Biology subjects

De Benedetti, A.

Publications and source records attributed to De Benedetti, A..

4 recordsLinked to original sources

bioRxiv2023

NEK1-Mediated Phosphorylation of YAP1 is key to Prostate Cancer Progression

Understanding how Androgen-dependent PCa cells progress to independence and modify accordingly their transcriptional repertoire is the key to preventing mCRPC progression. We recently identified a novel axis of the Hippo pathway characterized by the sequential kinase cascade induced by androgen deprivation: AR->TLK1B>NEK1>pYAP1-Y407 leading to CRPC adaptation. Phosphorylation of YAP-Y407 increases upon ADT or induction of DNA damage, correlated with the known increase in NEK1 expression/activity, and this is suppressed in the Y407F mutant. Dominant expression of YAP1-Y407F in Hek293 cells reprograms the YAP1-mediated transcriptome to reduced TEAD- and P73-regulated gene expression and mediates sensitivity to MMC. NEK1 haploinsufficient TRAMP mice display reduced YAP1 expression and if castrated fail to progress to overt prostate carcinomas, even while displaying reduced E-CAD expression in hyperplastic ductules. YAP1 overexpression, but not the Y407F mutant, transforms LNCaP cells to androgen independent growth and a mesenchymal morphology. Immunohistochemical examination of Prostate Cancer biopsies revealed that pYAP1-Y407 nuclear signal is low in samples of low-grade cancer but elevated in high GS specimens. We also found that J54, pharmacological inhibitor of the TLK1>NEK1>YAP1 nexus, leading to degradation of YAP1 can suppress the transcriptional reprogramming of LNCaP cells to Androgen-Independent growth and EMT progression even when YAP1-WT is overexpressed.

cancer biology↗

bioRxiv2022

The TLK1-MK5 axis regulates motility, invasion, and metastasis of prostate cancer cells

BackgroundMetastatic dissemination of prostate cancer (PCa) accounts for majority of PCa related deaths. However, the exact mechanism of PCa cell spread is still unknown. We uncovered a novel interaction between two unrelated promotility factors, tousled-like kinase 1 (TLK1) and MAPK-activated protein kinase 5 (MK5), which initiates a signaling cascade promoting metastasis. In PCa, TLK1-MK5 signaling might be crucial as androgen deprivation therapy (ADT) leads to increased expression of both TLK1 and MK5 in metastatic patients, but in this work, we directly investigated the motility, invasive, and metastatic capacity of PCa cells following impairment of the TLK1>MK5 axis. ResultsWe conducted scratch wound repair and 3D invasion assays with LNCaP and PC3 cells to determine if TLK1 and MK5 can regulate motility and invasion. Both genetic depletion and pharmacologic inhibition of TLK1 and MK5 resulted in reduced migration and invasion through a Matrigel plug. We further elucidated the potential mechanisms underlying these effects and found that that this is likely due to reorganization of the actin fibers at lamellipodia and the focal adhesions network, in conjunction with increased expression of some MMPs that can affect penetration through the ECM. PC3, a highly metastatic cell line when assayed in xenografts, was further tested in tail-vein injection/lung metastasis model, and we showed that following inoculation, treatment with GLPG (MK5 specific inhibitor) or J54 (TLK1 inhibitor) the resulting lung tumor nodules were greatly diminished in number, and for J54 also in size. ConclusionOur data support that TLK1-MK5 axis is functionally involved in driving PCa cell metastasis and clinical aggressiveness, hence, disruption of this axis may inhibit the metastatic capacity of PCa. SIMPLE SUMMARYRecent work by us and others has illustrated the critical importance of MK5/PRAK in the invasive and motility properties of several cancer cell lines and some mouse models. In our earlier work we also uncovered that TLK1 modulates the activity of MK5 by phosphorylating S354 and two additional sites (S160 and S386).. We have now expanded on the possible mechanisms of the TLK1>MK5 pro-motility and invasive activity, and report that this may be due to reorganization of the actin fibers at lamellipodia and the focal adhesions network, in conjunction with increased expression of some MMPs. Pharmacological or genetic manipulation of prostate cancer (PCa) cell lines, LNCaP and PC3, results in drastic loss of in vitro motility and invasive capacity of these cells concomitant with alterations of their general morphology and reorganization of the focal adhesions distribution. In addition, PC3 used in tail-vein experimental metastases studies show that the use of GLPG (MK5 inhibitor) or J54 (TLK1 inhibitor) results in a drastic reduction of metastatic lung nodules, macroscopically and histologically.

cancer biology↗

bioRxiv2022

SARS-CoV-2 Protein Nsp2 Stimulates Translation Under Normal and Hypoxic Conditions

When viruses like SARS-CoV-2 infect cells, they reprogram the repertoire of cellular and viral transcripts that are being translated to optimize their strategy of replication, often targeting host translation initiation factors, particularly eIF4F complex consisting of eIF4E, eIF4G and eIF4A. A proteomic analysis of SARS-CoV-2/human proteins interaction revealed viral Nsp2 and initiation factor eIF4E2, but a role of Nsp2 in regulating translation is still controversial. HEK293T cells stably expressing Nsp2 were tested for protein synthesis rates of synthetic and endogenous mRNAs known to be translated via cap- or IRES-dependent mechanism under normal and hypoxic conditions. Both cap- and IRES-dependent translation were increased in Nsp2-expressing cells under normal and hypoxic conditions, especially mRNAs that require high levels of eIF4F. This could be exploited by the virus to maintain high translation rates of both viral and cellular proteins, particularly in hypoxic conditions as may arise in SARS-CoV-2 patients with poor lung functioning.

physiology↗

bioRxiv2022

Evidence that Nek1 does not phosphorylate Rad54-S572 during recovery from IR

A main focus of the work in our lab is on the activity of Tousled Like Kinase 1 (TLK1) in the area of DNA Damage and Repair. As one of its key interactor, TLK1 phosphorylates NIMA related kinase 1 (Nek1), and Nek1 was reported by Spies et al. to phosphorylate and regulate the activity of the key HRR protein Rad541, which suggested an intriguing signal transduction pathway: TLK1>Nek1>Rad54. In an effort to confirm such relations, we now report that we have not been able to reproduce key findings from that study. Specifically, we found that Nek1 does not phosphorylate RAD54-S572 as was reported. We generated Nek1-KO mouse NT1 cells2 and Nek1-Knock-down in Hek293 (same cells as in Spies et al.), and the pRAD54-S572 signal does not change with our custom Ab, with or w/o IR. When we used an Ab from the Lobrich lab, it detected an immunoreactive band of wrong size for RAD54, which also did not change after IR even in synchronized G2 cells, contrary to their report. We also note that their P-assignment was based on guessing a weak consensus Nek1 sequence, and that site-directed mutagenesis of RAD54-S572 failed to yield biological effects in their in in vitro studies1. To conclusively establish that S572 is not a site of phosphorylation of Nek1, we carried out a IVK with purified Nek1 and RAD54 followed by MS analysis of the phosphatides, which revealed several but not S572. We also could not reproduce their copurification of Nek1-RAD54 by coIP, calling into question this interaction. Neither we could reproduce their results demonstrating the importance of Nek1 for HRR using the same SceI-mediated DR-GFP conversion assays.

molecular biology↗