Evolutionarily Conserved Protein ERH Controls CENP-E Mrna Splicing and Is Required for the Survival of KRAS Mutant Cancer Cells
Total Page：16
File Type：pdf, Size：1020Kb
Evolutionarily conserved protein ERH controls CENP-E PNAS PLUS mRNA splicing and is required for the survival of KRAS mutant cancer cells Meng-Tzu Wenga,b,c, Jih-Hsiang Leea, Shu-Chen Weid, Qiuning Lia, Sina Shahamatdara, Dennis Hsua, Aaron J. Schettere, Stephen Swatkoskif, Poonam Mannang, Susan Garfieldg, Marjan Gucekf, Marianne K. H. Kima, Christina M. Annunziataa, Chad J. Creightonh, Michael J. Emanuelei, Curtis C. Harrise, Jin-Chuan Sheud, Giuseppe Giacconea, and Ji Luoa,1 aMedical Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; bGraduate Institute of Clinical Medicine, National Taiwan University, Taipei 100, Taiwan; cFar-Eastern Memorial Hospital, Taipei 220, Taiwan; dDepartment of Internal Medicine, National Taiwan University Hospital and College of Medicine, Taipei 100, Taiwan; eLaboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; fProteomics Core Facility, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892; gConfocal Microscopy Core Facility, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; hDepartment of Medicine and Dan L. Duncan Cancer Center Division of Biostatistics, Baylor College of Medicine, Houston, TX 77030; and iDepartment of Genetics, Harvard Medical School and Brigham and Women’s Hospital, Boston, MA 02115 Edited by Bert Vogelstein, Johns Hopkins University, Baltimore, MD, and approved November 12, 2012 (received for review June 1, 2012) Cancers with Ras mutations represent a major therapeutic prob- anaphase-promoting complex (APC/C) that coordinately maintain lem. Recent RNAi screens have uncovered multiple nononcogene the fidelity of chromosome segregation (6). Symmetrical distribu- addiction pathways that are necessary for the survival of Ras mu- tion of chromosomes during mitosis is critical for genomic stability tant cells. Here, we identify the evolutionarily conserved gene en- and cell survival (8, 9). During metaphase, chromosomes con- hancer of rudimentary homolog (ERH), in which depletion causes gression from spindle poles to the metaphase midplate is driven by greater toxicity in cancer cells with mutations in the small GTPase the plus end-directed kinesin centromere protein E (CENP-E) (10). KRAS compared with KRAS WT cells. ERH interacts with the spliceo- Unattached kinetochores activate spindle assembly checkpoint some protein SNRPD3 and is required for the mRNA splicing of the proteins such as budding uninhibited by benzimidazoles 1 homolog CELL BIOLOGY mitotic motor protein CENP-E. Loss of ERH leads to loss of CENP-E (Bub1), MAD3/BUB1-related protein kinase (BubR1), and mitotic and consequently, chromosome congression defects. Gene expres- arrest deficient 2-like protein 1 (MAD2), which in turn, inhibit the sion profiling indicates that ERH is required for the expression of activity of APC/C to delay anaphase onset until all sister chromatids multiple cell cycle genes, and the gene expression signature result- are bioriented and properly attached to opposite spindle poles (11). ing from ERH down-regulation inversely correlates with KRAS sig- Many mitotic proteins are degraded by APC/C on mitotic exit. natures. Clinically, tumor ERH expression is inversely associated CENP-E is one such protein, and it is degraded on mitosis exit and with survival of colorectal cancer patients whose tumors harbor resynthesized in the next S-phase (12). Thus, the proper expression KRAS mutations. Together, these findings identify a role of ERH in and turnover of CENP-E during each cell cycle is necessary for mRNA splicing and mitosis, and they provide evidence that KRAS chromosome congression and genomic stability (13, 14). mutant cancer cells are dependent on ERH for their survival. In this report, we identify a candidate Ras synthetic lethal gene, enhancer of rudimentary homolog (ERH). ERH is a highly synthetic lethality | spliceosome conserved gene originally identified in Drosophila (15), and it has been implicated to play a role in nuclear gene expression (16– he Ras family of small GTPases is mutated in a significant 18). Here, we show that ERH interacts with the Sm protein Tfraction of human cancers, with high frequencies of muta- SNRPD3, and it plays a critical role in the mRNA splicing and tions in v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog therefore, expression of CENP-E. KRAS mutant colorectal (KRAS) found in colon, lung, and pancreatic cancers (1–4). Ras cancer (CRC) cells are more sensitive to the depletion of ERH proteins are activated by growth factor receptors, and they, in turn, protein. Consistent with this finding, low ERH expression is as- activate a number of downstream effector pathways to coordinate sociated with better survival in cancer patients whose tumors cell proliferation, survival, and migration. Somatic mutations in harbor KRAS mutations. Our findings suggest that targeted in- Ras frequently lead to its constitutive activation, which in turn, activation of splicing machinery could be exploited to thera- drives malignant growth. Cancer cells harboring mutations in peutically restrict the malignancy of Ras-driven cancer. KRAS often exhibit the classic behavior of oncogene addiction: they become dependent on the KRAS oncogene for growth and Results survival and therefore, are hypersensitive to the loss of KRAS Ras Mutant Cells Are Hypersensitive to ERH Depletion. We identified protein (5). Efforts to pharmacologically inactivate mutant KRAS ERH as a candidate KRAS synthetic lethal gene from a genome- have been unsuccessful thus far. To identify additional genetic de- pendencies in Ras mutant cells, we previously conducted a genome- wide shRNA synthetic lethal screen in isogenic KRAS mutant and WTcells(6).Inthisscreen,weidentified a surprisingly diverse set of Author contributions: M.-T.W. and J.L. designed research; M.-T.W., J.-H.L., S.-C.W., Q.L., genes whose depletion causes greater toxicity in KRAS mutant cells S. Shahamatdar, D.H., and S. Swatkoski performed research; S.-C.W., S. Swatkoski, P.M., compared with KRAS WT cells. Surprisingly, many of these genes S.G., M.G., C.M.A., C.C.H., J.-C.S., and G.G. contributed new reagents/analytic tools; do not directly partake in the Ras signaling network, but rather, they M.-T.W., J.-H.L., A.J.S., M.K.H.K, C.J.C., M.J.E., G.G., and J.L. analyzed the data; and act to maintain cell viability by alleviating the stress phenotypes in M.-T.W., J.H.L., and J.L. wrote the paper. cancer cells. We, therefore, proposed the concept of nononcogene The authors declare no conflict of interest. addiction to explain the heightened dependency of Ras mutant cells This article is a PNAS Direct Submission. on stress relief pathways for survival (7). Freely available online through the PNAS open access option. In the aforementioned screen, we identified genetic interactions 1To whom correspondence should be addressed. E-mail: [email protected]. between mutant KRAS and a network of mitotic genes, including This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the mitotic kinase polo-like kinase 1 (PLK1) and the E3 ligase 1073/pnas.1207673110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1207673110 PNAS Early Edition | 1of9 Downloaded by guest on September 26, 2021 − wide RNAi screen (6). ERH is a protein with 104 aa, and its breviated as KRAS mutant line, and the KRAS /wt derivative molecular function is poorly understood. To validate the genetic cells are abbreviated as the KRAS WT line. We found that, in interaction between ERH and the KRAS oncogene, we first each isogenic pair, the KRAS mutant cells showed less viability tested an shRNA targeting ERH that scored in the screen using on ERH knockdown compared with their respective KRAS WT DLD-1 and HCT116 isogenic cells that are either WT or mutant counterpart (Fig. 1A). Western blot and quantitative RT-PCR for KRAS. These isogenic cells were derived by targeted deletion (RT-qPCR) revealed a partial knockdown of ERH by this of the mutant KRASG13D allele (6, 19, 20). For simplicity, the shRNA (Fig. 1B and Fig. S1A). Because this shRNA is the only parental cell line with KRASG13D/wt genotype is hereafter ab- effective shRNA against ERH in our library, we identified two Fig. 1. Synthetic lethal interactions between ERH and the KRAS oncogene. (A) DLD-1 and HCT116 KRAS mutant cells show less viability compared with their respective WT control after retroviral ERH shRNA infection. Cell viability was assessed 4 d post-shRNA infection (error bars indicated SD of three independent experiments in all figures unless otherwise indicated). (B) Confirmation of ERH protein knockdown by ERH shRNA at 4 d post-shRNA infection. The number below each band indicates relative ERH protein level. (C) DLD-1 and HCT116 KRAS mutant cells show less viability compared with their respective KRAS WT control after ERH siRNA transfection. Cell viability was assessed 3 d post-siRNA transfection (assessment was the same for all siRNA viability experiment unless otherwise stated). (D) Confirmation of ERH protein knockdown by ERH siRNAs at 3 d post-siRNA transfection. (E) An HA-ERH cDNA rescue construct lacking the UTR regions of endogenous ERH remains sensitive to siERH-3, but it is resistant to siERH-5, which was confirmed by Western blot. (F) Stable expression of HA-ERH rescues the toxicity of siERH 5 in both DLD-1 and HCT116 KRAS mutant cells. (G) ERH depletion strongly decreases the ability of DLD-1 KRAS mutant cells to form colonies in soft agarose. Cells were transfected with indicated siRNAs and plated in soft agarose. Colonies were counted 14 d later. (H)Correlationof sensitivities to KRAS and ERH siRNAs in a panel of KRAS mutant (SW1116, SW620, SW403, LS123, and LOVO) and WT (RKO, CACO2, and SW48) CRC cell lines. 2of9 | www.pnas.org/cgi/doi/10.1073/pnas.1207673110 Weng et al.