RAS transformation requires CUX1-dependent repair of oxidative DNA damage.

The Cut homeobox 1 (CUX1) gene is a target of loss-of-heterozygosity in many cancers, yet elevated CUX1 expression is frequently observed and is associated with shorter disease-free survival. The dual role of CUX1 in cancer is illustrated by the fact that most cell lines with CUX1 LOH display amplification of the remaining allele, suggesting that decreased CUX1 expression facilitates tumor development while increased CUX1 expression is needed in tumorigenic cells. Indeed, CUX1 was found in a genome-wide RNAi screen to identify synthetic lethal interactions with oncogenic RAS. Here we show that CUX1 functions in base excision repair as an ancillary factor for the 8-oxoG-DNA glycosylase, OGG1. Single cell gel electrophoresis (comet assay) reveals that Cux1⁺/⁻ MEFs are haploinsufficient for the repair of oxidative DNA damage, whereas elevated CUX1 levels accelerate DNA repair. In vitro base excision repair assays with purified components demonstrate that CUX1 directly stimulates OGG1's enzymatic activity. Elevated reactive oxygen species (ROS) levels in cells with sustained RAS pathway activation can cause cellular senescence. We show that elevated expression of either CUX1 or OGG1 prevents RAS-induced senescence in primary cells, and that CUX1 knockdown is synthetic lethal with oncogenic RAS in human cancer cells. Elevated CUX1 expression in a transgenic mouse model enables the emergence of mammary tumors with spontaneous activating Kras mutations. We confirmed cooperation between Kras(G12V) and CUX1 in a lung tumor model. Cancer cells can overcome the antiproliferative effects of excessive DNA damage by inactivating a DNA damage response pathway such as ATM or p53 signaling. Our findings reveal an alternate mechanism to allow sustained proliferation in RAS-transformed cells through increased DNA base excision repair capability. The heightened dependency of RAS-transformed cells on base excision repair may provide a therapeutic window that could be exploited with drugs that specifically target this pathway.

Ras effector mutant expression suggest a negative regulator inhibits lung tumor formation.

Lung cancer is currently the most deadly malignancy in industrialized countries and accounts for 18% of all cancer-related deaths worldwide. Over 70% of patients with non-small cell lung cancer (NSCLC) are diagnosed at a late stage, with a 5-year survival below 10%. KRAS and the EGFR are frequently mutated in NSCLC and while targeted therapies for patients with EGFR mutations exist, oncogenic KRAS is thus far not druggable. KRAS activates multiple signalling pathways, including the PI3K/Akt pathway, the Raf-Mek-Erk pathway and the RalGDS/Ral pathway. Lung-specific expression of BrafV600E, the most prevalent BRAF mutation found in human tumors, results in Raf-Mek-Erk pathway activation and in the formation of benign adenomas that undergo widespread senescence in a Cre-activated Braf mouse model (Braf(CA)). However, oncogenic KRAS expression in mice induces adenocarcinomas, suggesting additional KRAS-activated pathways cooperate with sustained RAF-MEK-ERK signalling to bypass the oncogene-induced senescence proliferation arrest. To determine which KRAS effectors were responsible for tumor progression, we created four effector domain mutants (S35, G37, E38 and C40) in G12V-activated KRAS and expressed these alone or with BrafV600E in mouse lungs... The S35 and E38 mutants bind to Raf proteins but not PI3K or RalGDS; the G37 mutant binds to RalGDS and not Raf or PI3K and the C40 mutant is specific to PI3K. We designed lentiviral vectors to code for Cre recombinase along with KRAS mutants (V12, V12/S35, V12/G37, V12/E38 or V12/C40) or EGFP as a negative control.. These lentiviruses were used to infect Braf(CA) and wild-type mice. Surprisingly there was a significant decrease in tumor number and penetrance with each KRAS effector domain mutant relative to controls, suggesting that KRAS directly activates effectors with tumor suppressive functions.

A modular lentiviral and retroviral construction system to rapidly generate vectors for gene expression and gene knockdown in vitro and in vivo.

The ability to express exogenous cDNAs while suppressing endogenous genes via RNAi represents an extremely powerful research tool with the most efficient non-transient approach being accomplished through stable viral vector integration. Unfortunately, since traditional restriction enzyme based methods for constructing such vectors are sequence dependent, their construction is often difficult and not amenable to mass production. Here we describe a non-sequence dependent Gateway recombination cloning system for the rapid production of novel lentiviral (pLEG) and retroviral (pREG) vectors. Using this system to recombine 3 or 4 modular plasmid components it is possible to generate viral vectors expressing cDNAs with or without inhibitory RNAs (shRNAmirs). In addition, we demonstrate a method to rapidly produce and triage novel shRNAmirs for use with this system. Once strong candidate shRNAmirs have been identified they may be linked together in tandem to knockdown expression of multiple targets simultaneously or to improve the knockdown of a single target. Here we demonstrate that these recombinant vectors are able to express cDNA and effectively knockdown protein expression using both cell culture and animal model systems.

SHP-1 inhibits ß-catenin function by inducing its degradation and interfering with its association with TATA-binding protein.

ß-catenin plays a dual role both as a key effector in the regulation of adherens junctions and as a transcriptional coactivator. Tyrosine phosphorylation of ß-catenin is implicated as a means for its release from E-cadherin complexes and correlates with enhanced transcriptional activity. However, it remains unclear whether or not tyrosine phosphorylated ß-catenin degrades slower or faster than its unphosphorylated form or transactivates the downstream target genes differently. We have recently demonstrated that tyrosine phosphatase SHP-1 negatively regulates the nuclear transcriptional function of ß-catenin. The mechanism by which SHP-1 specifically inhibits ß-catenin/TCF transcriptional activity remains, however, to be elucidated. Herein, we demonstrate that inhibition of tyrosine phosphatases with pervanadate induced both c-src-dependent tyrosine phosphorylation and nuclear translocation of ß-catenin. Moreover, ectopic expression of SHP-1 but not the inactive form of SHP-1 (C453S) inhibited src-induced tyrosine phosphorylation of ß-catenin on tyrosines 86 and 654. SHP-1 expression and mutations of tyrosine-86 and tyrosine-654 to phenylalanine significantly and similarly decreased the transactivation potential of ß-catenin on the TOPFLASH reporter. SHP-1 expression as well as mutations of tyrosine-86 and tyrosine-654 to phenylalanine also significantly interfered with the association of ß-catenin with TBP. Mutations of tyrosine-86 and/or tyrosine-654 did not markedly alter ß-catenin stability whereas SHP-1 expression promoted proteasomal ß-catenin degradation through a GSK3ß-dependent mechanism. In conclusion, SHP-1 negatively regulates ß-catenin transcriptional activity i) by dephosphorylating ß-catenin on tyrosines 86 and 654, ii) by impairing its capacity to interact with the basal transcriptional factor TBP and iii) by promoting ß-catenin degradation in a GSK3ß-dependent manner.

Mutation G827R in matriptase causing autosomal recessive ichthyosis with hypotrichosis yields an inactive protease.

Matriptase is a member of the novel family of type II transmembrane serine proteases. It was recently shown that a rare genetic disorder, autosomal recessive ichthyosis with hypotrichosis, is caused by a mutation in the coding region of matriptase. However, the biochemical and functional consequences of the G827R mutation in the catalytic domain of the enzyme have not been reported. Here we expressed the G827R-matriptase mutant in bacterial cells and found that it did not undergo autocatalytic cleavage from its zymogen to its active form as did the wild-type matriptase. Enzymatic activity measurements showed that the G827R mutant was catalytically inactive. When expressed in HEK293 cells, G827R-matriptase remained inactive but was shed as a soluble form, suggesting that another protease cleaved the full-length mature form of matriptase. Molecular modeling based on the crystal structure of matriptase showed that replacing Gly(827) by Arg blocks access to the binding/catalytic cleft of the enzyme thereby preventing autocatalysis of the zymogen form. Our study, thus, provides direct evidence that the G827R mutation in patients with autosomal recessive ichthyosis with hypotrichosis leads to the expression of an inactive protease.