Inhibition of cytoplasmic EZH2 induces antitumor activity through stabilization of the DLC1 tumor suppressor protein.

mRNA expression of the DLC1 tumor suppressor gene is downregulated in many lung cancers and their derived cell lines, with DLC1 protein levels being low or absent. Although the role of increased EZH2 methyltransferase in cancer is usually attributed to its histone methylation, we unexpectedly observed that post-translational destabilization of DLC1 protein is common and attributable to its methylation by cytoplasmic EZH2, leading to CUL-4A ubiquitin-dependent proteasomal degradation of DLC1. Furthermore, siRNA knockdown of KRAS in several lines increases DLC1 protein, associated with a drastic reduction in cytoplasmic EZH2. Pharmacologic inhibition of EZH2, CUL-4A, or the proteasome can increase the steady-state level of DLC1 protein, whose tumor suppressor activity is further increased by AKT and/or SRC kinase inhibitors, which reverse the direct phosphorylation of DLC1 by these kinases. These rational drug combinations induce potent tumor growth inhibition, with markers of apoptosis and senescence, that is highly dependent on DLC1 protein.

Gain-of-Function RHOA Mutations Promote Focal Adhesion Kinase Activation and Dependency in Diffuse Gastric Cancer.

Diffuse gastric cancer (DGC) is a lethal malignancy lacking effective systemic therapy. Among the most provocative recent results in DGC has been that of highly recurrent missense mutations in the GTPase RHOA. The function of these mutations has remained unresolved. We demonstrate that RHOAY42C, the most common RHOA mutation in DGC, is a gain-of-function oncogenic mutant, and that expression of RHOAY42C with inactivation of the canonical tumor suppressor Cdh1 induces metastatic DGC in a mouse model. Biochemically, RHOAY42C exhibits impaired GTP hydrolysis and enhances interaction with its effector ROCK. RHOA Y42C mutation and Cdh1 loss induce actin/cytoskeletal rearrangements and activity of focal adhesion kinase (FAK), which activates YAP-TAZ, PI3K-AKT, and ß-catenin. RHOAY42C murine models were sensitive to FAK inhibition and to combined YAP and PI3K pathway blockade. These results, coupled with sensitivity to FAK inhibition in patient-derived DGC cell lines, nominate FAK as a novel target for these cancers. SIGNIFICANCE: The functional significance of recurrent RHOA mutations in DGC has remained unresolved. Through biochemical studies and mouse modeling of the hotspot RHOAY42C mutation, we establish that these mutations are activating, detail their effects upon cell signaling, and define how RHOA-mediated FAK activation imparts sensitivity to pharmacologic FAK inhibitors.See related commentary by Benton and Chernoff, p. 182.This article is highlighted in the In This Issue feature, p. 161.

SRC and ERK cooperatively phosphorylate DLC1 and attenuate its Rho-GAP and tumor suppressor functions.

SRC and ERK kinases control many cell biological processes that promote tumorigenesis by altering the activity of oncogenic and tumor suppressor proteins. We identify here a physiological interaction between DLC1, a focal adhesion protein and tumor suppressor, with SRC and ERK. The tumor suppressor function of DLC1 is attenuated by phosphorylation of tyrosines Y451 and Y701 by SRC, which down-regulates DLC1's tensin-binding and Rho-GAP activities. ERK1/2 phosphorylate DLC1 on serine S129, which increases both the binding of SRC to DLC1 and SRC-dependent phosphorylation of DLC1. SRC inhibitors exhibit potent antitumor activity in a DLC1-positive transgenic cancer model and a DLC1-positive tumor xenograft model, due to reactivation of the tumor suppressor activities of DLC1. Combined treatment of DLC1-positive tumors with SRC plus AKT inhibitors has even greater antitumor activity. Together, these findings indicate cooperation between the SRC, ERK1/2, and AKT kinases to reduce DLC1 Rho-GAP and tumor suppressor activities in cancer cells, which can be reactivated by the kinase inhibitors.

RIT1 oncoproteins escape LZTR1-mediated proteolysis.

RIT1 oncoproteins have emerged as an etiologic factor in Noonan syndrome and cancer. Despite the resemblance of RIT1 to other members of the Ras small guanosine triphosphatases (GTPases), mutations affecting RIT1 are not found in the classic hotspots but rather in a region near the switch II domain of the protein. We used an isogenic germline knock-in mouse model to study the effects of RIT1 mutation at the organismal level, which resulted in a phenotype resembling Noonan syndrome. By mass spectrometry, we detected a RIT1 interactor, leucine zipper-like transcription regulator 1 (LZTR1), that acts as an adaptor for protein degradation. Pathogenic mutations affecting either RIT1 or LZTR1 resulted in incomplete degradation of RIT1. This led to RIT1 accumulation and dysregulated growth factor signaling responses. Our results highlight a mechanism of pathogenesis that relies on impaired protein degradation of the Ras GTPase RIT1.

Receptor tyrosine kinase activation of RhoA is mediated by AKT phosphorylation of DLC1.

We report several receptor tyrosine kinase (RTK) ligands increase RhoA-guanosine triphosphate (GTP) in untransformed and transformed cell lines and determine this phenomenon depends on the RTKs activating the AKT serine/threonine kinase. The increased RhoA-GTP results from AKT phosphorylating three serines (S298, S329, and S567) in the DLC1 tumor suppressor, a Rho GTPase-activating protein (RhoGAP) associated with focal adhesions. Phosphorylation of the serines, located N-terminal to the DLC1 RhoGAP domain, induces strong binding of that N-terminal region to the RhoGAP domain, converting DLC1 from an open, active dimer to a closed, inactive monomer. That binding, which interferes with the interaction of RhoA-GTP with the RhoGAP domain, reduces the hydrolysis of RhoA-GTP, the binding of other DLC1 ligands, and the colocalization of DLC1 with focal adhesions and attenuates tumor suppressor activity. DLC1 is a critical AKT target in DLC1-positive cancer because AKT inhibition has potent antitumor activity in the DLC1-positive transgenic cancer model and in a DLC1-positive cancer cell line but not in an isogenic DLC1-negative cell line.

CDK5 is a major regulator of the tumor suppressor DLC1.

DLC1 is a tumor suppressor protein whose full activity depends on its presence at focal adhesions, its Rho-GTPase activating protein (Rho-GAP) function, and its ability to bind several ligands, including tensin and talin. However, the mechanisms that regulate and coordinate these activities remain poorly understood. Here we identify CDK5, a predominantly cytoplasmic serine/threonine kinase, as an important regulator of DLC1 functions. The CDK5 kinase phosphorylates four serines in DLC1 located N-terminal to the Rho-GAP domain. When not phosphorylated, this N-terminal region functions as an autoinhibitory domain that places DLC1 in a closed, inactive conformation by efficiently binding to the Rho-GAP domain. CDK5 phosphorylation reduces this binding and orchestrates the coordinate activation DLC1, including its localization to focal adhesions, its Rho-GAP activity, and its ability to bind tensin and talin. In cancer, these anti-oncogenic effects of CDK5 can provide selective pressure for the down-regulation of DLC1, which occurs frequently in tumors, and can contribute to the pro-oncogenic activity of CDK5 in lung adenocarcinoma.

Full activity of the deleted in liver cancer 1 (DLC1) tumor suppressor depends on an LD-like motif that binds talin and focal adhesion kinase (FAK).

The deleted in liver cancer 1 (DLC1) tumor suppressor gene, which is frequently inactivated in cancer, encodes a Rho-GAP (GTPase activating protein) focal adhesion protein whose negative regulation of Rho-GTPases is necessary but not sufficient for its full tumor suppressor activity. Here, we report that DLC1 forms a complex with two prooncogenic focal adhesion proteins, talin and the focal adhesion kinase (FAK). We identified an 8-aa sequence (residues 469LDDILYHV476) in DLC1 and designated it an LD-like motif, because it shares homology with the LD motifs of paxillin. This motif was necessary for DLC1 binding to talin and FAK, because a DLC1 mutant, from which six of the residues have been deleted, and another mutant carrying amino acid substitutions in three of the residues are deficient for binding both proteins and localization of DLC1 to focal adhesions. FAK binding was independent of talin and vice versa. In bioassays, both DLC1 mutants were less active than wild-type (WT) DLC1, although the ability of the mutants to negatively regulate overall Rho-GTP was not impaired. We conclude that the LD-like motif, which binds talin and FAK, is required for the full tumor suppressor activity of DLC1 and contributes to the association of DLC1 with focal adhesions.

Rrp1b, a new candidate susceptibility gene for breast cancer progression and metastasis.

A novel candidate metastasis modifier, ribosomal RNA processing 1 homolog B (Rrp1b), was identified through two independent approaches. First, yeast two-hybrid, immunoprecipitation, and functional assays demonstrated a physical and functional interaction between Rrp1b and the previous identified metastasis modifier Sipa1. In parallel, using mouse and human metastasis gene expression data it was observed that extracellular matrix (ECM) genes are common components of metastasis predictive signatures, suggesting that ECM genes are either important markers or causal factors in metastasis. To investigate the relationship between ECM genes and poor prognosis in breast cancer, expression quantitative trait locus analysis of polyoma middle-T transgene-induced mammary tumor was performed. ECM gene expression was found to be consistently associated with Rrp1b expression. In vitro expression of Rrp1b significantly altered ECM gene expression, tumor growth, and dissemination in metastasis assays. Furthermore, a gene signature induced by ectopic expression of Rrp1b in tumor cells predicted survival in a human breast cancer gene expression dataset. Finally, constitutional polymorphism within RRP1B was found to be significantly associated with tumor progression in two independent breast cancer cohorts. These data suggest that RRP1B may be a novel susceptibility gene for breast cancer progression and metastasis.

Oncogenic inhibition by a deleted in liver cancer gene requires cooperation between tensin binding and Rho-specific GTPase-activating protein activities.

The three deleted in liver cancer genes (DLC1-3) encode Rho-GTPase-activating proteins (RhoGAPs) whose expression is frequently down-regulated or silenced in a variety of human malignancies. The RhoGAP activity is required for full DLC-dependent tumor suppressor activity. Here we report that DLC1 and DLC3 bind to human tensin1 and its chicken homolog. The binding has been mapped to the tensin Src homology 2 (SH2) and phosphotyrosine binding (PTB) domains at the C terminus of tensin proteins. Distinct DLC1 sequences are required for SH2 and PTB binding. DCL binding to both domains is constitutive under basal conditions. The SH2 binding depends on a tyrosine in DCL1 (Y442) but is phosphotyrosine-independent, a highly unusual feature for SH2 binding. DLC1 competed with the binding of other proteins to the tensin C terminus, including beta 3-integrin binding to the PTB domain. Point mutation of a critical tyrosine residue (Y442F) in DLC1 rendered the protein deficient for binding the tensin SH2 domain and binding full-length tensin. The Y442F protein was diffusely cytoplasmic, in contrast to the localization of wild-type DLC1 to focal adhesions, but it retained the ability to reduce the intracellular levels of Rho-GTP. The Y442F mutant displayed markedly reduced biological activity, as did a mutant that was RhoGAP-deficient. The results suggest that DLC1 is a multifunctional protein whose biological activity depends on cooperation between its tensin binding and RhoGAP activities, although neither activity depends on the other.