Inhibition of UCH-L1 Deubiquitinating Activity with Two Forms of LDN-57444 Has Anti-Invasive Effects in Metastatic Carcinoma Cells

Abstract

Normally ubiquitin C-terminal hydrolase L1 (UCH-L1) is expressed in the central nervous and reproductive systems of adults, but its de novo expression has been detected in many human cancers. There is a growing body of evidence that UCH-L1 de-ubiquitinating (DUB) activity plays a major pro-metastatic role in certain carcinomas. Here we tested anti-metastatic effects of the small-molecule inhibitor of UCH-L1 DUB activity, LDN-57444, in cell lines from advanced oral squamous cell carcinoma (OSCC) as well as invasive nasopharyngeal (NP) cell lines expressing the major pro-metastatic gene product of Epstein–Barr virus (EBV) tumor virus, LMP1. To overcome the limited aqueous solubility of LDN-57444 we developed a nanoparticle formulation of LDN-57444 by incorporation of the compound in polyoxazoline micellear nanoparticles (LDN-POx). LDN-POx nanoparticles were equal in effects as the native compound in vitro. Our results demonstrate that inhibition of UCH-L1 DUB activity with LDN or LDN-POx inhibits secretion of exosomes and reduces levels of the pro-metastatic factor in exosomal fractions. Both forms of UCH-L1 DUB inhibitor suppress motility of metastatic squamous carcinoma cells as well as nasopharyngeal cells expressing EBV pro-metastatic Latent membrane protein 1 (LMP1) in physiological assays. Moreover, treatment with LDN and LDN-POx resulted in reduced levels of pro-metastatic markers, a decrease of carcinoma cell adhesion, as well as inhibition of extra-cellular vesicle (ECV)-mediated transfer of viral invasive factor LMP1. We suggest that soluble inhibitors of UCH-L1 such as LDN-POx offer potential forms of treatment for invasive carcinomas including EBV-positive malignancies.

The role of ubiquitination in tumorigenesis and targeted drug discovery

“…452 Due to the limited aqueous solubility of LDN-57444, its soluble form, LDN-Pox, was then developed and proved to have the potential to treat invasive carcinomas, including EBV-positive malignancies. 453 In addition, a cell-based screening was also used to select compounds inducing cathepsindependent apoptosis. Intriguingly, b-AP15 was identified to induce the accumulation of high-molecular-mass Ub complexes in cells ( Fig.…”

The roles of extracellular vesicles in the development, microenvironment, anticancer drug resistance, and therapy of head and neck squamous cell carcinoma

“…Studies have shown that ubiquitin C-terminal hydrolase L1 (UCH-L1) promotes motility of metastatic HNSCC as well as of extracellular vesicle-mediated transfer of the viral invasive factor LMP1 [66]. Further, Kobayashi et al proved that soluble inhibitors of UCH-L1 are effective in reducing lymph node metastasis of HNSCC; therefore, soluble inhibitors of UCH-L1 offer potential forms of treatment for invasive carcinomas, including EBVpositive malignancies [67].…”

Advances in Deubiquitinating Enzyme Inhibition and Applications in Cancer Therapeutics

“…LDN57444 and LDN91946 are examples of UCHL1-inhibiting isatin O-acyl oximes [219]. Inhibition of UCHL1 activity using LDN5744 or its nanoparticle formulation LDN-POx in vitro shows inhibition of exosome secretions and reduced levels of pro-metastatic factors in exosomal fractions while suppressing the motility of metastatic squamous carcinoma cells and nasopharyngeal cells expressing EBV pro-metastatic latent membrane protein 1 (LMP1) [220]. LDN and LDN-POx treatment also showed decreased carcinoma cell adhesion, reduced pro-metastatic markers, and inhibition of extracellular vesicle-mediated transfer of invasive factor LMP1 [220].…”