CRL4A-FBXW5–mediated degradation of DLC1 Rho GTPase-activating protein tumor suppressor promotes non-small cell lung cancer cell growth

Abstract

DLC1 encodes a RhoA GTPase-activating protein and tumor suppressor lost in cancer by genomic deletion or epigenetic silencing and loss of DLC1 gene transcription. We unexpectedly identified nonsmall cell lung cancer (NSCLC) cell lines and tumor tissue that expressed DLC1 mRNA yet lacked DLC1 protein expression. We determined that DLC1 was ubiquitinated and degraded by cullin 4A-RING ubiquitin ligase (CRL4A) complex interaction with DDB1 and the FBXW5 substrate receptor. siRNA-mediated suppression of cullin 4A, DDB1, or FBXW5 expression restored DLC1 protein expression in NSCLC cell lines. FBXW5 suppression-induced DLC1 reexpression was associated with a reduction in the levels of activated RhoA-GTP and in RhoA effector signaling. Finally, FBXW5 suppression caused a DLC1-dependent decrease in NSCLC anchorage-dependent and -independent proliferation. In summary, we identify a posttranslational mechanism for loss of DLC1 and a linkage between CRL4A-FBXW5-associated oncogenesis and regulation of RhoA signaling.Rho-selective GTPase-activating protein | Rho GTPase-activating protein 7 | STARD12 R ho family small GTPases function as extracellular signalregulated on-off switches that cycle between an active GTPbound state and an inactive GDP-bound state. Of the 20 human Rho family GTPases, the best studied are RhoA, Rac1, and Cdc42 (1). Rho-selective guanine nucleotide exchange factors (RhoGEFs) promote GDP-GTP exchange and formation of active Rho-GTP, whereas Rho-selective GTPase-activating proteins (RhoGAPs) stimulate hydrolysis of the bound GTP to return the GTPase to its inactive Rho-GDP form (2, 3). Rho-GTP binds preferentially to its downstream effectors, stimulating a diversity of cytoplasmic signaling cascades that control actin organization, cell morphology and polarity, cell cycle progression and cell proliferation, cell survival and migration, and gene expression (4).In light of their key role in regulating fundamental processes in cell behavior, it is not surprising that the aberrant activation of Rho family small GTPases contributes to cancer and other human disorders (5-8). However, in contrast to the Ras small GTPase, where direct mutational activation leads to insensitivity to inactivation by Ras-selective GTPase-activating proteins (RasGAPs), Rho GTPases are more commonly activated through indirect mechanisms (2, 3). In human cancers, persistent RhoGEF activation or loss of RhoGAP stimulation are common mechanisms leading to aberrant Rho activation. For example, we determined that the P-Rex1 RhoGEF was up-regulated transcriptionally in melanoma through persistent activation of the ERK mitogen-activated protein kinase pathway and the related P-Rex2 isoform was found mutationally activated in melanoma (9, 10).With regard to RhoGAPs, one of the most frequent and common mechanisms involves loss of expression of Deleted in Liver Cancer 1 (DLC1) in liver, breast, lung, ovarian, kidney, colon, stomach, prostate, and other cancers (3,11,12). DLC1 encodes a GAP primarily for RhoA and related isoforms. Initially ...

Rho regulation: DLC proteins in space and time

“…Another recent study identified the serine-threonine kinase CKD5 to phosphorylate DLC1 on multiple residues within the N-terminus, thereby inducing an open conformation that leads to activation of GAP function [80]. Finally, GAP regulation can be switched off irreversibly by ubiquitindependent protein degradation of DLC1, adding an additional layer of complexity to the post-translational regulation of DLC1 [38]. However, none of these studies has been able to define the specific DLC1 pool that is subject to such regulation and determine the biological context during which this occurs.…”

Regulating Rho GTPases and their regulators

“…The GAP activity of several RhoGAPs is regulated by phosphorylation (TABLE 2). A number of phosphorylation sites have been characterized on the RhoGAP DLC1 (deleted in liver cancer 1), the product of a tumour suppressor gene that is lost in several cancers through either epigenetic silencing or gene deletion 99 . PKA-mediated phosphorylation of DLC1 on Ser549 induces its homodimerization, which increases its RhoGAP activity 100 .…”

“…Ubiquitylation of DLC1 by a CUL4 FBXW5 ubiquitin ligase complex leads to its degradation in nonsmall-cell lung cancer cell lines 99 . This provides another mechanism, in addition to gene deletion or epigenetic silencing, to remove this tumour suppressor.…”

Roles of F-box proteins in cancer

“…However, FBXW5 was recently reported to control the stability of deleted in liver cancer 1 (DLC1; also known as RHO GTPase-activating protein 7) 122 and tuberous sclerosis 2 (TSC2) 123 tumour suppressors (see Supplementary information S2 (table)), which suggests a possible oncogenic role for FBXW5, but this requires knockout or knock-in mouse models for further validation.…”