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RETRACTED ARTICLE: Ras-ERK1/2 signaling contributes to the development of colorectal cancer via regulating H3K9ac

RETRACTED ARTICLE: Ras-ERK1/2 signaling contributes to the development of colorectal cancer via... Backgrounds/aims: Ras is a control switch of ERK1/2 pathway, and hyperactivation of Ras-ERK1/2 signaling appears frequently in human cancers. However, the molecular regulation following by Ras-ERK1/2 activation is still unclear. This work aimed to reveal whether Ras-ERK1/2 promoted the development of colorectal cancer via regulating H3K9ac. Methods: A vector for expression of K-Ras mutated at G12 V and T35S was transfected into SW48 cells, and the acetylation of H3K9 was measured by Western blot analysis. MTT assay, colony formation assay, transwell assay, chromatin immunoprecipitation and RT-qPCR were performed to detect whether H3K9ac was contributed to K-Ras-mediated cell growth and migration. Furthermore, whether HDAC2 and PCAF involved in modification of H3K9ac following Ras-ERK1/2 activation were studied. Results: K-Ras mutated at G12 V and T35S induced a significant activation of ERK1/2 signaling and a significant down- regulation of H3K9ac. Recovering H3K9 acetylation by using a mimicked H3K9ac expression vector attenuated the promoting effects of Ras-ERK1/2 on tumor cells growth and migration. Besides, H3K9ac can be deacetylated by HDAC2 and MDM2-depedent degradation of PCAF. Conclusion: H3K9ac was a specific target for Ras-ERK1/2 signaling pathway. H3K9 acetylation can be modulated by HDAC2 and MDM2-depedent degradation of PCAF. The revealed regulation provides a better understanding of Ras-ERK1/ 2 signaling in tumorigenesis. Keywords: Ras, ERK1/2, H3K9ac, HDAC2, PCAF, Colorectal cancer Background carcinogenesis when mutated at codon 12 [4]. In this Ras is a small GTPase. It has been considered as a control process, Glycine (G) in position of codon 12 is replaced by switch of ERK/MAPK pathway, and both of Ras and its Cysteine (C), Asparticacid (D), Serine (S), Arginine (R) or downstream signaling effector ERK/MAPK can modulate Valine (V). Of note, G12 V together with T35S and V45E the activation of PI3K, mTOR and AMPK pathways [1]. are most widely studied mutation sites of Ras.As reported Because of that, Ras plays a pivotal role in regulating mul- by Catalogue Of Somatic Mutations In Cancer (COSMIC), tiple cellular responses, such as proliferation, differenti- the incidence of K-Ras point mutation in human cancers ation, apoptosis, senescence, metabolism [2], and even is approximately 30%, with pancreatic cancer accounting cancer initiation and progression [3]. Ras protein is for 90%, colorectal cancer for 45%, and non-small cell encoded by three ubiquitously expressed genes: H-Ras, lung cancer for 35% (https://cancer.sanger.ac.uk/cosmic). K-Ras and N-Ras, among which K-Ras shows significant In eukaryotic cells, nucleosomes are composed of two each of histones H2A, H2B, H3 and H4. Recently, his- tone modification has been widely explored since its im- * Correspondence: huanzhou0002@sina.com Peng Tian and Yanfei Zhu contributed equally to this work. portant role in regulating tumorigenesis [5]. In the field Department of General Surgery, Zhengzhou University People’s Hospital of histone modification, histone acetylases (HATs) and (Henan Provincial People’s Hospital), No.7, Weiwu Road, Zhengzhou 450003, histone deacetylases (HDACs) are involved in alteration Henan, China Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Tian et al. BMC Cancer (2018) 18:1286 Page 2 of 10 of the chromatin structure, of which modulating gene plasmid (Clontech Inc., Palo Alto, CA). The amplified PCAF transcription. HDAC2, one type of HDACs, locates in and HDAC2 were subcloned into pcDNA6.0/HA-tag vector G12V/ nucleus and can function alone. It modulates gene ex- (Invitrogen, Carlsbad, CA, USA). The pEGFP-K-Ras T35S pression by deacetylating the N-terminal tails of the core construct was mutated using site-directed mutagenesis. histones, resulting in the tightening of the chromatin, The pEGFP-H3K9Q construct was constructed using the which reduces its accessibility for the transcriptional ma- TaKaRa MutanBEST Kit (#D401, TaKaRa, Dalian, China). chinery [6]. Recent years, acetylation of histone H3 has Two different sequences of siRNAs specific for HDAC2 become a hot topic in epigenetic regulation [7]. One of (si-HDAC2–1and si-HDAC2–2) were purchased from the widely studied acetylation site of histone H3 tails is GenePharma (Shanghai, China). A non-targeting sequence histone H3 lysine 9 (H3K9), produces the acetylated ly- was used as a negative control (si-con). MDM2-MU for ex- C464A sine 9 of histone H3 (H3K9ac). H3K9ac is also essen- pression of MDM2 was constructed and recombination tially related to transcriptional activation in human cells, into pIB/V5-His Vector (Invitrogen). and its hypoactivation is closely associated with the oc- SW48 cells were seeded in 6-well plates with a density of currence and development of multiple types of cancer 1×10 cells/well. When 50% confluence was researched, [7, 8]. More interestingly, H3K9ac can be specifically the cells were transfected with plasmids or siRNAs by modulated by HDAC2 in oligodendrocyte [9]. However, using lipofectamine 3000 (Invitrogen). At 48 h of transfec- the role of H3K9ac in colorectal cancer has not been tion, the culture medium was replaced by the complete well-studied yet. medium to stop transfection. Transfection efficiency was Previous studies have suggested that deregulation of Ras confirmed by using Western blot and/or RT-qPCR. signaling led to aberrant histone modification, resulting in cancer development. For instance, Ras-PI3K-AKT pathway Cell viability regulated histone H3 acetylation at lysine 56 (H3K56ac) The transfected SW48 cells were collected by using tryp- via the MDM2-dependent degradation, and thus regulat- sin (Sigma-Aldrich) and were seeded in 96-well plates ing tumor cells activity [10]. Another study demonstrated with a density of 5 × 10 cells/well. After 48 h of incuba- that Ras signaling showed oncogenic role through regulat- tion at 37 °C, 20 μL of MTT solution (Sigma-Aldrich) ing histone covalent modifications [11]. In this study, we with a final concentration of 5 mg/mL was added into established a link between Ras signaling and H3K9ac each well and the plates were incubated at 37 °C for an- modification, aiming to reveal one of the underlying mech- other 4 h. Then, the culture medium was removed and anisms of which K-Ras point mutation contributed to 100 μL dimethyl sulfoxide (DMSO, Sigma-Aldrich) was colorectal cancer cells growth and migration. added. Following 10 min of shaking in an ELISA reader (Bio-Rad Laboratories, Hercules, CA, USA), the absorb- Methods ance of each well was recorded at 570 nm. Cell culture and treatment Human colorectal cancer cell line SW48 purchased Transwell migrGation assay from American Type Culture Collection (Catalogue The transfected SW48 cells were collected and seeded in number: CCL-231™,ATCC,Manassas, VA,USA)was the upper side of 24-well transwell chamber with 8-μm cultured in Dulbecco’s Modified Eagle’s Medium pore filters (Costar, Boston, MA). The cells were main- (DMEM, Gibco, Grand Island, NY, USA) supplemented tained at serum-free culture medium. The lower side of with 10% heat-inactivated fetal bovine serum (FBS, the chamber was filled with 600 μL complete culture Gibco). The cells were maintained at 37 °C in a humidi- medium. After 12 h of incubation at 37 °C, the cells mi- fied atmosphere with 5% CO . grated into the lower side were fixed with methanol and MG132 (≥95% HPLC), an inhibitor of proteasome, was stained with 0.5% crystal violet (Beyotime, Shanghai, purchased from Sigma-Aldrich (St. Louis, MO, USA). China). The absorbance of cells that had been washed SW48 cells were treated by 25 μM MG132 for 0–12 h. with acetic acid was measured at 570 nm. SCH772984, SB203580, LY294002 and SP600125, the specific inhibitors for ERK1/2, MAPK, PI3K and JNK RNA extraction and RT-qPCR pathways were all purchased from Selleck Chemicals Total RNAs were extracted from the transfected cells by (Houston, TX). These inhibitors were used with concen- using the TRIzol reagent (Invitrogen). Five micrograms trations of 4 nM, 5 μM, 0.5 μM and 40 nM for treating of total RNA was subjected to reverse transcription cells for 12 h. using the Transcriptor First Strand cDNA Synthesis Kit (Roche, Basel, Switzerland). FastStart Universal SYBR Plasmid construction and cell transfection Green Master (Roche) was used in qPCR and each qPCR The coding regions of human wild-type K-Ras and H3 were was carried out in triplicate for a total of 20 μL reaction amplified by PCR and were subcloned into pEGFP-N1 mixtures on ABI PRISM 7500 Real-time PCR System Tian et al. BMC Cancer (2018) 18:1286 Page 3 of 10 (Applied Biosystems, Foster City, CA, USA). GAPDH were all purchased from Abcam (Cambridge, MA). served as an internal control. Data were analyzed ac- Followed by incubation with the secondary antibody for 1 h −ΔΔCt cording to the classic 2 method. at room temperature, the positive signal was detected by using enhanced chemiluminescence (ECL) and analyzed Soft-agar colony formation assay with ImageJ 1.49 (National Institutes of Health, Bethesda, Low melting agarose (Thermo Scientific®, Rockford, IL, MD, USA). USA) with concentration of 0.5% was placed in 6-well plates, and the plates were incubated at 4 °C for 30 min. Flow cytometric analysis of cell cycle distribution The transfected SW48 cells were seeded in 6-well with a The transfected SW48 cells in 6-well plates were cultured density of 600 cells/well, and were cultured in DMEM in serum-deprived medium for 12 h to synchronize cells containing 0.33% agarose at 37 °C for 2 weeks. The num- to G0-phase. Then, the cells were harvested by trypsinisa- ber of the colonies was counted microscopically. tion, washed twice with PBS and fixed in 70% ethanol at 4 °C overnight. The cells were re-suspended in the solution Western blot containing 0.2 mg/mL PI (Sigma-Aldrich), 0.1% Triton Total protein was extracted from the transfected SW48 cells X-100 (Invitrogen), and 20 μg/mL RNase A (Roche) for by using 1% Triton X-100 (Invitrogen) and 1 mM PMSF 30 min at room temperature in the dark. The percentage (pH 7.4) (Solarbio, Beijing, China) over ice for 30 min. The of cells in the G0/G1, S and G2/M phases of the cell cycle protein concentration was determined by BCA protein assay were analyzed by flow cytometry (FACS Calibur, Becton kit (Novagen, Madison, WI, USA). Equal amount of protein Dickson, San Jose, CA, USA) and ModFit software (Verity samples were subjected to SDS-PAGE and proteins were Software House, Topsham, ME, USA). transferred onto PVDF membranes (Millipore, Bedford, MA, USA). The membranes were blocked in 130 mM NaCl, 2.5 mM KCl, 10 mM Na HPO ,1.5mM KH PO ,0.1% Chromatin immunoprecipitation (ChIP) 2 4 2 4 Tween-20 and 5% BSA (pH 7.4) for 1 h at room The transfected SW48 cells (3 × 10 cells per sample) were temperature. Then the membranes were probed by primary incubated in 1% formaldehyde for 10 min at room antibodies overnight at 4 °C. Anti-p-ERK1/2 (MA5–15173) temperature, and the cells were collected and lysed in and anti-PCAF (MA5–11186) were purchased from Invitro- 200 μL SDS Lysis Buffer (Beyotime). After ultrasonication, gen; anti-ERK1/2 (ab17942), anti-H3K9ac (ab4441), anti-H3 the DNA was sheered to an average length of 200–800 bp. (ab12079), anti-HA (ab1424), anti-GFP (ab6556), anti-actin The samples were centrifuged at 10,000 g at 4 °C for 10 (ab8227), anti-His (ab197049), and anti-MDM2 (ab38618) min, and the supernatant was probed by anti-H3K9ac Fig. 1 Ras-ERK1/2 repressed H3K9 acetylation in SW48 cells. SW48 cells were transfected with empty pEGFP vector, pEGFP-K-Ras-WT (wild type) or G12V/T35S pEGFP-K-Ras construct. Protein levels of a p-ERK1/2 and b and c H3K9ac were measured by Western blot analysis. ** P < 0.01. Four inhibitors specific for ERK1/2, MAPK, PI3K and JNK pathways, i.e., SCH772984, SB203580, LY294002 and SP600125 were used to treat cells. Protein levels of d H3K9ac and e p-ERK1/2 were measured by Western blot analysis Tian et al. BMC Cancer (2018) 18:1286 Page 4 of 10 (ab4441, Abcam) and anti-PCAF (MA5–11186, Invitro- and transfected into SW48 cells. Figure 1a showed that gen) at 4 °C overnight. The sample treated by anti-IgG phosphorylation levels of ERK1/2 were remarkably G12V/T35S (ab190475, Abcam) was used as a blank control. After in- up-regulated in Ras group as compared to pEGFP cubation, 60 μL ProteinA Agarose/SalmonSperm DNA group (transfected with an empty plasmid), indicating (Thermo Scientific®, Rockford, IL, USA) was added, and ERK1/2 pathway was activated by K-Ras mutated at G12 V the samples were incubated at 4 °C for 2 h. The beads and T35S. Then, the expression changes of H3K9ac were washed sequentially for 10 min in low-salt wash buf- were measured by performing Western blot analysis. fer, high-salt wash buffer, LiCl wash buffer and TE buffer, Results in Fig. 1b and c displayed that, K-Ras mutated as previously described [12]. Lastly, the beads were at G12 V and T35S significantly down-regulated washed in 100 μL 10% SDS, 100 μL1M NaHCO ,and H3K9ac expression (P < 0.01), but has no effects on 800 μLddH O. 20 μL 5 M NaCl was added, and the cross- H3 expression. These data suggested that Ras-ERK1/2 links were reversed for 6 h at 65 °C. RT-qPCR was per- activation repressed the acetylation of H3 at lysine 9. formed to analyze the amount of immunoprecipitated In order to reveal whether H3K9 acetylation is specif- DNA and input DNA. ically mediated by ERK1/2, four inhibitors specific for ERK1/2, MAPK, PI3K and JNK pathways were used, Statistical analysis and the expression of H3K9ac was reassessed. As a Data presented as mean ± SEM. Statistical difference be- result, we found that only the inhibitor of ERK1/2 tween groups was analyzed by ANOVA following by Dun- (SCH772984) could recover H3K9ac expression fol- can post-hoc in SPSS 19.0 software (SPSS Inc., Chicago, lowing K-Ras mutation at G12 V and T35S (Fig. 1d IL, USA). A P-value of < 0.05 was considered significant. and e). No such effects were observed in cells treated with theinhibitorsspecificfor MAPK,PI3Kand JNK, Results i.e., SB203580, LY294002 and SP600125. These find- Ras-ERK1/2 repressed H3K9 acetylation in SW48 cells ings suggested that H3K9 acetylation was specifically To examine whether H3K9ac can be modulated by mediated by ERK1/2, rather than MAPK, PI3K and G12V/T35S Ras-ERK1/2 pathway, pEGFP-K-Ras was construct JNK. Fig. 2 Ras-ERK1/2 repressed H3K9 acetylation to promote the growth and migration of SW48 cells. a SW48 cells were transfected with pEGFP-H3, G12V/T35S G12V/T35S pEGFP-K-Ras plus pEGFP-H3, or pEGFP-K-Ras plus pEGFP-H3K9Q (with increasing amount 0.5, 1, and 2 g). a Transfection efficiency was G12V/T35S tested by Western blot. b MTT assay was performed to assess cell viability. Subsequently, pEGFP-H3, pEGFP-K-Ras plus pEGFP-H3, or pEGFP-K- G12V/T35S Ras plus pEGFP-H3K9Q (2 g) was transfected into cell, and c number of colonies and d cell migration were respectively determined by colony formation assay and transwell assay. ** P < 0.01; *** P <0.001 Tian et al. BMC Cancer (2018) 18:1286 Page 5 of 10 Ras-ERK1/2 repressed H3K9 acetylation to promote the growth and migration of SW48 cells Next, pEGFP-H3K9Q plasmid was constructed to mimic theacetylatedH3K9, andthe plasmidwas co-transfected G12V/T35S with pEGFP-K-Ras into SW48 cells. Transfection efficiency tested by western blotting revealed that H3K9ac expression was remarkably up-regulated by transfection with pEGFP-H3K9Q, in the presence and absence of G12V/T35S pEGFP-K-Ras (Fig. 2a). MTT assay result showed G12V/T35S that, co-transfection of cells with pEGFP-K-Ras and pEGFP-H3 significantly increased OD-value, compared to the transfection of pEGFP-H3 alone (P < 0.001, Fig. 2b). G12V/ Of note, co-transfection of cells with pEGFP-K-Ras T35S G12V/T35S and pEGFP-H3K9Q attenuated Ras -induced evaluation of OD-value (P < 0.001). Same trends were ob- served in Fig. 2c and d, colony number and OD-value in migration assay were both significantly increased in Ras +H3 group compared to GFP + H3 group (P <0.001). And they were both significantly decreased in Ras+H3K9Q group, as compared to Ras+H3 group (P < 0.01). Taken to- gether, recovering the acetylation of H3K9 attenuated the promoting effects of Ras-ERK1/2 on tumor cells growth Fig. 3 Ras-ERK1/2 repressed H3K9 acetylation to affect the transcription and migration. of Ras downstream genes. SW48 cells were transfected with pEGFP-H3, G12V/T35S G12V/T35S pEGFP-K-Ras plus pEGFP-H3, or pEGFP-K-Ras plus pEGFP- H3K9Q. a RT-qPCR was performed to assess the expression levels of Ras-ERK1/2 repressed H3K9 acetylation to affect the these genes. b ChIP was conducted to assess the levels of H3K9ac when transcription of Ras downstream genes different genes were expressed. * P <0.05; ** P < 0.01; *** P < 0.001 Next, the involvement of H3K9ac in the transcription of Ras downstream genes was addressed. RT-qPCR data in Fig. 3a showed that the mRNA levels of CYR61 cycle progression were tested to see the effect of HDAC2 (P < 0.01), IGFBP3 (P <0.01) and WNT16B (P < 0.05) silence on SW48 cells growth and migration. Figure 4c-e were significantly up-regulated, while the mRNA levels of demonstrated that both si-HDAC2–1 and si-HDAC2–2 NT5E (P < 0.001), GDF15 (P < 0.01), and CDC14A (P < 0.01) remarkably attenuated the effects of Ras-ERK1/2 ac- were significantly down-regulated in Ras+H3 group, as tivation on SW48 cells viability (P <0.01), migration compared to GFP + H3 group. However, the alteration (P < 0.001) and S-phase arrest. And also, the tran- of these mRNAs induced in Ras+H3 group were scription of CYR61 (P <0.01), IGFBP3 (P < 0.05), abolished in Ras+H3K9Q group. ChIP assay results WNT16B (P <0.05), NT5E (P <0.001), GDF15 (P < 0.001), in Fig. 3b showed that H3K9ac level was reduced at and CDC14A (P < 0.01) altered by Ras-ERK1/2 activation the promoters of these genes (P < 0.05, P <0.01 or P < 0.001) were attenuated by si-HDAC2–1 plus si-HDAC2–2 following the activation of Ras-ERK1/2. Based on (Fig. 4f). These data suggested that silence of HDAC2 these data, we speculated that Ras-ERK1/2 mediated recovered the acetylation of H3K9, which in turn the transcription of its downstream genes also via inhibited the growth and migratory capacities of regulating H3K9 acetylation. SW48 cells. Silence of HDAC2 recovered H3K9 acetylation and SW48 Ras-ERK1/2 repressed H3K9 acetylation in SW48 cells via cells phenotype degradation of PCAF Two different sequences of siRNAs specific for In order to reveal how Ras-ERK1/2 repressed H3K9 HDAC2 (si-HDAC2–1and si-HDAC2–2) were trans- acetylation, we focused on investigating PCAF,are- fected into SW48 cells. The mRNA levels of HDAC2 ported upstream gene of H3K9ac [13]. Figure 5adis- were remarkably silenced by siRNA transfection played that mRNA levels of PCAF and HDAC2 were G12V/T35S (Fig. 4a). Additionally, protein levels of H3K9ac were both unaffected by K-Ras .Figure 5b results indi- obviously up-regulated by siRNA transfection, even cated that the protein level of PCAF was down-regulated G12V/T35S G12V/T35S in K-Ras -expressing cells (Fig. 4b), suggest- by K-Ras , but the protein level of HDAC2 was ing H3K9 acetylation could be recovered by HDAC2 unaffected. These results implied that Ras-ERK1/2 silence. Subsequently, cell viability, migration, and cell post-transcriptionally down-regulated PCAF expression. Tian et al. BMC Cancer (2018) 18:1286 Page 6 of 10 Fig. 4 Silence of HDAC2 recovered H3K9 acetylation and SW48 cells phenotype. a The efficiency of siRNA-mediated HDAC2 silence was determined. b SW40 cells were transfected as indicated. The expression changes of H3K9ac were detected by Western blot analysis. c Cell viability, d migration, e cell cycle progression, and f several gene transcription were respectively assessed by MTT assay, transwell assay, flow cytometry and RT-qPCR. * P < 0.05; ** P < 0.01; *** P < 0.001 This was also confirmed in Fig. 5c, that both PCAF and Ras-ERK1/2 regulated H3K9ac via MDM2-mediated PCAF G12V/T35S H3K9ac protein expression was repressed in Ras degradation group. To further confirmed whether PCAF involved in It has been reported that the E3 ubiquitin ligase MDM2 the transcription of Ras downstream genes, ChIP was could bind to acetylases, such as p300/CBP or PCAF performed. Results from Fig. 5d showed that all of these [14]. Thus, we explored whether MDM2 was implicated G12V/T35S genes that exhibited reduced H3K9ac following in PCAF degradation in K-Ras -expressing cells. Ras-ERK1/2 activation also exhibited significant reduction Figure 6a and b showed that PCAF expression was grad- in PCAF binding, suggesting PCAF was responsible for ually repressed with MDM2 expression. However, in Ras-ERK1/2-repressed H3K9 acetylation. MDM2-mutant (MDM2-MU) transfected cells, no such Finally, SW48 cells were treated with MG132 (a prote- down-regulations were observed in PCAF expression asome inhibitor) to confirm whether PCAF regulated (Fig. 6c and d), indicating MDM2 was responsible for H3K9ac post-transcriptionally. Figure 5e showed that PCAF degradation. MG132 remarkably reversed the reduction of PCAF in Next, we established a link between H3K9ac expression G12V/T35S K-Ras -expressing cells. Results in Fig. 5f and MDM2 activity to reveal whether MDM2-mediated showed that H3K9ac levels were down-regulated by PCAF degradation was required to modulate Ras-ERK1/ G12V/T35S Ras after 48 h of transfection in absence of 2-repressed H3K9 acetylation. Figure 6eshowedthat, MG132. However, treating cells with 25 μM MG132 MDM2 was up-regulated, while H3K9ac was G12V/T35S gradually recovered the expression of H3K9ac (Fig. 5g). down-regulated in K-Ras -expressing cells. There- G12V/ Thus, it is possible that Ras-ERK1/2 pathway repressed after, the expression of MDM2 was repressed in K-Ras T35S H3K9 acetylation through regulating PCAF. -expressing cells by siRNA transfection (Fig. 6f). As a Tian et al. BMC Cancer (2018) 18:1286 Page 7 of 10 Fig. 5 Ras-ERK1/2 repressed H3K9 acetylation in SW48 cells via degradation of PCAF. a The mRNA level of PCAF after the indicated transfected was tested. b and c Western blot analysis was performed to measure the expression of PCAF, HDAC2, and H3K9ac following the indicated transfection. Anti-HA antibody was used for testing the exogenous levels of PCAF and HDAC2. d ChIP analysis for testing PCAF levels when different genes were expressed. e 25 μM of MG132 was used to treat cells, after which Western blot was performed to reassess PCAF level. Protein expression of H3K9ac was monitored in the f absence or g presence of MG132. ** P < 0.01; *** P < 0.001 result, we found that silence of MDM2 resulted in an cells growth, migration, and the transcription of several G12V/T35S up-regulation of H3K9ac in K-Ras -expressing cells tumor-associated genes. (Fig. 6g). Collectively, these data implied that Ras-ERK1/2 Histone H3 acetylation is a well-known modification regulates H3K9ac via MDM2-mediated PCAF degradation. process, which is often marks for the open up of chro- matin and activation of gene transcription [17]. To date, Discussion five isoforms of acetylated histone H3 proteins have In physiological conditions, inactive Ras (GDP-bound) been found. Depending on the acetylation sites of his- switches to active form (GTP-bound), and activates MEK tone H3, they are named as histone H3 acetylation at kinase, which in turn activates ERK kinase. The activation lysine 9 (H3K9ac), 14 (H3K14ac), 18 (H3K18ac), 23 of ERK subsequently phosphorylates a number of sub- (H3K23ac) and 27 (H3K27ac). The acetylation of histone strates, and thereby modulates cell fate [15]. Although Ras H3 has clinical diagnostic significance in many cancers, acted as a control switch in the activation of many signal- including epithelial ovarian tumor [18], hepatocellular ing pathways, it seems that ERK is one of the most im- carcinoma [19], oral cancer [8], and cervical cancer [20]. portant pathways which can be activated by Ras point Among these acetylated histone H3, H3K9ac is the most mutation [16]. This was also confirmed in this study, that widely studied one in cancer and other diseases. It has K-Ras mutated at G12 V and T35S induced a significant been suggested that H3K9 acetylation can be triggered activation of ERK1/2 signaling. Since hyperactivation of by external stimuli, such as long-term alcohol consump- Ras-ERK signaling pathway appears frequently in cancers, tion [21], and traffic-related air pollution [22]. Our study this signaling has been considered as a promising target for the first time suggested that H3K9 acetylation can be for controlling of cancers [15]. However, the molecular specifically catalyzed by Ras-ERK1/2 signaling, rather regulation following by Ras-ERK activation is still unclear. than MAPK, PI3K and JNK signaling. This work demonstrated that activation of Ras-ERK could The role of H3K9ac in colorectal cancer has been significantly repress the acetylation of H3K9 through sporadically studied. Lutz et al., demonstrated that high MDM2-dependent PCAF degradation. And also, the re- levels of H3K9ac were frequently occurred in patients pressed H3K9ac contributed to colorectal cancer SW48 with colorectal cancer [23]. Another study demonstrated Tian et al. BMC Cancer (2018) 18:1286 Page 8 of 10 G12V/T35S Fig. 6 Ras-ERK1/2 regulated H3K9ac via MDM2-mediated PCAF degradation. SW48 cells were transfected with pEGFP-K-Ras , PCAF-HA, MDM2-His (with increasing amount 0.5, 1, and 2 g) and pEGFP-N1. a Exogenous and b endogenous expression of PCAF was measured by Western blot. SW48 cells were transfected either by MDM2-His (2 g) or by MDM2 mutated type (MDM2-MU), then c exogenous and d endogenous expression of PCAF were reassessed. Anti-His and anti-HA antibodies were used for testing the exogenous levels of MDM2 and G12V/T35S PCAF, respectively. e MDM2 and H3K9ac expression in cells transfected with pEGFP-K-Ras or pEGFP-N1. f The protein level of MDM2 after siRNA transfection was tested. g After the indicated transfection, H3K9ac level was tested by Western blot that the expression pattern of H3K9ac was altered dur- colorectal cancer SW48 cells growth and migration fol- ing aging, which is a prime risk factor of the develop- lowing Ras-ERK1/2 activation. ment of colorectal cancer [24]. These two studies There are several genes have been found to be transcrip- suggested H3K9ac as a potential target for novel treat- tionally regulated by H3K9ac following Ras-ERK1/2 activa- ment option of colorectal cancer. However, a contrary tion in this study, further suggested H3K9ac as a finding was reported by Nakazawa et al., who demon- downstream effector of Ras-ERK1/2 signaling. All of the strated that H3K9ac expression was unchanged between studied genes are known to be closely related with tumor normal and neoplastic cell nulei in the colorectal cancers cells growth and migration. CYR61 expression was associ- [25]. Based on these previous studies, the role of ated with poor prognosis in patients with colorectal cancer H3K9ac in colorectal cancer is confusing. Herein, we [26] and it promotes cancer cells proliferation, invasion, attempted to study the in vitro effects of H3K9ac on survival, and metastasis [27, 28]. In addition to CYR61, colorectal cancer cells growth and migration, in order to IGFBP3 [29]and GDF15 [30] are also effective predictors reveal the exact function of H3K9ac in this cancer. By of outcomes in patients with colorectal cancer. WNT16B using a mimicked H3K9ac expression vector (H3K9Q), [31], NT5E [32], GPF15 [33], and CDC14A [34] are impli- G12V/T35S the expression of H3K9ac in K-Ras -transfected cated in tumorigenesis via regulating tumor growth and SW48 cells was recovered. As a result, the growth and EMT process. According to the findings reported else- migratory capacities of SW48 cells were both reduced, where, CYR61 [35], NT5E [36], WNT16B [37]and GDF15 suggesting the acetylation of H3K9 contributed to [38] were identified as oncogenes, while IGFBP3 [39]and Tian et al. BMC Cancer (2018) 18:1286 Page 9 of 10 CDC14A were found to be tumor-suppressive genes. In Consent for publication Not applicable. the current study, the expression of CYR61, IGFBP3, WNT16B was found to be down-regulated, whereas the ex- Competing interests pression of NT5E, GDF15, CDC14A was found to be The authors declare that they have no competing interests. up-regulated by H3K9ac. This phenomenon indicates the impacts of H3K9ac on colorectal cancer cells are complex, Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in since both oncogenes and tumor-suppressive genes can be published maps and institutional affiliations. up-regulated or down-regulated by H3K9ac. Additional in- vestigations are required to further analyze the pleiotropic Author details Department of Gastrointestinal Surgery, Zhengzhou University People’s effects of H3K9ac on cancer. Hospital (Henan Provincial People’s Hospital), Zhengzhou 450003, China. HATs and HDACs are two kind of key enzymes in 2 Department of General Surgery, Wuxi People’s Hospital of Nanjing Medical catalyzing the acetylation and deacetylation of H3. Hu- University, Wuxi 214023, China. Department of General Surgery, Zhengzhou University People’s Hospital (Henan Provincial People’s Hospital), No.7, Weiwu man HDACs can be divided into several large families: Road, Zhengzhou 450003, Henan, China. HDAC I (HDAC1, 2, 3 and 8), HDAC II (HDAC4, 5, 6, 7, 9 and 10), HDAC III (Sirt1-Sirt7), and HDAC IV Received: 14 August 2018 Accepted: 9 December 2018 (HDAC11) [40]. Human HATs include TIP60, MOZ/ MYST3, MORF/MYST4, HBO1/MYST2, MOF/MYST1, References P300, CBP, GCN5, PCAF, and ELP3 [41]. HDAC2 is 1. Slack C. Ras signaling in aging and metabolic regulation. Nutr Healthy Aging. 2017;4(3):195–205. highly expressed in patients with early stages of colorec- 2. Schlessinger J. Cell signaling by receptor tyrosine kinases. Cell. 2000;103(2): tal cancer, and the elevated HDAC2 expression is associ- 211–25. ated with the poor prognosis [42, 43]. In this study, we 3. Khan AQ, Kuttikrishnan S, Siveen KS, Prabhu KS, Shanmugakonar M, Al- Naemi HA, Haris M, Dermime S, Uddin S. 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RETRACTED ARTICLE: Ras-ERK1/2 signaling contributes to the development of colorectal cancer via regulating H3K9ac

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Springer Journals
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Copyright © The Author(s). 2018
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Biomedicine; Cancer Research; Oncology; Surgical Oncology; Health Promotion and Disease Prevention; Biomedicine, general; Medicine/Public Health, general
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10.1186/s12885-018-5199-3
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Abstract

Backgrounds/aims: Ras is a control switch of ERK1/2 pathway, and hyperactivation of Ras-ERK1/2 signaling appears frequently in human cancers. However, the molecular regulation following by Ras-ERK1/2 activation is still unclear. This work aimed to reveal whether Ras-ERK1/2 promoted the development of colorectal cancer via regulating H3K9ac. Methods: A vector for expression of K-Ras mutated at G12 V and T35S was transfected into SW48 cells, and the acetylation of H3K9 was measured by Western blot analysis. MTT assay, colony formation assay, transwell assay, chromatin immunoprecipitation and RT-qPCR were performed to detect whether H3K9ac was contributed to K-Ras-mediated cell growth and migration. Furthermore, whether HDAC2 and PCAF involved in modification of H3K9ac following Ras-ERK1/2 activation were studied. Results: K-Ras mutated at G12 V and T35S induced a significant activation of ERK1/2 signaling and a significant down- regulation of H3K9ac. Recovering H3K9 acetylation by using a mimicked H3K9ac expression vector attenuated the promoting effects of Ras-ERK1/2 on tumor cells growth and migration. Besides, H3K9ac can be deacetylated by HDAC2 and MDM2-depedent degradation of PCAF. Conclusion: H3K9ac was a specific target for Ras-ERK1/2 signaling pathway. H3K9 acetylation can be modulated by HDAC2 and MDM2-depedent degradation of PCAF. The revealed regulation provides a better understanding of Ras-ERK1/ 2 signaling in tumorigenesis. Keywords: Ras, ERK1/2, H3K9ac, HDAC2, PCAF, Colorectal cancer Background carcinogenesis when mutated at codon 12 [4]. In this Ras is a small GTPase. It has been considered as a control process, Glycine (G) in position of codon 12 is replaced by switch of ERK/MAPK pathway, and both of Ras and its Cysteine (C), Asparticacid (D), Serine (S), Arginine (R) or downstream signaling effector ERK/MAPK can modulate Valine (V). Of note, G12 V together with T35S and V45E the activation of PI3K, mTOR and AMPK pathways [1]. are most widely studied mutation sites of Ras.As reported Because of that, Ras plays a pivotal role in regulating mul- by Catalogue Of Somatic Mutations In Cancer (COSMIC), tiple cellular responses, such as proliferation, differenti- the incidence of K-Ras point mutation in human cancers ation, apoptosis, senescence, metabolism [2], and even is approximately 30%, with pancreatic cancer accounting cancer initiation and progression [3]. Ras protein is for 90%, colorectal cancer for 45%, and non-small cell encoded by three ubiquitously expressed genes: H-Ras, lung cancer for 35% (https://cancer.sanger.ac.uk/cosmic). K-Ras and N-Ras, among which K-Ras shows significant In eukaryotic cells, nucleosomes are composed of two each of histones H2A, H2B, H3 and H4. Recently, his- tone modification has been widely explored since its im- * Correspondence: huanzhou0002@sina.com Peng Tian and Yanfei Zhu contributed equally to this work. portant role in regulating tumorigenesis [5]. In the field Department of General Surgery, Zhengzhou University People’s Hospital of histone modification, histone acetylases (HATs) and (Henan Provincial People’s Hospital), No.7, Weiwu Road, Zhengzhou 450003, histone deacetylases (HDACs) are involved in alteration Henan, China Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Tian et al. BMC Cancer (2018) 18:1286 Page 2 of 10 of the chromatin structure, of which modulating gene plasmid (Clontech Inc., Palo Alto, CA). The amplified PCAF transcription. HDAC2, one type of HDACs, locates in and HDAC2 were subcloned into pcDNA6.0/HA-tag vector G12V/ nucleus and can function alone. It modulates gene ex- (Invitrogen, Carlsbad, CA, USA). The pEGFP-K-Ras T35S pression by deacetylating the N-terminal tails of the core construct was mutated using site-directed mutagenesis. histones, resulting in the tightening of the chromatin, The pEGFP-H3K9Q construct was constructed using the which reduces its accessibility for the transcriptional ma- TaKaRa MutanBEST Kit (#D401, TaKaRa, Dalian, China). chinery [6]. Recent years, acetylation of histone H3 has Two different sequences of siRNAs specific for HDAC2 become a hot topic in epigenetic regulation [7]. One of (si-HDAC2–1and si-HDAC2–2) were purchased from the widely studied acetylation site of histone H3 tails is GenePharma (Shanghai, China). A non-targeting sequence histone H3 lysine 9 (H3K9), produces the acetylated ly- was used as a negative control (si-con). MDM2-MU for ex- C464A sine 9 of histone H3 (H3K9ac). H3K9ac is also essen- pression of MDM2 was constructed and recombination tially related to transcriptional activation in human cells, into pIB/V5-His Vector (Invitrogen). and its hypoactivation is closely associated with the oc- SW48 cells were seeded in 6-well plates with a density of currence and development of multiple types of cancer 1×10 cells/well. When 50% confluence was researched, [7, 8]. More interestingly, H3K9ac can be specifically the cells were transfected with plasmids or siRNAs by modulated by HDAC2 in oligodendrocyte [9]. However, using lipofectamine 3000 (Invitrogen). At 48 h of transfec- the role of H3K9ac in colorectal cancer has not been tion, the culture medium was replaced by the complete well-studied yet. medium to stop transfection. Transfection efficiency was Previous studies have suggested that deregulation of Ras confirmed by using Western blot and/or RT-qPCR. signaling led to aberrant histone modification, resulting in cancer development. For instance, Ras-PI3K-AKT pathway Cell viability regulated histone H3 acetylation at lysine 56 (H3K56ac) The transfected SW48 cells were collected by using tryp- via the MDM2-dependent degradation, and thus regulat- sin (Sigma-Aldrich) and were seeded in 96-well plates ing tumor cells activity [10]. Another study demonstrated with a density of 5 × 10 cells/well. After 48 h of incuba- that Ras signaling showed oncogenic role through regulat- tion at 37 °C, 20 μL of MTT solution (Sigma-Aldrich) ing histone covalent modifications [11]. In this study, we with a final concentration of 5 mg/mL was added into established a link between Ras signaling and H3K9ac each well and the plates were incubated at 37 °C for an- modification, aiming to reveal one of the underlying mech- other 4 h. Then, the culture medium was removed and anisms of which K-Ras point mutation contributed to 100 μL dimethyl sulfoxide (DMSO, Sigma-Aldrich) was colorectal cancer cells growth and migration. added. Following 10 min of shaking in an ELISA reader (Bio-Rad Laboratories, Hercules, CA, USA), the absorb- Methods ance of each well was recorded at 570 nm. Cell culture and treatment Human colorectal cancer cell line SW48 purchased Transwell migrGation assay from American Type Culture Collection (Catalogue The transfected SW48 cells were collected and seeded in number: CCL-231™,ATCC,Manassas, VA,USA)was the upper side of 24-well transwell chamber with 8-μm cultured in Dulbecco’s Modified Eagle’s Medium pore filters (Costar, Boston, MA). The cells were main- (DMEM, Gibco, Grand Island, NY, USA) supplemented tained at serum-free culture medium. The lower side of with 10% heat-inactivated fetal bovine serum (FBS, the chamber was filled with 600 μL complete culture Gibco). The cells were maintained at 37 °C in a humidi- medium. After 12 h of incubation at 37 °C, the cells mi- fied atmosphere with 5% CO . grated into the lower side were fixed with methanol and MG132 (≥95% HPLC), an inhibitor of proteasome, was stained with 0.5% crystal violet (Beyotime, Shanghai, purchased from Sigma-Aldrich (St. Louis, MO, USA). China). The absorbance of cells that had been washed SW48 cells were treated by 25 μM MG132 for 0–12 h. with acetic acid was measured at 570 nm. SCH772984, SB203580, LY294002 and SP600125, the specific inhibitors for ERK1/2, MAPK, PI3K and JNK RNA extraction and RT-qPCR pathways were all purchased from Selleck Chemicals Total RNAs were extracted from the transfected cells by (Houston, TX). These inhibitors were used with concen- using the TRIzol reagent (Invitrogen). Five micrograms trations of 4 nM, 5 μM, 0.5 μM and 40 nM for treating of total RNA was subjected to reverse transcription cells for 12 h. using the Transcriptor First Strand cDNA Synthesis Kit (Roche, Basel, Switzerland). FastStart Universal SYBR Plasmid construction and cell transfection Green Master (Roche) was used in qPCR and each qPCR The coding regions of human wild-type K-Ras and H3 were was carried out in triplicate for a total of 20 μL reaction amplified by PCR and were subcloned into pEGFP-N1 mixtures on ABI PRISM 7500 Real-time PCR System Tian et al. BMC Cancer (2018) 18:1286 Page 3 of 10 (Applied Biosystems, Foster City, CA, USA). GAPDH were all purchased from Abcam (Cambridge, MA). served as an internal control. Data were analyzed ac- Followed by incubation with the secondary antibody for 1 h −ΔΔCt cording to the classic 2 method. at room temperature, the positive signal was detected by using enhanced chemiluminescence (ECL) and analyzed Soft-agar colony formation assay with ImageJ 1.49 (National Institutes of Health, Bethesda, Low melting agarose (Thermo Scientific®, Rockford, IL, MD, USA). USA) with concentration of 0.5% was placed in 6-well plates, and the plates were incubated at 4 °C for 30 min. Flow cytometric analysis of cell cycle distribution The transfected SW48 cells were seeded in 6-well with a The transfected SW48 cells in 6-well plates were cultured density of 600 cells/well, and were cultured in DMEM in serum-deprived medium for 12 h to synchronize cells containing 0.33% agarose at 37 °C for 2 weeks. The num- to G0-phase. Then, the cells were harvested by trypsinisa- ber of the colonies was counted microscopically. tion, washed twice with PBS and fixed in 70% ethanol at 4 °C overnight. The cells were re-suspended in the solution Western blot containing 0.2 mg/mL PI (Sigma-Aldrich), 0.1% Triton Total protein was extracted from the transfected SW48 cells X-100 (Invitrogen), and 20 μg/mL RNase A (Roche) for by using 1% Triton X-100 (Invitrogen) and 1 mM PMSF 30 min at room temperature in the dark. The percentage (pH 7.4) (Solarbio, Beijing, China) over ice for 30 min. The of cells in the G0/G1, S and G2/M phases of the cell cycle protein concentration was determined by BCA protein assay were analyzed by flow cytometry (FACS Calibur, Becton kit (Novagen, Madison, WI, USA). Equal amount of protein Dickson, San Jose, CA, USA) and ModFit software (Verity samples were subjected to SDS-PAGE and proteins were Software House, Topsham, ME, USA). transferred onto PVDF membranes (Millipore, Bedford, MA, USA). The membranes were blocked in 130 mM NaCl, 2.5 mM KCl, 10 mM Na HPO ,1.5mM KH PO ,0.1% Chromatin immunoprecipitation (ChIP) 2 4 2 4 Tween-20 and 5% BSA (pH 7.4) for 1 h at room The transfected SW48 cells (3 × 10 cells per sample) were temperature. Then the membranes were probed by primary incubated in 1% formaldehyde for 10 min at room antibodies overnight at 4 °C. Anti-p-ERK1/2 (MA5–15173) temperature, and the cells were collected and lysed in and anti-PCAF (MA5–11186) were purchased from Invitro- 200 μL SDS Lysis Buffer (Beyotime). After ultrasonication, gen; anti-ERK1/2 (ab17942), anti-H3K9ac (ab4441), anti-H3 the DNA was sheered to an average length of 200–800 bp. (ab12079), anti-HA (ab1424), anti-GFP (ab6556), anti-actin The samples were centrifuged at 10,000 g at 4 °C for 10 (ab8227), anti-His (ab197049), and anti-MDM2 (ab38618) min, and the supernatant was probed by anti-H3K9ac Fig. 1 Ras-ERK1/2 repressed H3K9 acetylation in SW48 cells. SW48 cells were transfected with empty pEGFP vector, pEGFP-K-Ras-WT (wild type) or G12V/T35S pEGFP-K-Ras construct. Protein levels of a p-ERK1/2 and b and c H3K9ac were measured by Western blot analysis. ** P < 0.01. Four inhibitors specific for ERK1/2, MAPK, PI3K and JNK pathways, i.e., SCH772984, SB203580, LY294002 and SP600125 were used to treat cells. Protein levels of d H3K9ac and e p-ERK1/2 were measured by Western blot analysis Tian et al. BMC Cancer (2018) 18:1286 Page 4 of 10 (ab4441, Abcam) and anti-PCAF (MA5–11186, Invitro- and transfected into SW48 cells. Figure 1a showed that gen) at 4 °C overnight. The sample treated by anti-IgG phosphorylation levels of ERK1/2 were remarkably G12V/T35S (ab190475, Abcam) was used as a blank control. After in- up-regulated in Ras group as compared to pEGFP cubation, 60 μL ProteinA Agarose/SalmonSperm DNA group (transfected with an empty plasmid), indicating (Thermo Scientific®, Rockford, IL, USA) was added, and ERK1/2 pathway was activated by K-Ras mutated at G12 V the samples were incubated at 4 °C for 2 h. The beads and T35S. Then, the expression changes of H3K9ac were washed sequentially for 10 min in low-salt wash buf- were measured by performing Western blot analysis. fer, high-salt wash buffer, LiCl wash buffer and TE buffer, Results in Fig. 1b and c displayed that, K-Ras mutated as previously described [12]. Lastly, the beads were at G12 V and T35S significantly down-regulated washed in 100 μL 10% SDS, 100 μL1M NaHCO ,and H3K9ac expression (P < 0.01), but has no effects on 800 μLddH O. 20 μL 5 M NaCl was added, and the cross- H3 expression. These data suggested that Ras-ERK1/2 links were reversed for 6 h at 65 °C. RT-qPCR was per- activation repressed the acetylation of H3 at lysine 9. formed to analyze the amount of immunoprecipitated In order to reveal whether H3K9 acetylation is specif- DNA and input DNA. ically mediated by ERK1/2, four inhibitors specific for ERK1/2, MAPK, PI3K and JNK pathways were used, Statistical analysis and the expression of H3K9ac was reassessed. As a Data presented as mean ± SEM. Statistical difference be- result, we found that only the inhibitor of ERK1/2 tween groups was analyzed by ANOVA following by Dun- (SCH772984) could recover H3K9ac expression fol- can post-hoc in SPSS 19.0 software (SPSS Inc., Chicago, lowing K-Ras mutation at G12 V and T35S (Fig. 1d IL, USA). A P-value of < 0.05 was considered significant. and e). No such effects were observed in cells treated with theinhibitorsspecificfor MAPK,PI3Kand JNK, Results i.e., SB203580, LY294002 and SP600125. These find- Ras-ERK1/2 repressed H3K9 acetylation in SW48 cells ings suggested that H3K9 acetylation was specifically To examine whether H3K9ac can be modulated by mediated by ERK1/2, rather than MAPK, PI3K and G12V/T35S Ras-ERK1/2 pathway, pEGFP-K-Ras was construct JNK. Fig. 2 Ras-ERK1/2 repressed H3K9 acetylation to promote the growth and migration of SW48 cells. a SW48 cells were transfected with pEGFP-H3, G12V/T35S G12V/T35S pEGFP-K-Ras plus pEGFP-H3, or pEGFP-K-Ras plus pEGFP-H3K9Q (with increasing amount 0.5, 1, and 2 g). a Transfection efficiency was G12V/T35S tested by Western blot. b MTT assay was performed to assess cell viability. Subsequently, pEGFP-H3, pEGFP-K-Ras plus pEGFP-H3, or pEGFP-K- G12V/T35S Ras plus pEGFP-H3K9Q (2 g) was transfected into cell, and c number of colonies and d cell migration were respectively determined by colony formation assay and transwell assay. ** P < 0.01; *** P <0.001 Tian et al. BMC Cancer (2018) 18:1286 Page 5 of 10 Ras-ERK1/2 repressed H3K9 acetylation to promote the growth and migration of SW48 cells Next, pEGFP-H3K9Q plasmid was constructed to mimic theacetylatedH3K9, andthe plasmidwas co-transfected G12V/T35S with pEGFP-K-Ras into SW48 cells. Transfection efficiency tested by western blotting revealed that H3K9ac expression was remarkably up-regulated by transfection with pEGFP-H3K9Q, in the presence and absence of G12V/T35S pEGFP-K-Ras (Fig. 2a). MTT assay result showed G12V/T35S that, co-transfection of cells with pEGFP-K-Ras and pEGFP-H3 significantly increased OD-value, compared to the transfection of pEGFP-H3 alone (P < 0.001, Fig. 2b). G12V/ Of note, co-transfection of cells with pEGFP-K-Ras T35S G12V/T35S and pEGFP-H3K9Q attenuated Ras -induced evaluation of OD-value (P < 0.001). Same trends were ob- served in Fig. 2c and d, colony number and OD-value in migration assay were both significantly increased in Ras +H3 group compared to GFP + H3 group (P <0.001). And they were both significantly decreased in Ras+H3K9Q group, as compared to Ras+H3 group (P < 0.01). Taken to- gether, recovering the acetylation of H3K9 attenuated the promoting effects of Ras-ERK1/2 on tumor cells growth Fig. 3 Ras-ERK1/2 repressed H3K9 acetylation to affect the transcription and migration. of Ras downstream genes. SW48 cells were transfected with pEGFP-H3, G12V/T35S G12V/T35S pEGFP-K-Ras plus pEGFP-H3, or pEGFP-K-Ras plus pEGFP- H3K9Q. a RT-qPCR was performed to assess the expression levels of Ras-ERK1/2 repressed H3K9 acetylation to affect the these genes. b ChIP was conducted to assess the levels of H3K9ac when transcription of Ras downstream genes different genes were expressed. * P <0.05; ** P < 0.01; *** P < 0.001 Next, the involvement of H3K9ac in the transcription of Ras downstream genes was addressed. RT-qPCR data in Fig. 3a showed that the mRNA levels of CYR61 cycle progression were tested to see the effect of HDAC2 (P < 0.01), IGFBP3 (P <0.01) and WNT16B (P < 0.05) silence on SW48 cells growth and migration. Figure 4c-e were significantly up-regulated, while the mRNA levels of demonstrated that both si-HDAC2–1 and si-HDAC2–2 NT5E (P < 0.001), GDF15 (P < 0.01), and CDC14A (P < 0.01) remarkably attenuated the effects of Ras-ERK1/2 ac- were significantly down-regulated in Ras+H3 group, as tivation on SW48 cells viability (P <0.01), migration compared to GFP + H3 group. However, the alteration (P < 0.001) and S-phase arrest. And also, the tran- of these mRNAs induced in Ras+H3 group were scription of CYR61 (P <0.01), IGFBP3 (P < 0.05), abolished in Ras+H3K9Q group. ChIP assay results WNT16B (P <0.05), NT5E (P <0.001), GDF15 (P < 0.001), in Fig. 3b showed that H3K9ac level was reduced at and CDC14A (P < 0.01) altered by Ras-ERK1/2 activation the promoters of these genes (P < 0.05, P <0.01 or P < 0.001) were attenuated by si-HDAC2–1 plus si-HDAC2–2 following the activation of Ras-ERK1/2. Based on (Fig. 4f). These data suggested that silence of HDAC2 these data, we speculated that Ras-ERK1/2 mediated recovered the acetylation of H3K9, which in turn the transcription of its downstream genes also via inhibited the growth and migratory capacities of regulating H3K9 acetylation. SW48 cells. Silence of HDAC2 recovered H3K9 acetylation and SW48 Ras-ERK1/2 repressed H3K9 acetylation in SW48 cells via cells phenotype degradation of PCAF Two different sequences of siRNAs specific for In order to reveal how Ras-ERK1/2 repressed H3K9 HDAC2 (si-HDAC2–1and si-HDAC2–2) were trans- acetylation, we focused on investigating PCAF,are- fected into SW48 cells. The mRNA levels of HDAC2 ported upstream gene of H3K9ac [13]. Figure 5adis- were remarkably silenced by siRNA transfection played that mRNA levels of PCAF and HDAC2 were G12V/T35S (Fig. 4a). Additionally, protein levels of H3K9ac were both unaffected by K-Ras .Figure 5b results indi- obviously up-regulated by siRNA transfection, even cated that the protein level of PCAF was down-regulated G12V/T35S G12V/T35S in K-Ras -expressing cells (Fig. 4b), suggest- by K-Ras , but the protein level of HDAC2 was ing H3K9 acetylation could be recovered by HDAC2 unaffected. These results implied that Ras-ERK1/2 silence. Subsequently, cell viability, migration, and cell post-transcriptionally down-regulated PCAF expression. Tian et al. BMC Cancer (2018) 18:1286 Page 6 of 10 Fig. 4 Silence of HDAC2 recovered H3K9 acetylation and SW48 cells phenotype. a The efficiency of siRNA-mediated HDAC2 silence was determined. b SW40 cells were transfected as indicated. The expression changes of H3K9ac were detected by Western blot analysis. c Cell viability, d migration, e cell cycle progression, and f several gene transcription were respectively assessed by MTT assay, transwell assay, flow cytometry and RT-qPCR. * P < 0.05; ** P < 0.01; *** P < 0.001 This was also confirmed in Fig. 5c, that both PCAF and Ras-ERK1/2 regulated H3K9ac via MDM2-mediated PCAF G12V/T35S H3K9ac protein expression was repressed in Ras degradation group. To further confirmed whether PCAF involved in It has been reported that the E3 ubiquitin ligase MDM2 the transcription of Ras downstream genes, ChIP was could bind to acetylases, such as p300/CBP or PCAF performed. Results from Fig. 5d showed that all of these [14]. Thus, we explored whether MDM2 was implicated G12V/T35S genes that exhibited reduced H3K9ac following in PCAF degradation in K-Ras -expressing cells. Ras-ERK1/2 activation also exhibited significant reduction Figure 6a and b showed that PCAF expression was grad- in PCAF binding, suggesting PCAF was responsible for ually repressed with MDM2 expression. However, in Ras-ERK1/2-repressed H3K9 acetylation. MDM2-mutant (MDM2-MU) transfected cells, no such Finally, SW48 cells were treated with MG132 (a prote- down-regulations were observed in PCAF expression asome inhibitor) to confirm whether PCAF regulated (Fig. 6c and d), indicating MDM2 was responsible for H3K9ac post-transcriptionally. Figure 5e showed that PCAF degradation. MG132 remarkably reversed the reduction of PCAF in Next, we established a link between H3K9ac expression G12V/T35S K-Ras -expressing cells. Results in Fig. 5f and MDM2 activity to reveal whether MDM2-mediated showed that H3K9ac levels were down-regulated by PCAF degradation was required to modulate Ras-ERK1/ G12V/T35S Ras after 48 h of transfection in absence of 2-repressed H3K9 acetylation. Figure 6eshowedthat, MG132. However, treating cells with 25 μM MG132 MDM2 was up-regulated, while H3K9ac was G12V/T35S gradually recovered the expression of H3K9ac (Fig. 5g). down-regulated in K-Ras -expressing cells. There- G12V/ Thus, it is possible that Ras-ERK1/2 pathway repressed after, the expression of MDM2 was repressed in K-Ras T35S H3K9 acetylation through regulating PCAF. -expressing cells by siRNA transfection (Fig. 6f). As a Tian et al. BMC Cancer (2018) 18:1286 Page 7 of 10 Fig. 5 Ras-ERK1/2 repressed H3K9 acetylation in SW48 cells via degradation of PCAF. a The mRNA level of PCAF after the indicated transfected was tested. b and c Western blot analysis was performed to measure the expression of PCAF, HDAC2, and H3K9ac following the indicated transfection. Anti-HA antibody was used for testing the exogenous levels of PCAF and HDAC2. d ChIP analysis for testing PCAF levels when different genes were expressed. e 25 μM of MG132 was used to treat cells, after which Western blot was performed to reassess PCAF level. Protein expression of H3K9ac was monitored in the f absence or g presence of MG132. ** P < 0.01; *** P < 0.001 result, we found that silence of MDM2 resulted in an cells growth, migration, and the transcription of several G12V/T35S up-regulation of H3K9ac in K-Ras -expressing cells tumor-associated genes. (Fig. 6g). Collectively, these data implied that Ras-ERK1/2 Histone H3 acetylation is a well-known modification regulates H3K9ac via MDM2-mediated PCAF degradation. process, which is often marks for the open up of chro- matin and activation of gene transcription [17]. To date, Discussion five isoforms of acetylated histone H3 proteins have In physiological conditions, inactive Ras (GDP-bound) been found. Depending on the acetylation sites of his- switches to active form (GTP-bound), and activates MEK tone H3, they are named as histone H3 acetylation at kinase, which in turn activates ERK kinase. The activation lysine 9 (H3K9ac), 14 (H3K14ac), 18 (H3K18ac), 23 of ERK subsequently phosphorylates a number of sub- (H3K23ac) and 27 (H3K27ac). The acetylation of histone strates, and thereby modulates cell fate [15]. Although Ras H3 has clinical diagnostic significance in many cancers, acted as a control switch in the activation of many signal- including epithelial ovarian tumor [18], hepatocellular ing pathways, it seems that ERK is one of the most im- carcinoma [19], oral cancer [8], and cervical cancer [20]. portant pathways which can be activated by Ras point Among these acetylated histone H3, H3K9ac is the most mutation [16]. This was also confirmed in this study, that widely studied one in cancer and other diseases. It has K-Ras mutated at G12 V and T35S induced a significant been suggested that H3K9 acetylation can be triggered activation of ERK1/2 signaling. Since hyperactivation of by external stimuli, such as long-term alcohol consump- Ras-ERK signaling pathway appears frequently in cancers, tion [21], and traffic-related air pollution [22]. Our study this signaling has been considered as a promising target for the first time suggested that H3K9 acetylation can be for controlling of cancers [15]. However, the molecular specifically catalyzed by Ras-ERK1/2 signaling, rather regulation following by Ras-ERK activation is still unclear. than MAPK, PI3K and JNK signaling. This work demonstrated that activation of Ras-ERK could The role of H3K9ac in colorectal cancer has been significantly repress the acetylation of H3K9 through sporadically studied. Lutz et al., demonstrated that high MDM2-dependent PCAF degradation. And also, the re- levels of H3K9ac were frequently occurred in patients pressed H3K9ac contributed to colorectal cancer SW48 with colorectal cancer [23]. Another study demonstrated Tian et al. BMC Cancer (2018) 18:1286 Page 8 of 10 G12V/T35S Fig. 6 Ras-ERK1/2 regulated H3K9ac via MDM2-mediated PCAF degradation. SW48 cells were transfected with pEGFP-K-Ras , PCAF-HA, MDM2-His (with increasing amount 0.5, 1, and 2 g) and pEGFP-N1. a Exogenous and b endogenous expression of PCAF was measured by Western blot. SW48 cells were transfected either by MDM2-His (2 g) or by MDM2 mutated type (MDM2-MU), then c exogenous and d endogenous expression of PCAF were reassessed. Anti-His and anti-HA antibodies were used for testing the exogenous levels of MDM2 and G12V/T35S PCAF, respectively. e MDM2 and H3K9ac expression in cells transfected with pEGFP-K-Ras or pEGFP-N1. f The protein level of MDM2 after siRNA transfection was tested. g After the indicated transfection, H3K9ac level was tested by Western blot that the expression pattern of H3K9ac was altered dur- colorectal cancer SW48 cells growth and migration fol- ing aging, which is a prime risk factor of the develop- lowing Ras-ERK1/2 activation. ment of colorectal cancer [24]. These two studies There are several genes have been found to be transcrip- suggested H3K9ac as a potential target for novel treat- tionally regulated by H3K9ac following Ras-ERK1/2 activa- ment option of colorectal cancer. However, a contrary tion in this study, further suggested H3K9ac as a finding was reported by Nakazawa et al., who demon- downstream effector of Ras-ERK1/2 signaling. All of the strated that H3K9ac expression was unchanged between studied genes are known to be closely related with tumor normal and neoplastic cell nulei in the colorectal cancers cells growth and migration. CYR61 expression was associ- [25]. Based on these previous studies, the role of ated with poor prognosis in patients with colorectal cancer H3K9ac in colorectal cancer is confusing. Herein, we [26] and it promotes cancer cells proliferation, invasion, attempted to study the in vitro effects of H3K9ac on survival, and metastasis [27, 28]. In addition to CYR61, colorectal cancer cells growth and migration, in order to IGFBP3 [29]and GDF15 [30] are also effective predictors reveal the exact function of H3K9ac in this cancer. By of outcomes in patients with colorectal cancer. WNT16B using a mimicked H3K9ac expression vector (H3K9Q), [31], NT5E [32], GPF15 [33], and CDC14A [34] are impli- G12V/T35S the expression of H3K9ac in K-Ras -transfected cated in tumorigenesis via regulating tumor growth and SW48 cells was recovered. As a result, the growth and EMT process. According to the findings reported else- migratory capacities of SW48 cells were both reduced, where, CYR61 [35], NT5E [36], WNT16B [37]and GDF15 suggesting the acetylation of H3K9 contributed to [38] were identified as oncogenes, while IGFBP3 [39]and Tian et al. BMC Cancer (2018) 18:1286 Page 9 of 10 CDC14A were found to be tumor-suppressive genes. In Consent for publication Not applicable. the current study, the expression of CYR61, IGFBP3, WNT16B was found to be down-regulated, whereas the ex- Competing interests pression of NT5E, GDF15, CDC14A was found to be The authors declare that they have no competing interests. up-regulated by H3K9ac. This phenomenon indicates the impacts of H3K9ac on colorectal cancer cells are complex, Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in since both oncogenes and tumor-suppressive genes can be published maps and institutional affiliations. up-regulated or down-regulated by H3K9ac. Additional in- vestigations are required to further analyze the pleiotropic Author details Department of Gastrointestinal Surgery, Zhengzhou University People’s effects of H3K9ac on cancer. Hospital (Henan Provincial People’s Hospital), Zhengzhou 450003, China. HATs and HDACs are two kind of key enzymes in 2 Department of General Surgery, Wuxi People’s Hospital of Nanjing Medical catalyzing the acetylation and deacetylation of H3. Hu- University, Wuxi 214023, China. 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Journal

BMC Cancer – Springer Journals

Published: Dec 22, 2018

Keywords: Ras; ERK1/2; H3K9ac; HDAC2; PCAF; Colorectal cancer

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