OTUB 1 triggers lung cancer development by inhibiting RAS monoubiquitination
2016; Springer Nature; Volume: 8; Issue: 3 Linguagem: Inglês
10.15252/emmm.201505972
ISSN1757-4684
AutoresMaria Francesca Baietti, Michal Šimíček, Layka Abbasi Asbagh, Enrico Radaelli, Sam Lievens, Jonathan Crowther, Mikhail Steklov, Vasily N. Aushev, David Martínez‐García, Jan Tavernier, Anna Sablina,
Tópico(s)RNA Research and Splicing
ResumoResearch Article8 February 2016Open Access Source DataTransparent process OTUB1 triggers lung cancer development by inhibiting RAS monoubiquitination Maria Francesca Baietti Maria Francesca Baietti Center for the Biology of Disease, VIB, Leuven, Belgium Center for Human Genetics, KU Leuven, Leuven, Belgium Search for more papers by this author Michal Simicek Michal Simicek Center for the Biology of Disease, VIB, Leuven, Belgium Center for Human Genetics, KU Leuven, Leuven, Belgium Search for more papers by this author Layka Abbasi Asbagh Layka Abbasi Asbagh Center for the Biology of Disease, VIB, Leuven, Belgium Center for Human Genetics, KU Leuven, Leuven, Belgium Search for more papers by this author Enrico Radaelli Enrico Radaelli Center for the Biology of Disease, VIB, Leuven, Belgium Center for Human Genetics, KU Leuven, Leuven, Belgium Search for more papers by this author Sam Lievens Sam Lievens Department of Medical Protein Research, VIB, Leuven, Belgium Department of Biochemistry, Gent University, Gent, Belgium Search for more papers by this author Jonathan Crowther Jonathan Crowther Center for the Biology of Disease, VIB, Leuven, Belgium Center for Human Genetics, KU Leuven, Leuven, Belgium Search for more papers by this author Mikhail Steklov Mikhail Steklov Center for the Biology of Disease, VIB, Leuven, Belgium Center for Human Genetics, KU Leuven, Leuven, Belgium Search for more papers by this author Vasily N Aushev Vasily N Aushev Center for the Biology of Disease, VIB, Leuven, Belgium Center for Human Genetics, KU Leuven, Leuven, Belgium Institute of Carcinogenesis, Blokhin Russian Cancer Research Center, Moscow, Russia Search for more papers by this author David Martínez García David Martínez García Center for the Biology of Disease, VIB, Leuven, Belgium Center for Human Genetics, KU Leuven, Leuven, Belgium Search for more papers by this author Jan Tavernier Jan Tavernier Department of Medical Protein Research, VIB, Leuven, Belgium Department of Biochemistry, Gent University, Gent, Belgium Search for more papers by this author Anna A Sablina Corresponding Author Anna A Sablina Center for the Biology of Disease, VIB, Leuven, Belgium Center for Human Genetics, KU Leuven, Leuven, Belgium Search for more papers by this author Maria Francesca Baietti Maria Francesca Baietti Center for the Biology of Disease, VIB, Leuven, Belgium Center for Human Genetics, KU Leuven, Leuven, Belgium Search for more papers by this author Michal Simicek Michal Simicek Center for the Biology of Disease, VIB, Leuven, Belgium Center for Human Genetics, KU Leuven, Leuven, Belgium Search for more papers by this author Layka Abbasi Asbagh Layka Abbasi Asbagh Center for the Biology of Disease, VIB, Leuven, Belgium Center for Human Genetics, KU Leuven, Leuven, Belgium Search for more papers by this author Enrico Radaelli Enrico Radaelli Center for the Biology of Disease, VIB, Leuven, Belgium Center for Human Genetics, KU Leuven, Leuven, Belgium Search for more papers by this author Sam Lievens Sam Lievens Department of Medical Protein Research, VIB, Leuven, Belgium Department of Biochemistry, Gent University, Gent, Belgium Search for more papers by this author Jonathan Crowther Jonathan Crowther Center for the Biology of Disease, VIB, Leuven, Belgium Center for Human Genetics, KU Leuven, Leuven, Belgium Search for more papers by this author Mikhail Steklov Mikhail Steklov Center for the Biology of Disease, VIB, Leuven, Belgium Center for Human Genetics, KU Leuven, Leuven, Belgium Search for more papers by this author Vasily N Aushev Vasily N Aushev Center for the Biology of Disease, VIB, Leuven, Belgium Center for Human Genetics, KU Leuven, Leuven, Belgium Institute of Carcinogenesis, Blokhin Russian Cancer Research Center, Moscow, Russia Search for more papers by this author David Martínez García David Martínez García Center for the Biology of Disease, VIB, Leuven, Belgium Center for Human Genetics, KU Leuven, Leuven, Belgium Search for more papers by this author Jan Tavernier Jan Tavernier Department of Medical Protein Research, VIB, Leuven, Belgium Department of Biochemistry, Gent University, Gent, Belgium Search for more papers by this author Anna A Sablina Corresponding Author Anna A Sablina Center for the Biology of Disease, VIB, Leuven, Belgium Center for Human Genetics, KU Leuven, Leuven, Belgium Search for more papers by this author Author Information Maria Francesca Baietti1,2,‡, Michal Simicek1,2,6,‡, Layka Abbasi Asbagh1,2, Enrico Radaelli1,2, Sam Lievens3,4, Jonathan Crowther1,2, Mikhail Steklov1,2, Vasily N Aushev1,2,5, David Martínez García1,2, Jan Tavernier3,4 and Anna A Sablina 1,2 1Center for the Biology of Disease, VIB, Leuven, Belgium 2Center for Human Genetics, KU Leuven, Leuven, Belgium 3Department of Medical Protein Research, VIB, Leuven, Belgium 4Department of Biochemistry, Gent University, Gent, Belgium 5Institute of Carcinogenesis, Blokhin Russian Cancer Research Center, Moscow, Russia 6Present address: PNAC, MRC, LMB, Cambridge, UK ‡These authors equally contributed to this work *Corresponding author. Tel: +32 16330790; Fax: +32 16330145; E-mail: [email protected] EMBO Mol Med (2016)8:288-303https://doi.org/10.15252/emmm.201505972 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Activation of the RAS oncogenic pathway, frequently ensuing from mutations in RAS genes, is a common event in human cancer. Recent reports demonstrate that reversible ubiquitination of RAS GTPases dramatically affects their activity, suggesting that enzymes involved in regulating RAS ubiquitination may contribute to malignant transformation. Here, we identified the de-ubiquitinase OTUB1 as a negative regulator of RAS mono- and di-ubiquitination. OTUB1 inhibits RAS ubiquitination independently of its catalytic activity resulting in sequestration of RAS on the plasma membrane. OTUB1 promotes RAS activation and tumorigenesis in wild-type RAS cells. An increase of OTUB1 expression is commonly observed in non-small-cell lung carcinomas harboring wild-type KRAS and is associated with increased levels of ERK1/2 phosphorylation, high Ki67 score, and poorer patient survival. Our results strongly indicate that dysregulation of RAS ubiquitination represents an alternative mechanism of RAS activation during lung cancer development. Synopsis The regulation of RAS mono-ubiquitination by OTUB1 is an alternative mechanism of RAS activation during lung cancer development. OTUB1 negatively regulates RAS mono-ubiquitination resulting in sequestration of RAS on the plasma membrane. OTUB1 promotes RAS signaling and tumorigenic growth of wild-type KRAS lung adenocarcinoma cells. OTUB1 up-regulation is associated with poorer survival of wild-type KRAS lung adenocarcinoma patients. Introduction The RAS small GTPases (HRAS, NRAS, and KRAS) are essential regulators of diverse eukaryotic cellular processes, such as cell proliferation, cytoskeletal assembly and organization, and intracellular membrane trafficking [for review (Colicelli, 2004)]. The RAS family members function as molecular switches alternating between an inactive GDP-bound and active GTP-bound state. The transition between GDP- and GTP-bound forms is tightly regulated by guanine nucleotide exchange factors (GEF) and GTPase-activating proteins (GAP). The RAS small GTPases play a major role in the development of human cancer. Oncogenic RAS mutations occur in up to 30% of non-small-cell lung carcinomas (NSCLC), mostly adenocarcinomas (Karnoub & Weinberg, 2008). The intrinsic GTP hydrolysis activity of RAS is the predominant target of most common somatic mutations found in the oncogenic variants of RAS alleles (Pylayeva-Gupta et al, 2011). KRAS-mutant lung adenocarcinomas have higher levels of the MAPK pathway activation than wild-type (wt) KRAS tumors. However, the MAPK cascade is also hyperactivated in a significant proportion of wt KRAS tumors, suggesting that RAS proteins may be frequently activated by alternative mechanisms not yet fully elucidated (Network, 2014). Beyond oncogenic mutations of RAS, up-regulation of RAS-specific GEFs and functional loss of GAPs also have been shown to contribute to cancer development and progression (Vigil et al, 2010). In addition, several post-translational modifications, such as phosphorylation (Bivona et al, 2006), lipidation (Hancock, 2003), and acetylation (Yang et al, 2013), are known to regulate the functions of RAS GTPases. RAS stability is controlled by the E3 ubiquitin ligases, β-TrCP1, and Nedd4-1, that directly polyubiquitinate RAS proteins triggering degradation (Shukla et al, 2014; Zeng et al, 2014). In addition, we and others have recently demonstrated that RAS family members can undergo reversible mono- and di-ubiquitination (Jura et al, 2006; Xu et al, 2010; Sasaki et al, 2011; Baker et al, 2013a; Simicek et al, 2013). However, how reversible ubiquitination affects RAS activity and its tumorigenic properties remains very much controversial. Earlier studies reported that reversible ubiquitination restricts the activity of HRAS and NRAS, but not that of KRAS, whereas more recent reports demonstrated that KRAS can also undergo mono- and di-ubiquitination (Jura et al, 2006). Xu et al (2010) demonstrated that di-ubiquitination of HRAS and NRAS by the E3 ubiquitin ligase RABEX5 (RABGEF1) induces their re-localization to the endomembranes, leading to a decrease in RAS activity and downstream signaling. On the other hand, two other groups demonstrated that monoubiquitination of HRAS at Lys117 accelerates intrinsic nucleotide exchange and promotes GTP loading, whereas monoubiquitination of KRAS at Lys147 impaired NF1-mediated GTP hydrolysis (Sasaki et al, 2011; Baker et al, 2013a,b). Moreover, the KRAS gene fusion with the ubiquitin-conjugating enzyme UBE2L3 has been identified in metastatic prostate cancer. The UBE2L3-KRAS fusion protein is highly ubiquitinated and exhibits transforming activity via specific activation of AKT and p38 MAPK pathways (Wang et al, 2011). Taken together, these studies strongly highlight the importance of reversible ubiquitination of RAS-like GTPases governing downstream signaling. These results also suggest that enzymes involved in RAS ubiquitination may contribute to tumorigenic transformation by modulating RAS activity. In this study, we focused on the identification of specific RAS de-ubiquitinating enzymes (DUBs) and their role in cancer development and progression. We found that OTUB1 up-regulation contributes specifically to the development of wt KRAS lung adenocarcinomas by inhibiting reversible ubiquitination of RAS proteins. Results OTUB1 controls RAS ubiquitination To identify DUBs involved in the control of RAS ubiquitination, we utilized a targeted mammalian protein–protein interaction (MAPPIT) screen, a two-hybrid technology for the detection of protein-protein interactions in intact mammalian cells (Lievens et al, 2009, 2012). As a proof of concept, we applied the MAPPIT system to examine the interactions between HRAS and its known downstream effectors (Fig EV1). GTPase-deficient HRAS G12V-mutant bait gave rise to the robust MAPPIT signals with each of the tested effector preys (Fig EV1), confirming the feasibility of the MAPPIT approach to identify novel RAS regulators. Click here to expand this figure. Figure EV1. MAPPIT assay to identify interacting partners of the RAS GTPasesMAPPIT assay detected interactions between HRAS G12V and its downstream targets. A MAPPIT assay was performed with REM2 and EFHA1 as negative controls, and RAS effectors, BRAF, c-RAF RBD, PIK3CA RBD, PIK3CG, and RGL2, as preys, screened against HRAS G12V as bait. pSEL(+2L) vectors coding HRAS G12V or NKP30 negative control was expressed into HEK293T cells together with the indicated prey. The results are expressed as a mean of normalized luciferase activity ± s.e.m (leptin-treated cells vs leptin-untreated cells). n = 2. Download figure Download PowerPoint A targeted MAPPIT screen, in which the HRAS G12V bait was screened against a library of 55 DUBs, identified four DUBs, USP12, JOSD2, UCHL5, and OTUB1, as potential interactors of HRAS G12V (Fig 1A). We next assessed whether the candidate DUBs could also interact with KRAS G12V and NRAS Q61K. The MAPPIT assay revealed that, in contrast to other tested DUBs, OTUB1 demonstrated a much higher affinity for both NRAS and KRAS compared to random non-specific baits, MAL and eDHFR (Fig 1B). Altogether, the MAPPIT experiments identified OTUB1 (OTU de-ubiquitinase, ubiquitin aldehyde binding 1), a member of the ovarian tumor domain protease (OTU) family of DUBs (Wang et al, 2009; Iglesias-Gato et al, 2015), as a putative binding partner of RAS proteins (Fig 1A and B). Figure 1. The de-ubiquitinase OTUB1 interacts with the RAS GTPases A targeted MAPPIT screen identifies several DUBs as putative RAS interactors. A MAPPIT array containing DUB prey library was screened with HRAS G12V as bait. pSEL(+2L)-HRAS G12V was expressed in HEK293T cells together with the indicated prey. BRAF served as a positive control. Each measurement was done in triplicate. The results are expressed as a mean of normalized luciferase activity (leptin-treated cells vs leptin-untreated cells). The overall mean value + 2 s.d. served as a threshold. MAPPIT assay confirms the interaction between OTUB1 and RAS proteins. pSEL(+2L) vectors coding RAS proteins were expressed in HEK293T cells together with the indicated prey. Empty vector and two random baits, MAL and eDHFR, were used as negative controls. REM2 and EFHA1 preys that bind to the bait receptor itself were used to evaluate the expression of the RAS baits. The results are expressed as a mean of normalized luciferase activity ± s.e.m (leptin-treated cells vs leptin-untreated cells), n = 3. NRAS Q61K mutant co-immunoprecipitates with OTUB1. At 48 h post-transfection with Flag-tagged NRAS Q61K and HA-tagged OTUB1 expression constructs, HA-tagged OTUB1 was immunoprecipitated with anti-HA-agarose followed by immunoblotting using anti-Flag or anti-HA antibodies. OTUB1 interacts with wt NRAS and active NRAS-mutant. Flag-tagged NRAS proteins were immunoprecipitated using anti-Flag (M2) agarose from HEK293T cells overexpressing HA-tagged OTUB1 or empty vector (V). GTP binding does not affect the complex formation between NRAS and OTUB1. Recombinant Flag-tagged NRAS was incubated with lysates derived from HEK293T cells expressing HA-tagged OTUB1 in the excess of GTP-γ-S or GDP, followed by immunoblotting with the indicated antibodies. OTUB1 interacts with wt KRAS. Flag-tagged KRAS was immunoprecipitated using anti-Flag (M2) agarose from HEK293T cells overexpressing HA-tagged OTUB1 or empty vector (V). OTUB1 co-localizes with KRAS at the plasma membrane. At 24 h after co-transfection with GFP-tagged KRAS and HA–tagged OTUB1, HeLa cells were immunostained with anti-HA antibody. The outlined areas are shown at higher magnification at the top of each image. Scale bar, 10 μm. Data information: (C–F) IP, immunoprecipitates; WCL, whole cell lysate. Source data are available online for this figure. Source Data for Figure 1 [emmm201505972-sup-0004-SDataFig1.pdf] Download figure Download PowerPoint Using a set of reciprocal immunoprecipitations, we confirmed that OTUB1 interacted with RAS proteins (Fig 1C–F). We found that OTUB1 formed a complex with either wt NRAS or constitutively active form of NRAS Q61K (Fig 1D). Consistently with this observation, both inactive GDP-bound and active GTP-γ-S-bound forms of NRAS interacted with OTUB1 (Fig 1E), indicating that GTP binding does not significantly affect the interaction between RAS and OTUB1. Furthermore, immunofluorescence analysis revealed that OTUB1 and wt KRAS co-localized at the plasma membrane (Fig 1G). These results strongly indicate that OTUB1 interacts with RAS proteins in a GTP-independent manner, suggesting that OTUB1 is an upstream regulator of RAS GTPases. We next investigated whether OTUB1 is implicated in the regulation of RAS ubiquitination. Consistently with previous reports (Jura et al, 2006; Sasaki et al, 2011), we found that all RAS proteins undergo mono- and di-ubiquitination (Fig 2A–D). Suppression of OTUB1 with two different shRNAs resulted in increased levels of NRAS monoubiquitination (Fig 2A), whereas overexpression of wt OTUB1 almost completely abolished ubiquitination of RAS proteins (Fig 2B–D). Figure 2. OTUB1 triggers membrane localization of RAS ubiquitination by inhibiting its ubiquitination A. Suppression of OTUB1 expression increases NRAS mono- and di-ubiquitination. 6×His-tagged ubiquitin and Flag-NRAS were introduced into HEK293T cells expressing shGFP or shRNAs against OTUB1. Ubiquitinated NRAS was purified by Co2+ metal affinity chromatography and detected by anti-Flag antibody. B–D. Catalytic activity of OTUB1 is not required to inhibit RAS ubiquitination. 6×His-tagged ubiquitin and RAS expression constructs were introduced into HEK293T cells expressing wt HA-OTUB1, the catalytically dead mutant HA-OTUB1 C91S, or empty vector (V). Ubiquitinated RAS was purified by Co2+ metal affinity chromatography and detected by anti-Flag antibody. E. OTUB1 induces membrane RAS re-localization. Confocal imaging of HeLa cells expressing the indicated constructs. For each sample, z-stacks obtained by scanning the sample from the apical to the basal layer. Step-size, 2 μm. Scale bar, 20 μm. F. RAS cellular distribution expressed as percentage of cells with specific RAS localization. For quantification of RAS localization, cells were randomly imaged using IN Cell Analyzer. RAS localization (> 200 cells) was scored as intracellular and diffused (IN), mostly at the plasma membrane (PM), or both intracellular and plasma membrane (PM/IN). P-value = 0.0005 as determined by chi-squared test, n = 3. Representative images of HeLa cells expressing GFP-tagged KRAS are shown in Appendix Fig S1. pull-down, PD. whole cell lysate, WCL. not modified, n.m. Source data are available online for this figure. Source Data for Figure 2 [emmm201505972-sup-0005-SDataFig2.pdf] Download figure Download PowerPoint OTUB1 has recently emerged as a unique DUB that binds to several classes of E2s, including Ubc13 and UbcH5C, and inhibits ubiquitination independently of its proteolytic activity (Nakada et al, 2010; Juang et al, 2012; Sato et al, 2012; Wiener et al, 2012). Therefore, we tested whether the catalytic activity of OTUB1 is essential to promote RAS de-ubiquitination. We found that catalytically inactive OTUB1 C91S-mutant (Edelmann et al, 2009) as well as wt OTUB1 dramatically decreased the ubiquitination levels of RAS, suggesting that OTUB1 may affect RAS ubiquitination by inhibiting the E2 ubiquitin-conjugating enzymes (Fig 2B and C). In fact, in vitro ubiquitination of RAS was abolished by wt OTUB1, but not by deltaN(1-30) OTUB1-mutant lacking binding to E2 (Fig EV2A). In contrast, incubation of ubiquitinated RAS with wt OTUB1 did not decrease levels of RAS ubiquitination, thus supporting the premise that OTUB1 functions via E2 inhibition independent of its catalytic activity (Fig EV2B). Click here to expand this figure. Figure EV2. OTUB1 inhibits RAS ubiquitination by suppressing E2 ligase activity OTUB1 blocks RAS ubiquitination by inhibiting E2 ligase activity. Recombinant Flag-tagged NRAS was incubated with UBE1, UbcH5C (UBE2D3), ubiquitin, RABEX5 (1-76), and either wild-type OTUB1 or deltaN(1-30)-OTUB1 mutant. Ubiquitination of recombinant NRAS was analyzed by immunoblotting using anti-Flag antibody. OTUB1 does not de-ubiquitinate NRAS in vitro. The whole cell lysate overexpressing of Flag-NRAS was incubated with HA-OTUB1 wt or C91S. Ubiquitinated RAS was purified by Co2+ metal affinity chromatography and detected by anti-Flag antibody. Source data are available online for this figure. Download figure Download PowerPoint Since previous reports demonstrated that reversible ubiquitination of RAS promotes its endosomal association (Jura et al, 2006), we tested whether OTUB1 affects the subcellular localization of RAS. Consistent with the observation that OTUB1 inhibits RAS ubiquitination, analysis of RAS localization revealed that OTUB1 overexpression augmented the presence of RAS proteins on the plasma membrane (Fig 2E and F; Appendix Fig S1). Hence, by inhibiting RAS ubiquitination, OTUB1 functions to hinder RAS re-localization from the plasma membrane thereby contributing to the spatial control of RAS-dependent cellular responses. OTUB1 triggers RAS activity and downstream signaling We next analyzed how OTUB1 affects RAS activity and signaling. We found that overexpression of OTUB1 in HEK293T cells led to hyperactivation of wt RAS upon serum stimulation (Fig 3A and B). In concordance with this result, OTUB1 overexpression triggered a significant increase in phospho-ERK1/2 levels at different time points after addition of serum (Fig 3C–E). We observed a similar overactivation of the MAPK pathway, when we overexpressed catalytically inactive OTUB1 C91S-mutant, indicating that catalytic activity of OTUB1 is not necessary to induce the MAPK pathway activation (Fig 3F). In contrast, OTUB1 overexpression did not dramatically affect phosphorylation levels of AKT1 (Appendix Fig S2A and B). The latter observation could be due to OTUB1-mediated inhibition of TRAF6 (Li et al, 2010) that plays a crucial role in AKT activation (Yang et al, 2009). Figure 3. OTUB1 increases RAS activity and enhances MAPK activation in wt RAS cells A, B. OTUB1 overexpression promotes serum-induced activation of endogenous wt RAS (A) or wt NRAS (B). GTP-bound RAS was pulled down from HEK293T cells expressing HA-tagged OTUB1 or empty vector (V) using recombinant RAF1 RBD conjugated to agarose beads. Input was controlled by immunoblotting using anti-panRAS or anti-Flag antibodies. C, D. OTUB1 enhances serum-induced ERK1/2 phosphorylation in endogenous wt RAS (C) or wt KRAS (D). Levels of phosphorylated ERK1/2 and total ERK1/2 in HEK293T cells expressing HA-tagged OTUB1 or empty vector (V) were analyzed by Meso Scale assay. The results are expressed as a mean of pERK1/2 levels relative to total ERK1/2 ± s.e.m. n = 2. P-values were determined by two-sided t-Test. E, F. Overexpression of wt OTUB1 (E) or the catalytically dead mutant OTUB1 C91S (F) promotes serum-induced MAPK activation in cell expressing wt NRAS. Whole cell lysates were analyzed by immunoblotting using the indicated antibodies. G, H. Overexpression of OTUB1 has no effect on serum-induced MAPK activation in cell expressing mutant NRAS Q61K or KRAS G12V. Whole cell lysates were analyzed by immunoblotting using antibodies as indicated. I. Immunoblot of OTUB1 overexpression in immortalized human embryonic kidney epithelial cells (HEK TEST), expressing empty vector (V), myristoylated AKT1 (myr-AKT), MEK1 D218, D222-mutant (MEKDD). J, K. OTUB1 cooperates with active AKT1 to promote anchorage-independent growth. Representative images of soft-agar colonies formed by HEK TEST cells expressing the indicated constructs. The number of soft agar colonies formed by cells expressing OTUB1 compared to cells expressing an empty vector. Data are presented as mean ± s.e.m. P-values were determined by two-sided t-Test, n = 2. Data information: (A–H) Serum-starved HEK293T cells expressing the indicated constructs were stimulated with 10% serum for the indicated time periods. Source data are available online for this figure. Source Data for Figure 3 [emmm201505972-sup-0006-SDataFig3.pdf] Download figure Download PowerPoint On the other hand, when we overexpressed OTUB1 in HEK293T cells expressing constitutively active RAS-mutants, NRAS Q61K or KRAS G12V, we did not observe any significant up-regulation of ERK1/2 phosphorylation, most likely because the MAPK pathway was already optimally active due to the introduction of the active RAS-mutants (Fig 3G and H). We also did not observe OTUB1-induced hyperactivation of the MAPK kinase pathway when we overexpressed a dominant-negative KRAS S17N-mutant, indicating that the effect of OTUB1 overexpression is RAS dependent (Appendix Fig S2C). Taken together, these data indicate that OTUB1 up-regulation leads to activation of wt RAS signaling. OTUB1 triggers cell transformation by inducing the MAPK cascade activation Hyperactivation of the MAPK signaling by OTUB1 overexpression suggests that OTUB1 overexpression may promote tumorigenic transformation. Multiple studies have demonstrated that the co-expression of the telomerase catalytic subunit (hTERT), the SV40 Large T (LT) and small t (ST) oncoproteins, and an activated allele of RAS (RAS G12V) renders a wide range of human cells tumorigenic (Zhao et al, 2004), while co-activation of the MAPK and PI3K pathways suffices to replace RAS G12V in human cell transformation (Boehm et al, 2007). We used immortalized, but non-malignant human embryonic kidney epithelial cells expressing hTERT, LT, and ST (HEK TEST cells) as a model to assess tumorigenic potential of OTUB1 (Boehm et al, 2007). Given that OTUB1 overexpression up-regulated the MAPK pathway, but did not affect AKT signaling, we hypothesized that OTUB1 could cooperate with myristoylated (myr) and therefore the constitutively active allele of AKT1 (myr-AKT) to promote cell transformation. In fact, overexpression of OTUB1 together with myr-AKT1 dramatically induced anchorage-independent growth, whereas OTUB1 alone was not sufficient to trigger soft agar colony formation (Fig 3I–K). On the other hand, OTUB1 did not further accelerate anchorage-independent colony formation of HEK TE cells overexpressing both a constitutively active MEK1 D218, D222 allele (MEKDD) and myr-AKT, further confirming that OTUB1 overexpression promotes tumorigenic transformation by inducing the MAPK cascade activation. OTUB1 is more frequently up-regulated in wt KRAS non-small-cell lung carcinomas Our results suggest that increased OTUB1 expression could be an alternative mechanism of RAS activation superseding that of RAS activating mutations. Analysis of The Cancer Genome Atlas (TCGA) revealed that gain of the 11q13.1 locus, where the OTUB1 gene resides, was commonly observed in both lung adenocarcinomas and lung squamous cell carcinomas (SCC) (Figs 4A and EV3A). Correlation analysis revealed a strong association between copy number variation of 11q13.1 locus and OTUB1 expression levels, suggesting that OTUB1 is commonly up-regulated in lung tumors due to gain of the 11q13 locus (Figs 4B and C, and EV3A and B). OTUB1 mRNA expression was also significantly up-regulated in about 50% of adenocarcinomas and about 80% of SCC compared to normal tissue samples (Fig 4D–F). These observations are further consolidated by the increase of OTUB1 in a majority of tumorigenic lesions compared to their respective matched normal samples (Fig EV3C and D). Figure 4. OTUB1 expression is up-regulated in wt KRAS lung tumors A. Gains of OTUB1 and KRAS mutation are mutually exclusive in lung adenocarcinomas. OncoPrint showing the distribution of KRAS somatic mutations and OTUB1 copy number alterations in TCGA lung adenocarcinomas and squamous cell carcinomas obtained from cBioPortal (Cerami et al, 2012; Gao et al, 2013). Co-occurrence analysis showing significant mutual exclusivity between KRAS mutation and OTUB1 gain. B, C. OTUB1 overexpression in TCGA lung carcinomas is associated with 11q13.1 copy number alteration. Pearson correlation of OTUB1 copy number (log2 ratio) with OTUB1 mRNA levels (RNAseq normalized read counts, log2 transformed) was analyzed. D–F. OTUB1 expression in TCGA lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (SCC) patients. Patients were stratified according to their OTUB1 mRNA levels and/or their KRAS status as described in 4. Box whisker plots represent OTUB1 mRNA expression levels in TCGA lung carcinoma patients determined by RNAseq analysis. P-values were determined by two-sided t-Test. Total number of patients, n. G. Gain of 11q13.1 locus is an early event in lung adenocarcinoma development. TCGA lung adenocarcinoma patients with diploid or gain of the OTUB1 locus were stratified according tumor stages (T1–T4). Total numbers of patients, n. Statistical comparison of the sample distributions were compared using Chi-square test. H. OTUB1 mRNA overexpression is an early event in lung adenocarcinoma development. TCGA lung adenocarcinoma patients were stratified by tumor stages (T1–T4) and OTUB1 expression levels as described in 4. Total numbers of patients, n. Statistical comparison of the sample distributions were compared using Chi-square test. I. KRAS mutation is a late event in lung adenocarcinoma progression. TCGA lung adenocarcinoma patients with different KRAS mutation status were stratified by tumor stages (T1–T4). Total numbers of patients, n. Statistical comparison of the sample distributions were compared using Chi-square test. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. OTUB1 expression is up-regulated in lung tumors A. Copy number alterations of the OTUB1 containing region in TCGA lung adenocarcinoma and squamous cell carcinoma. B. TCGA Lung adenocarcinoma and squamous cell carcinoma were stratified according their KRAS status and OTUB1 expression levels (neg/low,
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