinase-1 (FLK1) transcription regulates endothelial cell proliferation and angiogenesis

NANOG is a master transcription factor associated with the maintenance of stem cell pluripotency. Here, we demonstrate that transcription factor NANOG is expressed in cultured endothelial cells (ECs) and in a subset of tumor cell lines. Importantly, we provide evidence that WNT3A stimulation of ECs induces the transcription of NANOG which mediates the expression of vascular endothelial growth factor receptor-2, also known as fetal liver kinase-1 (FLK1). We defined ATTA as a minimal binding site for NANOG. Accordingly, a luciferase reporter assay showed that NANOG binds to and activates 4 ATTA binding sites identified in the FLK1 promoter after WNT3A stimulation. Consistent with this data, we found that, under basal conditions and in response to WNT3A stimulation, NANOG binding to these ATTA sequences markedly induced the expression of FLK1. Thus, our data indicate an essential role in angiogenesis for NANOG binding to these 4 ATTA sites. Surprisingly, NANOG depletion not only decreased FLK1 expression but also reduced cell proliferation and angiogenesis. These findings show the necessary and sufficient role of NANOG in inducing the transcription of FLK1 to regulate the angiogenic phenotypes of ECs.