ABSTRACT
We performed a small interfering RNA screen to identify targets for cutaneous squamous cell carcinoma (cSCC) therapy in the ubiquitin/ubiquitin-like system. We provide evidence for selective anti-cSCC activity of knockdown of the E3 ubiquitin ligase MARCH4, the ATPase p97/VCP, the deubiquitinating enzyme USP8, the cullin-RING ligase (CRL) 4 substrate receptor CDT2/DTL, and components of the anaphase-promoting complex/cyclosome (APC/C). Specifically attenuating CRL4CDT2 by CDT2 knockdown can be more potent in killing cSCC cells than targeting CRLs or CRL4s in general by RBX1 or DDB1 depletion. Suppression of the APC/C or forced APC/C activation by targeting its repressor EMI1 are both potential therapeutic approaches. We observed that cSCC cells can be selectively killed by small-molecule inhibitors of USP8 (DUBs-IN-3/compound 22c) and the NEDD8 E1 activating enzyme/CRLs (MLN4924/pevonedistat). A substantial proportion of cSCC cell lines are very highly MLN4924-sensitive. Pathways that respond to defects in proteostasis are involved in the anti-cSCC activity of p97 suppression. Targeting USP8 can reduce the expression of growth factor receptors that participate in cSCC development. EMI1 and CDT2 depletion can selectively cause DNA re-replication and DNA damage in cSCC cells.
Subject(s)
Carcinoma, Squamous Cell/drug therapy , Skin Neoplasms/drug therapy , Carcinoma, Squamous Cell/pathology , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Cyclopentanes/pharmacology , Cyclopentanes/therapeutic use , Drug Screening Assays, Antitumor , Endopeptidases/genetics , Endopeptidases/metabolism , Endosomal Sorting Complexes Required for Transport/antagonists & inhibitors , Endosomal Sorting Complexes Required for Transport/genetics , Endosomal Sorting Complexes Required for Transport/metabolism , F-Box Proteins/antagonists & inhibitors , F-Box Proteins/genetics , F-Box Proteins/metabolism , Gene Knockdown Techniques , Humans , Molecular Targeted Therapy/methods , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Pyrimidines/pharmacology , Pyrimidines/therapeutic use , RNA, Small Interfering/metabolism , Skin Neoplasms/pathology , Ubiquitin Thiolesterase/antagonists & inhibitors , Ubiquitin Thiolesterase/genetics , Ubiquitin Thiolesterase/metabolism , Ubiquitin-Activating Enzymes/antagonists & inhibitors , Ubiquitin-Activating Enzymes/genetics , Ubiquitin-Activating Enzymes/metabolismABSTRACT
We show that suppression of the spliceosome has potential for the treatment of cutaneous squamous cell carcinoma (cSCC). The small-molecule inhibitors of the spliceosome at the most advanced stage of development target the splicing factor SF3B1/SF3b155. The majority of cSCC cell lines are more sensitive than normal skin cells to death induced by the SF3B1 inhibitor pladienolide B. Knockdown of SF3B1 and a range of other splicing factors with diverse roles in the spliceosome can also selectively kill cSCC cells. We demonstrate that endogenous c-MYC participates in conferring sensitivity to spliceosome inhibition. c-MYC expression is elevated in cSCC lines and its knockdown reduces alterations in mRNA splicing and attenuates cell death caused by interference with the spliceosome. In addition, this study provides further support for a key role of the p53 pathway in the response to spliceosome disruption. SF3B1 inhibition causes wild-type p53 upregulation associated with altered mRNA splicing and reduced protein expression of both principal p53 negative regulators MDMX/MDM4 and MDM2. We observed that wild-type p53 can promote pladienolide B-induced death in tumour cells. However, p53 is commonly inactivated by mutation in cSCCs and p53 participates in killing normal skin cells at high concentrations of pladienolide B. This may limit the therapeutic window of SF3B1 inhibitors for cSCC. We provide evidence that, while suppression of SF3B1 has promise for treating cSCCs with mutant p53, inhibitors which target the spliceosome through SF3B1-independent mechanisms could have greater cSCC selectivity as a consequence of reduced p53 upregulation in normal cells.
