Int J Clin Exp Pathol 2021;14(7):795-810 www.ijcep.com /ISSN:1936-2625/IJCEP0132218

Original Article Sumoylation of ETV1 modulates its oncogenic potential in prostate

Sangphil Oh1,2*, Sook Shin1,2*, Ralf Janknecht1,2,3

1Department of Cell Biology, 2Stephenson Cancer Center, 3Department of Pathology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA. *Equal contributors. Received February 21, 2021; Accepted June 21, 2021; Epub July 15, 2021; Published July 30, 2021

Abstract: The transcription factor ETS variant 1 (ETV1) is capable of promoting prostate tumorigenesis. We dem- onstrate that ETV1 can be posttranslationally modified by covalent attachment of small -like modifier 1 (SUMO1) onto four different lysine residues. In human embryonic kidney 293T cells, mutation of these sumoylation sites stimulated the transactivation potential of ETV1 at the matrix metalloproteinase 1 (MMP1), but not Yes-asso- ciated protein 1 promoter, while ETV1 protein stability and intracellular localization remained unchanged. In stark contrast, sumoylation-deficient ETV1 was repressed in its ability to stimulate the MMP1 promoter and to coop- erate with a histone demethylase, JmjC domain-containing 2A (JMJD2A), in LNCaP prostate cancer cells. Mutation of sumoylation sites enhanced the ability of ETV1 to interact with the histone deacetylase (HDAC) 1, but had basically no impact on complex formation with HDAC3 or JMJD2A. Further, compared to non-sumoylated ETV1, its sumoylated forms were less able to bind to the transcription factor, SMAD family member 4. Lastly, in contrast to wild-type ETV1, sumoylation-deficient ETV1 repressed LNCaP . Altogether, these data suggest that sumoylation modulates ETV1 function in a cell type-specific manner, possibly by altering the spectrum of transcriptional cofac- tors being recruited. Notably, SUMO pathway components SUMO1, ubiquitin-like modifier activating 2 and ubiquitin conjugating enzyme 9 were upregulated in prostate tumors, implying that enhanced sumoylation indeed promotes ETV1’s oncogenic activity during prostate cancer formation.

Keywords: ETV1, posttranslational modification, prostate cancer, SUMO, transcription

Introduction ETV1, N-terminal deletions, or fusion proteins that retain most of the ETV1 amino acids, ETS variant 1 (ETV1), formerly also designated including the ETS domain [7, 8]. Mimicking this as ETS-related 81 (ER81), is a member of a overexpression of ETV1 in transgenic mouse transcription factor family that comprises 28 models led to the development of prostatic proteins in humans and is characterized by the intraepithelial neoplasia, which is a precursor ETS DNA-binding domain, which interacts with of prostate adenocarcinoma [9, 10]. In addi- GGAA-containing target sequences [1, 2]. tion, ETV1 overexpression synergized with Knockout of ETV1 in mice led to defective con- homozygous deletion of the PTEN tumor sup- nections between sensory and motor neurons, pressor in the induction of adenocarcinomas in which caused motor discoordination and even- the mouse prostate [11]. ETV1 overexpression tually death approximately one month after was also associated with higher Gleason score birth [3]. Further, ETV1 is required for rapid con- and increased disease recurrence after prosta- duction in the heart, and overexpression of tectomy, suggesting that ETV1 especially marks ETV1 induced atrial arrhythmias in mice and aggressive prostate tumors [10-12]. has been observed in respective human patients [4-6]. ETV1 has also been implicated Similar to prostate cancer, ETV1 has been as a promoter of oncogenesis. Most significant- found to be translocated to the EWS gene in a ly, the ETV1 gene was found to be translocated subset of Ewing tumors. This generates an in 5-10% of all human prostate carcinomas, EWS-ETV1 fusion protein that is endowed with which results in overexpression of full-length the potent N-terminal activation domain of EWS Regulation of ETV1 by SUMO1 modification and the DNA-binding domain of ETV1 [13, 14]. Materials and methods A consequence is the constitutive activation of ETV1 target and potentially of other Preparation of protein extracts genes regulated by GGAA microsatellites that may facilitate neoplastic transformation [15]. Human embryonic kidney 293T cells were gro- Moreover, amplification of the ETV1 gene was wn in 12-wells and transiently transfected by reported to occur in up to 40% of all melano- the calcium phosphate coprecipitation method mas and ETV1 synergized with the V600E [38]. 50 ng 6Myc-ER81/ETV1 construct [34] or mutant of BRAF, which is a major driver of mela- empty vector pCS3+-6Myc were cotransfected nomagenesis, in the transformation of melano- with 50 or 500 ng HA-SUMO1 or 500 ng Flag3- cytes [16]. Overexpression of ETV1 was also SUMO1 (these SUMO1 expression vectors noted in colorectal tumors and its downregula- encode for amino acids 1-97 of human SUMO1). tion compromised the growth of colon cancer Total DNA amount was adjusted with pBlue- cells [17, 18]. script KS+ (Stratagene) to 2.2 µg. 36 h after transfection, cells were washed once with A role of ETV1 in tumorigenesis has also been phosphate-buffered saline and then incubated implicated in the breast. Overexpression of the on ice with 800 µl of 40 mM HEPES (pH 7.4), 10 receptor tyrosine kinase HER2/ERBB2, which mM EDTA, 150 mM NaCl with and without 10 is an underlying cause of breast cancer in ~20% mM N-ethylmaleimide (NEM; diluted from a of all cases, led to the overexpression of ETV1 freshly prepared 200 mM stock solution in in respective transgenic mice, and ETV1 is also H2O). After centrifugation (3 min, 2500 rpm), highly expressed in several human breast can- the cell pellet was lysed for one hour on ice with cer cell lines [19, 20]. Moreover, ETV1 is capa- 100 µl of 10 mM Tris-HCl, 150 mM NaCl, 50 ble of stimulating the HER2/ERBB2 gene pro- mM NaF, 30 mM Na4P2O7, 1% Triton X-100 (pH moter, and a feed-forward mechanism of upreg- 7.1) supplemented with 1 mM PMSF, 10 µg/ml ulation for these two proteins has even been leupeptin, 2 µg/ml aprotinin, 1 µg/ml pepstatin proposed [21]. Consistently, ETV1 appears to A, 0.5 mM Na3VO4 and with or without 10 mM be co-overexpressed with HER2/ERBB2 in NEM. The lysate was cleared by centrifugation human breast tumors [22, 23]. Further, down- and then subjected to SDS polyacrylamide gel regulation of ETV1 caused decreased growth of electrophoresis followed by anti- western human breast cancer cells in vitro and in a blotting [39]. To test sumoylation of ETV1 xenograft model [24]. Another receptor tyrosine mutants, 300 ng HA-SUMO1 or empty vector kinase, KIT, may also elevate ETV1 activity in pEV3S, 100 ng 6Myc-ER81/ETV1 constructs gastrointestinal stromal tumors and this (wild-type and point mutants) and 1800 ng appears to facilitate their growth [25, 26]. pBluescript KS+ were employed for the trans- fection of 293T cells, which were lysed in the Biochemical analysis indicates that ETV1 is presence of 10 mM NEM as above. heavily modified by posttranslational modifica- tion. In particular, phosphorylation by MAP Anti-SUMO1 immunoprecipitations kinases, MAPKAP kinases, PKA and ATR occurs on various serine and threonine residues and HeLa and SW620 cells grown in 10 cm dishes can affect ETV1’s transactivation potential, were washed once with ice-cold phosphate- DNA-binding activity, and/or protein stability buffered saline plus 10 mM NEM and then [27-33]. Likewise, the interaction with and acet- lysed as above in the presence of 10 mM NEM. ylation by the cofactor p300 affects the func- One 10-cm dish was lysed in a volume of tion of ETV1 [34-36]. An in vitro screen for pro- approximately 1.2 ml and subjected to immu- teins that become modified with SUMO1 (small noprecipitation with control (Santa Cruz Bio- ubiquitin-like modifier 1) identified ETV1 as a technology sc-7966) or anti-SUMO1 (Santa potential substrate for sumoylation [37], but Cruz Biotechnology sc-5308) mouse monoclo- the lysine residues being modified and the nal antibody essentially as described before function of this posttranslational modification [40]. This was followed by SDS polyacrylamide have remained unexplored. Hence, we studied gel electrophoresis and western blotting [41] in this report the relevance of the covalent with rabbit anti-ER81/ETV1 antibody #959 attachment of SUMO1 onto ETV1. [33].

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Luciferase assays jected to SDS-PAGE and western blotting with rabbit anti-ETV1 (Abcam ab81086) and anti- Human 293T cells were grown in poly-L-lysine- actin (Sigma A2066) antibodies. Quantitation coated 12-wells to ~25% confluency [42] and of signal intensities was done with a LICOR then transfected with 100 ng indicated lucifer- Odyssey imager. Similarly transfected 293T ase reporter plasmid, 900 ng pBluescript KS+ cells were fractionated with the NE-PER nuclear as a carrier, 30 ng of empty vector pEV3S or and cytoplasmic extraction kit (Pierce) essen- indicated ETV1 expression plasmids, and 4 ng tially as described [51]. empty vector pQCXIP or pQCXIP-H-Ras-G12V utilizing 2 µg polyethylenimine [43]. After 8 h, Coimmunoprecipitation and GST pulldown as- cells were washed once with phosphate-buff- says ered saline and then incubated for another 36 h in appropriate media before lysis with 300 µl 293T cells, which were grown in poly-L-lysine- of 25 mM Tris, 2 mM EDTA (pH 7.8 adjusted coated 6-cm plates to ~30% confluency [52], were transfected with 0.5-1 µg of 6Myc-ER81/ with H3PO4), 10% glycerol, 1% Triton X-100, 2 mM DTT [44]. Luciferase activities were then ETV1 (wild-type or 4xR mutant), 1.5 µg Flag- measured in a Berthold Lumat LB9507 lumi- JMJD2A, or 3 µg Flag-HDAC1 or Flag-HDAC3 nometer as described [45]. In case of LNCaP expression vectors. Total DNA was made up to cells, they were grown in poly-L-lysine-coated 4 µg with pBluescript KS+ and then 8 µg poly- 6-wells to ~30% confluency and transfected ethylenimine was employed for transfection with 1 µg luciferase reporter plasmid, 1 µg [43]. 10 h later, cells were washed once with 3 pBluescript KS+, indicated amounts of pEV3S ml phosphate-buffered saline, incubated for or ETV1 expression plasmids, and indicated another 36 h, and then lysed as described [53]. amounts of pEV3S or JMJD2A expression vec- Immunoprecipitations were then conducted tor utilizing 6 µg polyethylenimine. with anti-Flag monoclonal antibody M2 (Sigma- Aldrich) and coprecipitated proteins detected RT-PCR by anti-Myc western blotting essentially as described [54]. For GST pulldown assays, GST 293T cells were transfected in 12-wells as and GST-SMAD4 proteins were produced in above utilizing the calcium phosphate copre- Escherichia coli and purified with the help of cipitation method and RNA prepared employing glutathione agarose [55]. Binding of ETV1 pres- 0.5 ml Trizol [46]. After isopropanol precipita- ent in protein extracts from transfected 293T tion and washing with 75% ethanol, RNA was cells was then assessed in the presence of 10 dried and dissolved in 40 µl H2O [47]. Then, mM NEM as previously described [56]. RT-PCR was conducted as described [48] with MMP1 (5’-GTTCAGGGACAGAATGTGCTA-3’ and Cell growth assay 5’-CTGCAGTTGAACCAGCTATTAG-3’) or GAPDH (5’-GAGCCACATCGCTCAGACACC-3’ and 5’-TG- LNCaP cells were seeded into poly-L-lysine ACAAGCTTCCCGTTCTCAGC-3’) primers. Resul- coated 12-wells [57] and infected thrice every tant DNA fragments were separated on aga- 12 h with retrovirus (pQCXIH system, Clontech), rose gels [49] and visualized by staining with which was produced in 293T cells essentially ethidium bromide [50]. as described [58] and expressed untagged wild-type or 4xR ER81/ETV1. Then, cells were Cycloheximide treatment and cellular fraction- cultivated for two days without selection before ation seeding at a density of 3000 cells/well into 96-well plates [59]. Relative cell growth was Human 293T cells grown in poly-L-lysine-coat- determined with the PrestoBlue cell viability kit ed 6-cm plates were transfected with 3.6 µg (Thermo Fisher) by measuring the difference of + pBluescript KS and either 0.4 µg wild-type or absorbance at 570 nm and 595 nm according 4xR ER81/ETV1 expression plasmid as above. to the manufacturer’s recommendations. The next day, cells were split into 12-wells and another day later treated with 100 µg/ml cyclo- Statistical analysis heximide (diluted from a 100 mg/ml stock in DMSO) for 0-6 h. Thereafter, cells were lysed Data were analyzed with Prism 6 for Mac OS X and boiled in Laemmli buffer and lysates sub- (GraphPad Software, Inc.) and the significance

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Figure 1. Sumoylation of ETV1. A. 6Myc-tagged ETV1 was coexpressed with HA- (two different amounts) or Flag3- tagged SUMO1 in 293T cells. Cell extracts were prepared in the presence and absence of NEM and anti-Myc west- ern blotting performed. Arrows point at non-sumoylated ETV1, while asterisks mark sumoylated forms of ETV1. B. 6Myc-tagged SMAD4, EWS-ETV1 and ETV1 were expressed without or with HA-SUMO1 in 293T cells and protein extracts prepared in the presence of NEM. Shown is an anti-Myc western blot. C. Sumoylation of endogenous ETV1 in HeLa and SW620 cells. Protein extracts were subjected to immunoprecipitation with indicated antibodies and the presence of ETV1 was revealed by anti-ETV1 western blotting. level was set at P<0.05. Averages with stan- in ETV1. Please note that each sumoylation dard deviations are displayed. Statistical tests event added approximately 30-45 kDa appar- being utilized are detailed in the figure ent molecular weight onto ETV1, which is much legends. more than expected from the molecular weight of ~10 kDa for the tagged SUMO1 proteins. Results Such a phenomenon has often been observed for sumoylation in the literature, as this large Sumoylation of ETV1 modification leads to branched polypeptide To analyze whether ETV1 is sumoylated, we chains whose shape greatly hampers their chose human embryonic kidney 293T cells as a mobility in SDS polyacrylamide gels [60]. study object since they robustly express all To evaluate how efficiently ETV1 became SUMO pathway components. We expressed sumoylated, we compared it to SMAD family ETV1 without and with HA-tagged SUMO1 in member 4 (SMAD4), a transcription factor that 293T cells. Cell extracts were then prepared in can be sumoylated on two lysine residues [61- the presence or absence of N-ethylmaleimide 63], and the EWS-ETV1 fusion protein that is (NEM), an alkylating reagent that blocks cyste- ine peptidases (including SUMO proteases). present in Ewing tumors and encompasses the Expression of HA-SUMO1 led to a dose-depen- C-terminal 164 ETV1 amino acids [13]. We dent appearance of higher molecular weight observed that ETV1 was much more sumoylat- forms of ETV1 (marked by asterisks in Figure ed than EWS-ETV1 or SMAD4 (Figure 1B), and 1A), whose quantities were considerably only mono-sumoylation was detectable for reduced in the absence of NEM. Likewise, SMAD4 despite the presence of two sumoyla- expression of SUMO1 carrying three copies of a tion sites in this protein. Next, we assessed if Flag-tag resulted in higher molecular weight endogenous ETV1 would be sumoylated. forms of ETV1 (Figure 1A), and these were Sumoylation is a highly dynamic process and slightly larger than the corresponding bands the ratio of SUMO-modified to non-sumoylated upon HA-SUMO1 expression, since the three endogenous protein is normally tiny [64]. Flag tags add more molecular weight onto Hence, we were never able to detect endoge- SUMO1 than one HA tag. The fact that up to nous sumoylated ETV1 in western blots of cell four higher molecular weight forms of ETV1 extracts, which is why we resorted to immuno- were observed upon SUMO1 expression sug- precipitation to enrich for sumoylated ETV1. gested the presence of four sumoylation sites And indeed, when we immunoprecipitated with

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249-477 displayed two and amino acids 333-477 none (Figure 2A). Also, the fact that EWS-ETV1, which encompa- sses ETV1 amino acids 314- 477, was seemingly mono- sumoylated (see Figure 1B) is consistent with K317 being a site for SUMO1 attachment. We then mutated K89, K228, K257 and/or K317 to arginine and determined how this would affect total sumoyla- tion of ETV1. With the excep- tion of the R257 mutant, mutation of individual lysine residues had barely an effect on sumoylation (Figure 2B). However, mutation of two Figure 2. Identification of sumoylation sites. A. Indicated ETV1 amino acids lysine residues together alwa- fused to a 6Myc-tag were coexpressed without and with SUMO1 in 293T ys had a noticeable effect on cells. Shown are anti-Myc western blots. Arrowheads point to unmodified and asterisks at sumoylated proteins. On the left, a sketch of the human reducing total sumoylation, ETV1 protein depicting its ETS DNA-binding domain as well as the two acidic most drastically in the R89/ domains (ac) that contribute to transactivation. Lysines being part of a su- 257 combination mutant that moylation consensus motif (YKXE) are pointed out. B. 6Myc-ETV1 that was was devoid of detectable su- mutated at the indicated site(s) from lysine to arginine was coexpressed with moylation. Of the triple mu- SUMO1 in 293T cells. Shown is an anti-Myc western blot. tants, only R89/228/317 and R228/257/317 were mono- anti-SUMO1 antibodies, we observed that ETV1 sumoylated, while the other two triple mutants was pulled down in both human HeLa cervical as well as the quadruple R89/228/257/317 carcinoma and SW620 colorectal cancer cells mutant were not sumoylated (Figure 2B). These (Figure 1C). The size of the pulled-down mole- results suggest that K257 and K89 are the cule at ~80 kDa is consistent with mono- most important sumoylation sites, and absence sumoylation of the ~60 kDa endogenous ETV1 of sumoylation at these two lysine residues (note that 6Myc-tagged ETV1 runs at ~80 kDa might prevent SUMO1 modification on K228 in Figure 1A, 1B due to its large N-terminal and K317. 6Myc tag). This suggests that under physiologic conditions, ETV1 is unlikely to be poly-sumoylat- Impact of sumoylated lysine residues on ETV1 ed, in contrast to when SUMO1 is overex- transcriptional activity pressed as shown in Figure 1A, 1B. The ΨKXE consensus sumoylation motif is very Identification of ETV1 sumoylation sites similar to the synergy control motif, (I/V)KXE, where additional proline residues are present Lysine residues that become sumoylated are on at least one side spaced by 0-3 amino acids often localized within a ΨKXE consensus motif, [65, 66]. Such synergy control motifs have where Ψ is a bulky aliphatic residue, frequently been demonstrated to be modified by SUMO, leucine, isoleucine or valine, and X any amino and the ability to repress transcription through acid [60]. Analysis of ETV1 revealed four such a synergy control motif was in some cases lysine residues at positions 89, 228, 257 and shown to be dependent on sumoylation [66, 317 (Figure 2A). Consistent with all four of 67]. Moreover, synergy control motifs require these lysine residues being sumoylation sites, for transcriptional inhibition the presence of ETV1 amino acids 1-182 displayed only one multiple DNA-binding sites that facilitate the higher molecular weight form upon SUMO1 interaction of several copies of a transcription overexpression, while amino acids 1-249 and factor at a particular gene promoter [67]. Apart

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type or indicated mutants) rela- tive to empty vector pEV3S are depicted. Differences compared to wild-type ETV1 were evalu- ated by one-way ANOVA (Bon- ferroni’s multiple comparisons test; n=4). **P<0.01; ***P<0.001; ****P<0.0001. Western blotting shows comparable expression of wild-type ETV1 and its su- moylation site mutants (bottom); actin levels served as loading controls. B. Analogous, in the presence of cotransfected onco- genic Ras.

from K228, all other ETV1 sumoylation sites are part of such a synergy control motif, suggesting that sumoylation may repress ETV1 transcrip- tional activity. To test this hypothesis, we utilized the

E74 3-tk80-luc luciferase repo- rter construct that contains three E74 sites to which ETV1 can bind [27]. When trans- fected into 293T cells, the

E74 3-tk80-luc reporter was not affected by cotransfected wild-type ETV1 or ETV1 mutat- ed at any single sumoylation site (lysine to arginine muta- tions; Figure 3A). However, three double and all four tri- ple mutants displayed tran- scriptional stimulation, and the highest activation was observed with the R89/228/ 257/317 quadruple mutant (4xR; Figure 3A). This indi- cates that sumoylation re- presses transcription mediat- ed by ETV1. Because ETV1 can be activated by pho- sphorylation triggered throu- gh the Ras oncoprotein [27], we also analyzed ETV1-de- pendent transcription upon cotransfection of oncogenic Ras (Figure 3B). Again, we observed that mutation of Figure 3. Activation of a promoter with multimerized ETV1 binding sites. A. ETV1 sumoylation sites led to transcriptional activation 293T cells were transfected with the E743-tk80-luc luciferase reporter con- struct. Changes in reporter gene activity upon cotransfection of ETV1 (wild- under this condition.

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Figure 4. Differential response of various promoters to abrogation of ETV1 sumoylation in 293T cells. (A) E74-tk80- luc, (B) MMP1 (-525/+15) or (C) YAP1 (-496/+22) luciferase reporter construct was cotransfected with pEV3S vector control or indicated ETV1 expression plasmids as well as without or with oncogenic Ras. One-way ANOVA (Bonferroni’s multiple comparisons test; n=4). **P<0.01; ***P<0.001; ****P<0.0001.

Since the transcription repression function of bly, mutation of all four sumoylation sites led to synergy control motifs is reportedly dependent superactivation of MMP1 gene transcription on multimerized binding sites [67], we assess- (Figure 5A). As sumoylation may affect protein ed how ETV1 sumoylation would affect tran- stability or intracellular localization [60, 69], we scription mediated by a single E74 site in the also analyzed whether this would be the case corresponding E74-tk80-luc luciferase reporter for ETV1. However, mutation of all four ETV1 (Figure 4A). Unexpectedly, we observed that sumoylation sites did not significantly affect inhibition of sumoylation had similar stimulato- protein stability nor ETV1 distribution between the cytoplasm and cell nucleus (Figure 5B, 5C). ry effects as with the E743-tk80-luc luciferase reporter in the absence and presence of onco- Next, we assessed the role of sumoylation in genic Ras, implying that ETV1 does not require LNCaP prostate cancer cells, since ETV1 espe- multimerization in order to be transcriptionally cially exerts pro-tumorigenic functions in pros- inhibited by sumoylation in 293T cells. We then tatic adenocarcinoma [8]. As expected from tested a proven target of ETV1, the matrix previous analyses showing MMP1 to be regu- metalloproteinase 1 (MMP1) gene [28], and lated by ETV1 in LNCaP cells [33], ETV1 overex- found that its promoter was also stimulated pression greatly increased the activity of an upon mutation of sumoylation sites, but only in MMP1 luciferase reporter construct (Figure the presence of cotransfected Ras (Figure 4B). 6A). Startlingly, simultaneous mutation of mul- Interestingly, another target of ETV1, the Yes- tiple sumoylation sites repressed rather than associated protein 1 (YAP1) gene promoter activated the MMP1 promoter, and the most [68], was unaffected by mutation of ETV1 drastic effect was observed with the 4xR qua- sumoylation sites (Figure 4C), implicating that druple mutant. A similar repression of lucifer- posttranslational modification of ETV1 with ase activity was observed with the 4xR mutant SUMO1 has promoter-specific effects on its at the promoter of the YAP1 gene (Figure 6B), transcriptional activity. We extended our analy- another previously demonstrated ETV1 target sis also to the endogenous MMP1 gene in 293T gene in LNCaP cells [68]. But no change com- cells and, as reported before [28], found activa- pared to wild-type ETV1 was observable for tion of MMP1 gene transcription by ETV1 only the 4xR mutant with the E743-tk80-luc lucifer- upon stimulation with the receptor tyrosine ase reporter in LNCaP cells (Figure 6C), which kinase HER2/ERBB2 (that induces phosphory- is different from 293T cells described above. lation of ETV1 similar to oncogenic Ras); nota- Moreover, the known transcriptional synergy

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Figure 5. Hyperactivation of endogenous MMP1 transcription by ETV1-4xR. A. 293T cells were transfected with wild- type or 4xR ETV1. Where indicated, oncogenic HER2/ERBB2 was additionally transfected. MMP1 mRNA levels were assessed by RT-PCR; GAPDH mRNA levels served as controls. B. 293T cells transfected with ETV1 or its 4xR mutant were treated with the protein synthesis inhibitor cycloheximide for up to 6 h and ETV1 protein levels (normalized to actin) monitored. Statistical significance was assessed by two-way ANOVA (Sidak’s multiple comparison test; n=3); n.s., not significant. C. Sumoylation does not affect intracellular distribution in transfected 293T cells. The ratio of cytoplasmic to nuclear signal for wild-type ETV1 was arbitrarily set to 1 and then compared to the 4xR mutant. Sta- tistical significance was assessed by an unpaired, two-tailed Student’st -test (n=3).

Figure 6. Transcriptional impact of ETV1 sumoylation in LNCaP prostate cancer cells. A. Response of the MMP1 (‑525/+15) luciferase reporter upon cotransfection of 30 ng wild-type ETV1 and indicated SUMO site mutant ex- pression vectors; pEV3S denotes the empty expression vector. Differences compared to wild-type ETV1 were evalu- ated by one-way ANOVA (Dunnett’s multiple comparisons test; n=4); n.s., not significant;** P<0.01; ****P<0.0001. B and C. Likewise for the YAP1 (-496/+22) and E743-tk80-luc reporter constructs, respectively. D. Synergy between ETV1 (15 ng expression vector) and JMJD2A (75 ng expression vector) was assessed with the MMP1 (‑525/+15) luciferase reporter. One-way ANOVA (Tukey’s multiple comparisons test; n=6).

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gated whether this posttrans- lational modification would affect the binding of ETV1 with transcriptional cofactors. No substantial change in the ability to interact with JMJD2A was noted upon mutation of the four ETV1 sumoylation sites (Figure 7A, left panels). But we also tested binding of ETV1 with two other pro- teins, the histone deacetyla- Figure 7. Sumoylation affects the ETV1 interactome. A. Coimmunoprecip- se (HDAC) 1 and HDAC3, tiation experiments. Wild-type (wt) 6Myc-ETV1 or its 4xR mutant was coex- which were hitherto unknown pressed with indicated Flag-tagged proteins. After anti-Flag immunoprecipi- as interaction partners of tation (IP), coprecipitated ETV1 was detected by anti-Myc western blotting. ETV1. We observed that, like IgH, immunoglobulin heavy chain. B. 6Myc-ETV1 was coexpressed with and JMJD2A, HDAC3 binding to without SUMO1 and its in vitro binding to purified GST or GST-SMAD4 as- ETV1 was basically unaffect- sessed. Shown are anti-Myc blots. Arrowheads point to non-sumoylated ETV1, while asterisks mark sumoylated forms of ETV1. ed upon mutation of the four sumoylation sites, whereas complex formation with HD- AC1 was greatly enhanced upon mutation of all four ETV1 sumoylation sites (Figure 7A, right panels). This is opposite to what was found for the p68/DDX5 and p72/DDX17 RNA helicases, whose inter- action with HDAC1 was in- creased upon sumoylation [71]. We also analyzed the known in vitro binding of ETV1 to another transcription fac- tor, SMAD4 [72]. Here, we observed that compared to Figure 8. Abrogation of ETV1 sumoylation leads to reduced LNCaP prostate the input control, the sumo- cancer cell growth. A. Western blots showing comparable overexpression of ylated forms of ETV1 were wild-type and 4xR ETV1 in retrovirally infected LNCaP cells. pQCXIH, empty depleted in the SMAD4-bound vector; arrow points to ETV1 band. B. Cell growth assay. Statistical signifi- fraction (Figure 7B), suggest- cance was assessed with two-way ANOVA (Tukey’s multiple comparison test; ing that sumoylation prevents n=3); n.s., not significant; ****P<0.0001. the binding of ETV1 to SMAD4. Altogether, these data sug- between ETV1 and its interaction partner, the gest that ETV1 sumoylation can alter its affinity histone demethylase and epigenetic regulator for other proteins. JmjC domain-containing 2A (JMJD2A) [68, 70], was reduced upon mutation of the four 4xR mutant represses LNCaP cell growth sumoylation sites (Figure 6D). These data indi- cate that sumoylation exerts an activating role ETV1 is required for maximal growth of LNCaP on ETV1-mediated transcription in LNCaP cells prostate cancer cells [33, 73]. Hence, we that is opposite to its repressive role in 293T employed this cell line to assess how ETV1 cells. sumoylation might affect the ability of ETV1 to influence cell growth. To this end, wild-type Sumoylation affects the ETV1 interactome ETV1 or its 4xR mutant was retrovirally overex- pressed in LNCaP cells at comparable levels To gain insight how sumoylation may affect the (Figure 8A). Then, cell growth was examined. transactivation potential of ETV1, we interro- Overexpression of wild-type ETV1 had no sig-

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Figure 9. Expression of SUMO1, UBA2, and UBC9 mRNA in prostate cancer analyzed with Oncomine. A. Upregulation of SUMO1 mRNA in prostate tumors compared to normal prostate gland tissue. Shown are log2 median-centered SUMO1 mRNA levels. Statistical analysis was done with an unpaired, two-tailed Student’s t-test. B, C. Analogous for UBA2 and UBC9 mRNA, respectively. D. Higher SUMO1 mRNA levels in metastases than in primary prostate tumors. E, F. Same for UBA2 and UBC9, respectively. nificant impact Figure ( 8B), probably because enzyme, one subunit of which is ubiquitin-like endogenous levels of ETV1 are already at satu- modifier activating enzyme 2 (UBA2), and con- rating levels in LNCaP cells. But the 4xR mutant tinues with the ubiquitin conjugating enzyme 9 significantly reduced cell growth. This is likely (UBC9) that mediates transfer of SUMO1 to its due to the fact that ectopic ETV1-4xR outcom- target proteins [60, 69]. Bioinformatics analy- petes endogenous ETV1 and thereby blunts the sis of published data [74-79] with Oncomine latter’s pro-oncogenic activity. These data indi- [80] revealed that SUMO1 (Figure 9A), UBA2 cate that sumoylation promotes the oncogenic (Figure 9B), and UBC9 (Figure 9C) are upregu- activity of ETV1 in prostate cancer cells. lated in prostate tumors compared to the nor- mal prostate gland. Moreover, available data Overexpression of SUMO pathway components from metastasized tumors [81, 82] showed a in prostate cancer higher expression of SUMO1, UBA2, as well as UBC9 in metastases compared to primary pros- SUMO1 is activated through an enzymatic tate tumors (Figure 9D-F). These data suggest cascade that starts with the SUMO activating that the SUMO pathway is hyperactivated in pri-

804 Int J Clin Exp Pathol 2021;14(7):795-810 Regulation of ETV1 by SUMO1 modification mary and even more so in metastasized pros- here have been shown to become sumoylated tate tumors. in ETV4 and ETV5, indicating that this post- translational modification is conserved. How- Discussion ever, while sumoylation of ETV5 solely re- pressed the activity of this transcription factor, In this study, we have made the following dis- repressing and activating consequences of coveries: (i) ETV1 can become sumoylated in SUMO modification were reported in case of cells on four lysine residues (K89, K228, K257, ETV4 [84-87]. Furthermore, the major sites of K317). (ii) Sumoylation of ETV1 can result into acetylation and sumoylation seemingly overlap transcriptional activation (LNCaP cells) or re- in ETV4, thereby making these two posttransla- pression (293T cells). (iii) A previously unknown tional modifications mutually exclusive [88], interaction of ETV1 with HDAC1 seems to be but this does not hold true for ETV1, whose two suppressed by sumoylation, and similarly su- acetylation sites, K33 and K116 [35], are dif- moylation reduces binding of ETV1 to SMAD4, ferent from its sumoylation sites. Hence, while binding to two other proteins (HDAC3, despite the fact that homologous lysine resi- JMJD2A) is basically unaffected by ETV1 dues become sumoylated in ETV1, ETV4 and sumoylation. (iv) In LNCaP prostate cancer ETV5, this may have different functional conse- cells, the quadruple SUMO1 site mutant of quences for these three related transcription ETV1 acts in a dominant-negative manner and factors. represses cell growth; this supports the notion that sumoylation enhances the growth-stimula- Our data indicate that sumoylation can change tory effect of ETV1. (v) Components of the the interactome of ETV1. In particular, binding SUMO pathway (SUMO1, UBA2, UBC9) are of HDAC1 and SMAD4 to ETV1 seems to be upregulated in prostate tumors, which may pro- suppressed by sumoylation. This may explain mote sumoylation of ETV1 and thereby stimu- how sumoylation raises the transactivation late its oncogenic function in this cancer. potential of ETV1 in LNCaP cells, since histone deacetylation (as catalyzed by HDAC1) is nor- Despite the fact that SUMO1 overexpression mally linked to gene repression [89] and SMAD4 enforced the sumoylation of more than half of binding attenuates the ability of ETV1 to stimu- the total ETV1 protein in our experiments, the late transcription [72]. However, HDACs have degree of endogenous sumoylation seems to also been found at active genes, and HDAC be much smaller. This is a common phenome- inhibitors were able to repress transcription at non observed with many other sumoylated pro- some genes [90, 91], indicating that HDACs do teins, but begs the question why sumoylation not always act as transcriptional corepressors would then have any significant effect on a pro- but also occasionally as coactivators. Such tein’s function [60, 69]. One proposed idea is may be the case in 293T cells for HDAC1, which that modification with SUMO1 is a highly could explain why sumoylation apparently dynamic process, where sumoylation and desu- diminishes the ETV1 transactivation potential moylation are rapidly occurring on the same in this cell line. Also, HDAC3 binding was unaf- molecule, leading to an overall low steady-state fected by ETV1 sumoylation. Thus, it is possible level of SUMO1 modification, but guaranteeing that SUMO1 modification of ETV1 leads to that a large fraction of a protein is becoming replacement of HDAC1 by HDAC3 as an interac- sumoylated at least for a short period of time tion partner if their binding to ETV1 is mutually and that this is sufficient for activity control. exclusive; and if HDAC3 has a higher corepres- Another proposed idea is that sumoylation sor potential than HDAC1, this may also cause induces a conformational change in a protein a reduction in ETV1-dependent transcription in that persists after desumoylation [60, 69]. 293T cells. We do not know how sumoylation Future studies should look into these and other affects the binding of ETV1 to many other inter- possibilities how SUMO1 modification of ETV1 actants, whose expression may be different can lead to profound changes in its behavior as between 293T and LNCaP cells and might thus observed in this report. lead to different outcomes with regard to ETV1- mediated gene regulation. There are two close homologs of ETV1: ETV4 and ETV5 [15, 83]. Lysine residues homologous The upregulation of SUMO1, UBA2, and UBC9 to the four ETV1 sumoylation sites identified in prostate tumors could lead to enhanced

805 Int J Clin Exp Pathol 2021;14(7):795-810 Regulation of ETV1 by SUMO1 modification sumoylation of ETV1, yet overexpression of the Institute (R01 CA154745 and R03 CA223615). SUMO-specific protease SENP1 that could facil- In addition, R.J. has been supported in part itate desumoylation of ETV1 has also been by the Oklahoma Tobacco Settlement Endow- found in prostate cancer [92]. As such, it is ment Trust through an award made to the unclear how or whether at all the degree of University of Oklahoma/Stephenson Cancer ETV1 sumoylation is changed during prostate Center. Further assistance was furnished by cancer formation. Regardless, we observed the Stephenson Cancer Center Molecular Bio- that blocking sumoylation on ETV1 reduced logy Core that was supported by National LNCaP prostate cancer cell growth, strongly Institutes of Health grants P30 CA225520 and suggesting that inhibiting sumoylation of ETV1 P20 GM103639. The content of this manu- would be beneficial in prostate cancer therapy. script is solely the responsibility of the authors However, the is also modi- and does not necessarily represent the official fied by SUMO and its activity was shown to be views of the granting agencies. increased at selective gene promoters upon mutation of its sumoylation sites or overexpres- Disclosure of conflict of interest sion of SENP1 [93-95]. This would suggest that generic inhibition of sumoylation may dampen None. ETV1’s tumor-promoting activity, but stimulate the pro-oncogenic androgen receptor, and the Address correspondence to: Ralf Janknecht, Uni- net effect on prostate tumorigenicity would versity of Oklahoma Health Sciences Center, 975 NE be uncertain. Further, overexpression of SENP1 10th Street, BRC-1464, Oklahoma City, OK 73104, reportedly induced prostatic intraepithelial USA. Tel: +1-405-2712377; E-mail: ralf-janknecht@ neoplasia and SENP1 was needed for prostate ouhsc.edu cancer cell metastasis [92, 96], which implies that sumoylation may be beneficial in constrain- References ing prostate tumor development. Definitely, [1] Brown TA and McKnight SL. Specificities of more studies are needed to investigate how the protein-protein and protein-DNA interaction of SUMO pathway is affected in prostate tumors GABP alpha and two newly defined ets-related and whether or not its modulation through proteins. Genes Dev 1992; 6: 2502-2512. SUMO pathway drugs could be translated into [2] Hollenhorst PC, McIntosh LP and Graves BJ. clinical care for prostate cancer patients [97]. Genomic and biochemical insights into the specificity of ETS transcription factors. Annu ETV1 not only plays a pro-tumorigenic role in Rev Biochem 2011; 80: 437-471. prostate, but also in other [15, 83]. [3] Arber S, Ladle DR, Lin JH, Frank E and Jessell Given that sumoylation has opposite effects on TM. ETS gene Er81 controls the formation of the transactivation potential of ETV1 in 293T functional connections between group Ia sen- compared to LNCaP cells, it is far from certain sory afferents and motor neurons. Cell 2000; that sumoylation will promote the oncogenic 101: 485-498. potential of ETV1 in non-prostatic tissues. Org- [4] Shekhar A, Lin X, Liu FY, Zhang J, Mo H, Basta- an-specific, opposite functions of sumoylation rache L, Denny JC, Cox NJ, Delmar M, Roden may also be present in case of the androgen DM, Fishman GI and Park DS. Transcription receptor, as a recent report suggests that factor ETV1 is essential for rapid conduction in the heart. J Clin Invest 2016; 126: 4444-4459. sumoylation of androgen receptor promotes [5] Rommel C, Rosner S, Lother A, Barg M, Schwa- metastasis in endocrine therapy resistant derer M, Gilsbach R, Bomicke T, Schnick T, breast cancer [98]. Hence, while our data indi- Mayer S, Doll S, Hesse M, Kretz O, Stiller B, cate that blocking ETV1 sumoylation in pros- Neumann FJ, Mann M, Krane M, Fleischmann tate cancer could suppress growth, this may or BK, Ravens U and Hein L. The transcription may not hold true for melanoma, breast, or factor ETV1 induces atrial remodeling and ar- colorectal cancer in which ETV1 is also impli- rhythmia. Circ Res 2018; 123: 550-563. cated as a tumor promoter. [6] Yamaguchi N, Xiao J, Narke D, Shaheen D, Lin X, Offerman E, Khodadadi-Jamayran A, Shek- Acknowledgements har A, Choy A, Wass SY, Van Wagoner DR, Chung MK and Park DS. Cardiac pressure over- This work was in part funded by grants from the load decreases ETV1 expression in the left National Institutes of Health/National Cancer atrium, contributing to atrial electrical and

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structural remodeling. Circulation 2021; 143: Pastinen T, Osborne CS, Taipale J and Houlston 805-820. RS. Promoter capture Hi-C-based identification [7] Tomlins SA, Rhodes DR, Perner S, Dhanasek- of recurrent noncoding mutations in colorectal aran SM, Mehra R, Sun XW, Varambally S, Cao cancer. Nat Genet 2018; 50: 1375-1380. X, Tchinda J, Kuefer R, Lee C, Montie JE, Shah [18] Oh S, Song H, Freeman WM, Shin S and RB, Pienta KJ, Rubin MA and Chinnaiyan AM. Janknecht R. Cooperation between ETS tran- Recurrent fusion of TMPRSS2 and ETS tran- scription factor ETV1 and histone demethylase scription factor genes in prostate cancer. Sci- JMJD1A in colorectal cancer. Int J Oncol 2020; ence 2005; 310: 644-648. 57: 1319-1332. [8] Nicholas TR, Strittmatter BG and Hollenhorst [19] Shepherd TG, Kockeritz L, Szrajber MR, Muller PC. Oncogenic ETS factors in prostate cancer. WJ and Hassell JA. The pea3 subfamily ets Adv Exp Med Biol 2019; 1210: 409-436. genes are required for HER2/Neu-mediated [9] Tomlins SA, Laxman B, Dhanasekaran SM, Hel- mammary oncogenesis. Curr Biol 2001; 11: geson BE, Cao X, Morris DS, Menon A, Jing X, 1739-1748. Cao Q, Han B, Yu J, Wang L, Montie JE, Rubin [20] Baert JL, Monte D, Musgrove EA, Albagli O, MA, Pienta KJ, Roulston D, Shah RB, Varambal- Sutherland RL and de Launoit Y. Expression of ly S, Mehra R and Chinnaiyan AM. Distinct the PEA3 group of ETS-related transcription classes of chromosomal rearrangements cre- factors in human breast-cancer cells. Int J Can- ate oncogenic ETS gene fusions in prostate cer 1997; 70: 590-597. cancer. Nature 2007; 448: 595-599. [21] Bosc DG and Janknecht R. Regulation of [10] Shin S, Kim TD, Jin F, van Deursen JM, Dehm HER2/Neu promoter activity by the ETS tran- SM, Tindall DJ, Grande JP, Munz JM, Vasmatzis scription factor, ER81. J Cell Biochem 2002; G and Janknecht R. Induction of prostatic in- 86: 174-183. traepithelial neoplasia and modulation of an- [22] Vageli D, Ioannou MG and Koukoulis GK. Tran- drogen receptor by ETS variant 1/ETS-related scriptional activation of hTERT in breast carci- protein 81. Cancer Res 2009; 69: 8102-8110. nomas by the Her2-ER81-related pathway. On- [11] Baena E, Shao Z, Linn DE, Glass K, Hamblen col Res 2009; 17: 413-423. MJ, Fujiwara Y, Kim J, Nguyen M, Zhang X, Go- [23] Wang Y, Wang L, Chen Y, Li L, Yang X, Li B, Song dinho FJ, Bronson RT, Mucci LA, Loda M, Yuan S, Yang L, Hao Y and Yang J. ER81 expression GC, Orkin SH and Li Z. ETV1 directs androgen in breast cancers and hyperplasia. Pathology metabolism and confers aggressive prostate Res Int 2011; 2011: 980513. cancer in targeted mice and patients. Genes [24] Shin S, Bosc DG, Ingle JN, Spelsberg TC and Dev 2013; 27: 683-698. Janknecht R. Rcl is a novel ETV1/ER81 target [12] Attard G, Clark J, Ambroisine L, Mills IG, Fisher gene upregulated in breast tumors. J Cell Bio- G, Flohr P, Reid A, Edwards S, Kovacs G, Berney chem 2008; 105: 866-874. D, Foster C, Massie CE, Fletcher A, De Bono JS, Scardino P, Cuzick J and Cooper CS. Heteroge- [25] Chi P, Chen Y, Zhang L, Guo X, Wongvipat J, neity and clinical significance of ETV1 translo- Shamu T, Fletcher JA, Dewell S, Maki RG, cations in human prostate cancer. Br J Cancer Zheng D, Antonescu CR, Allis CD and Sawyers 2008; 99: 314-320. CL. ETV1 is a lineage survival factor that coop- [13] Jeon IS, Davis JN, Braun BS, Sublett JE, Rous- erates with KIT in gastrointestinal stromal tu- sel MF, Denny CT and Shapiro DN. A variant mours. Nature 2010; 467: 849-853. Ewing’s sarcoma translocation (7;22) fuses [26] Ran L, Sirota I, Cao Z, Murphy D, Chen Y, Shuk- the EWS gene to the ETS gene ETV1. Onco- la S, Xie Y, Kaufmann MC, Gao D, Zhu S, Rossi gene 1995; 10: 1229-1234. F, Wongvipat J, Taguchi T, Tap WD, Mellinghoff [14] Janknecht R. EWS-ETS oncoproteins: the linch- IK, Besmer P, Antonescu CR, Chen Y and Chi P. pins of Ewing tumors. Gene 2005; 363: 1-14. Combined inhibition of MAP kinase and KIT [15] Oh S, Shin S and Janknecht R. ETV1, 4 and 5: signaling synergistically destabilizes ETV1 and an oncogenic subfamily of ETS transcription suppresses GIST tumor growth. Cancer Discov factors. Biochim Biophys Acta 2012; 1826: 2015; 5: 304-315. 1-12. [27] Janknecht R. Analysis of the ERK-stimulated [16] Jane-Valbuena J, Widlund HR, Perner S, John- ETS transcription factor ER81. Mol Cell Biol son LA, Dibner AC, Lin WM, Baker AC, Nazarian 1996; 16: 1550-1556. RM, Vijayendran KG, Sellers WR, Hahn WC, [28] Bosc DG, Goueli BS and Janknecht R. HER2/ Duncan LM, Rubin MA, Fisher DE and Gar- Neu-mediated activation of the ETS transcrip- raway LA. An oncogenic role for ETV1 in mela- tion factor ER81 and its target gene MMP-1. noma. Cancer Res 2010; 70: 2075-2084. 2001; 20: 6215-6224. [17] Orlando G, Law PJ, Cornish AJ, Dobbins SE, [29] Wu J and Janknecht R. Regulation of the ETS Chubb D, Broderick P, Litchfield K, Hariri F, transcription factor ER81 by the 90-kDa ribo-

807 Int J Clin Exp Pathol 2021;14(7):795-810 Regulation of ETV1 by SUMO1 modification

somal S6 kinase 1 and protein kinase A. J Biol by upstream stimulatory factor. Oncogene Chem 2002; 277: 42669-42679. 2003; 22: 8042-8047. [30] Janknecht R. Regulation of the ER81 transcrip- [43] Kim TD, Shin S and Janknecht R. ETS tran- tion factor and its coactivators by mitogen- and scription factor ERG cooperates with histone stress-activated protein kinase 1 (MSK1). On- demethylase KDM4A. Oncol Rep 2016; 35: cogene 2003; 22: 746-755. 3679-3688. [31] Goueli BS and Janknecht R. Upregulation of [44] Janknecht R and Hunter T. Activation of the the catalytic telomerase subunit by the tran- Sap-1a transcription factor by the c-Jun N-ter- scription factor ER81 and oncogenic HER2/ minal kinase (JNK) mitogen-activated protein Neu, Ras, or Raf. Mol Cell Biol 2004; 24: 25- kinase. J Biol Chem 1997; 272: 4219-4224. 35. [45] Janknecht R, Zinck R, Ernst WH and Nordheim [32] Xie L, Gazin C, Park SM, Zhu LJ, Debily MA, Kit- A. Functional dissection of the transcription tler EL, Zapp ML, Lapointe D, Gobeil S, Virba- factor Elk-1. Oncogene 1994; 9: 1273-1278. sius CM and Green MR. A synthetic interaction [46] Li X, Oh S, Song H, Shin S, Zhang B, Freeman screen identifies factors selectively required WM and Janknecht R. A potential common role for proliferation and TERT transcription in - of the Jumonji C domain-containing 1A histone deficient human cancer cells. PLoS Genet demethylase and chromatin remodeler ATRX in 2012; 8: e1003151. promoting colon cancer. Oncol Lett 2018; 16: [33] Oh S, Shin S, Lightfoot SA and Janknecht R. 6652-6662. 14-3-3 proteins modulate the ETS transcrip- [47] Shin S and Janknecht R. Activation of andro- tion factor ETV1 in prostate cancer. Cancer gen receptor by histone demethylases JMJD2A Res 2013; 73: 5110-5119. and JMJD2D. Biochem Biophys Res Commun [34] Papoutsopoulou S and Janknecht R. Phos- 2007; 359: 742-746. phorylation of ETS transcription factor ER81 in [48] DiTacchio L, Bowles J, Shin S, Lim DS, Koop- a complex with its coactivators CREB-binding man P and Janknecht R. Transcription factors protein and p300. Mol Cell Biol 2000; 20: ER71/ETV2 and SOX9 participate in a positive 7300-7310. feedback loop in fetal and adult mouse testis. [35] Goel A and Janknecht R. Acetylation-mediated J Biol Chem 2012; 287: 23657-23666. transcriptional activation of the ETS protein [49] Oh S and Janknecht R. Histone demethylase ER81 by p300, P/CAF, and HER2/Neu. Mol JMJD5 is essential for embryonic develop- Cell Biol 2003; 23: 6243-6254. ment. Biochem Biophys Res Commun 2012; [36] Goel A and Janknecht R. Concerted activation 420: 61-65. of ETS protein ER81 by p160 coactivators, the [50] Sui Y, Li X, Oh S, Zhang B, Freeman WM, Shin S acetyltransferase p300 and the receptor tyro- and Janknecht R. Opposite roles of the JMJ- sine kinase HER2/Neu. J Biol Chem 2004; D1A interaction partners MDFI and MDFIC in 279: 14909-14916. colorectal cancer. Sci Rep 2020; 10: 8710. [37] Gocke CB, Yu H and Kang J. Systematic identi- [51] Kim TD, Fuchs JR, Schwartz E, Abdelhamid D, fication and analysis of mammalian small Etter J, Berry WL, Li C, Ihnat MA, Li PK and ubiquitin-like modifier substrates. J Biol Chem Janknecht R. Pro-growth role of the JMJD2C 2005; 280: 5004-5012. histone demethylase in HCT-116 colon cancer [38] Dowdy SC, Mariani A and Janknecht R. HER2/ cells and identification of curcuminoids as Neu- and TAK1-mediated up-regulation of the JMJD2 inhibitors. Am J Transl Res 2014; 6: transforming growth factor beta inhibitor 236-247. Smad7 via the ETS protein ER81. J Biol Chem [52] Berry WL, Kim TD and Janknecht R. Stimula- 2003; 278: 44377-44384. tion of β-catenin and colon cancer cell growth [39] Mooney SM, Goel A, D’Assoro AB, Salisbury JL by the KDM4B histone demethylase. Int J On- and Janknecht R. Pleiotropic effects of p300- col 2014; 44: 1341-1348. mediated acetylation on p68 and p72 RNA he- [53] Li X, Moon G, Shin S, Zhang B and Janknecht licase. J Biol Chem 2010; 285: 30443-30452. R. Cooperation between ETS variant 2 and Ju- [40] Knebel J, De Haro L and Janknecht R. Repres- monji domain-containing 2 histone demethyl- sion of transcription by TSGA/Jmjd1a, a novel ases. Mol Med Rep 2018; 17: 5518-5527. interaction partner of the ETS protein ER71. J [54] Kim TD, Oh S, Shin S and Janknecht R. Regula- Cell Biochem 2006; 99: 319-329. tion of tumor suppressor p53 and HCT116 cell [41] Kim TD, Oh S, Lightfoot SA, Shin S, Wren JD physiology by histone demethylase JMJD2D/ and Janknecht R. Upregulation of PSMD10 KDM4D. PLoS One 2012; 7: e34618. caused by the JMJD2A histone demethylase. [55] Rossow KL and Janknecht R. The Ewing’s sar- Int J Clin Exp Med 2016; 9: 10123-10134. coma gene product functions as a transcrip- [42] Goueli BS and Janknecht R. Regulation of tional activator. Cancer Res 2001; 61: 2690- telomerase reverse transcriptase gene activity 2695.

808 Int J Clin Exp Pathol 2021;14(7):795-810 Regulation of ETV1 by SUMO1 modification

[56] Kim J, Shin S, Subramaniam M, Bruinsma E, transcription factor ETV1. J Clin Invest 2016; Kim TD, Hawse JR, Spelsberg TC and Janknecht 126: 706-720. R. Histone demethylase JARID1B/KDM5B is a [69] Celen AB and Sahin U. Sumoylation on its 25th corepressor of TIEG1/KLF10. Biochem Bio- anniversary: mechanisms, pathology, and phys Res Commun 2010; 401: 412-416. emerging concepts. FEBS J 2020; 287: 3110- [57] Shin S, Oh S, An S and Janknecht R. ETS vari- 3140. ant 1 regulates matrix metalloproteinase-7 [70] Berry WL and Janknecht R. KDM4/JMJD2 his- transcription in LNCaP prostate cancer cells. tone demethylases: epigenetic regulators in Oncol Rep 2013; 29: 306-314. cancer cells. Cancer Res 2013; 73: 2936- [58] Berry WL, Shin S, Lightfoot SA and Janknecht 2942. R. Oncogenic features of the JMJD2A histone [71] Mooney SM, Grande JP, Salisbury JL and demethylase in breast cancer. Int J Oncol Janknecht R. Sumoylation of p68 and p72 RNA 2012; 41: 1701-1706. helicases affects protein stability and transac- [59] Kim TD, Shin S, Berry WL, Oh S and Janknecht tivation potential. Biochemistry 2010; 49: R. The JMJD2A demethylase regulates apopto- 1-10. sis and proliferation in colon cancer cells. J Cell [72] Oh S, Shin S, Song H, Grande JP and Janknecht Biochem 2012; 113: 1368-1376. R. Relationship between ETS transcription fac- [60] Flotho A and Melchior F. Sumoylation: a regula- tor ETV1 and TGF-beta-regulated SMAD pro- tory protein modification in health and disease. teins in prostate cancer. Sci Rep 2019; 9: Annu Rev Biochem 2013; 82: 357-385. 8186. [61] Lee PS, Chang C, Liu D and Derynck R. Su- [73] Cai C, Hsieh CL, Omwancha J, Zheng Z, Chen moylation of Smad4, the common Smad me- SY, Baert JL and Shemshedini L. ETV1 is a nov- diator of transforming growth factor-beta fami- el androgen receptor-regulated gene that me- ly signaling. J Biol Chem 2003; 278: diates prostate cancer cell invasion. Mol Endo- 27853-27863. crinol 2007; 21: 1835-1846. [62] Lin X, Liang M, Liang YY, Brunicardi FC, Mel- [74] Vanaja DK, Cheville JC, Iturria SJ and Young CY. chior F and Feng XH. Activation of transforming Transcriptional silencing of zinc finger protein growth factor-beta signaling by SUMO-1 modifi- 185 identified by expression profiling is associ- cation of tumor suppressor Smad4/DPC4. J ated with prostate cancer progression. Cancer Biol Chem 2003; 278: 18714-18719. Res 2003; 63: 3877-3882. [63] Ohshima T and Shimotohno K. Transforming [75] Welsh JB, Sapinoso LM, Su AI, Kern SG, Wang- growth factor-beta-mediated signaling via the Rodriguez J, Moskaluk CA, Frierson HF Jr and p38 MAP kinase pathway activates Smad-de- Hampton GM. Analysis of pendent transcription through SUMO-1 modifi- identifies candidate markers and pharmaco- cation of Smad4. J Biol Chem 2003; 278: logical targets in prostate cancer. Cancer Res 50833-50842. 2001; 61: 5974-5978. [64] Gareau JR and Lima CD. The SUMO pathway: [76] Tomlins SA, Mehra R, Rhodes DR, Cao X, Wang emerging mechanisms that shape specificity, L, Dhanasekaran SM, Kalyana-Sundaram S, conjugation and recognition. Nat Rev Mol Cell Wei JT, Rubin MA, Pienta KJ, Shah RB and Biol 2010; 11: 861-871. Chinnaiyan AM. Integrative molecular concept [65] Iniguez-Lluhi JA and Pearce D. A common motif modeling of prostate cancer progression. Nat within the negative regulatory regions of mul- Genet 2007; 39: 41-51. tiple factors inhibits their transcriptional syn- [77] Wallace TA, Prueitt RL, Yi M, Howe TM, Gil- ergy. Mol Cell Biol 2000; 20: 6040-6050. lespie JW, Yfantis HG, Stephens RM, Caporaso [66] Subramanian L, Benson MD and Iniguez-Lluhi NE, Loffredo CA and Ambs S. Tumor immuno- JA. A synergy control motif within the attenua- biological differences in prostate cancer be- tor domain of CCAAT/enhancer-binding protein tween African-American and European-Ameri- alpha inhibits transcriptional synergy through can men. Cancer Res 2008; 68: 927-936. its PIASy-enhanced modification by SUMO-1 or [78] Grasso CS, Wu YM, Robinson DR, Cao X, Dha- SUMO-3. J Biol Chem 2003; 278: 9134-9141. nasekaran SM, Khan AP, Quist MJ, Jing X, Loni- [67] Holmstrom S, Van Antwerp ME and Iniguez- gro RJ, Brenner JC, Asangani IA, Ateeq B, Chun Lluhi JA. Direct and distinguishable inhibitory SY, Siddiqui J, Sam L, Anstett M, Mehra R, roles for SUMO isoforms in the control of tran- Prensner JR, Palanisamy N, Ryslik GA, Vandin scriptional synergy. Proc Natl Acad Sci U S A F, Raphael BJ, Kunju LP, Rhodes DR, Pienta KJ, 2003; 100: 15758-15763. Chinnaiyan AM and Tomlins SA. The mutation- [68] Kim TD, Jin F, Shin S, Oh S, Lightfoot SA, al landscape of lethal castration-resistant Grande JP, Johnson AJ, van Deursen JM, Wren prostate cancer. Nature 2012; 487: 239-243. JD and Janknecht R. Histone demethylase JM- [79] Singh D, Febbo PG, Ross K, Jackson DG, Mano- JD2A drives prostate tumorigenesis through la J, Ladd C, Tamayo P, Renshaw AA, D’Amico

809 Int J Clin Exp Pathol 2021;14(7):795-810 Regulation of ETV1 by SUMO1 modification

AV, Richie JP, Lander ES, Loda M, Kantoff PW, [89] Dawson MA and Kouzarides T. Cancer epi- Golub TR and Sellers WR. Gene expression genetics: from mechanism to therapy. Cell correlates of clinical prostate cancer behavior. 2012; 150: 12-27. Cancer Cell 2002; 1: 203-209. [90] Wang Z, Zang C, Cui K, Schones DE, Barski A, [80] Rhodes DR, Kalyana-Sundaram S, Mahavisno Peng W and Zhao K. Genome-wide mapping of V, Varambally R, Yu J, Briggs BB, Barrette TR, HATs and HDACs reveals distinct functions in Anstet MJ, Kincead-Beal C, Kulkarni P, active and inactive genes. Cell 2009; 138: Varambally S, Ghosh D and Chinnaiyan AM. 1019-1031. Oncomine 3.0: genes, pathways, and networks [91] Kim YJ, Greer CB, Cecchini KR, Harris LN, Tuck in a collection of 18,000 cancer gene expres- DP and Kim TH. HDAC inhibitors induce tran- sion profiles. Neoplasia 2007; 9: 166-180. scriptional repression of high copy number [81] Yu YP, Landsittel D, Jing L, Nelson J, Ren B, Liu genes in breast cancer through elongation L, McDonald C, Thomas R, Dhir R, Finkelstein blockade. Oncogene 2013; 32: 2828-2835. S, Michalopoulos G, Becich M and Luo JH. [92] Bawa-Khalfe T, Cheng J, Lin SH, Ittmann MM Gene expression alterations in prostate cancer and Yeh ET. SENP1 induces prostatic intraepi- predicting tumor aggression and preceding de- thelial neoplasia through multiple mecha- velopment of malignancy. J Clin Oncol 2004; nisms. J Biol Chem 2010; 285: 25859-25866. 22: 2790-2799. [93] Poukka H, Karvonen U, Janne OA and Palvimo [82] LaTulippe E, Satagopan J, Smith A, Scher H, JJ. Covalent modification of the androgen- re Scardino P, Reuter V and Gerald WL. Compre- ceptor by small ubiquitin-like modifier 1 hensive gene expression analysis of prostate (SUMO-1). Proc Natl Acad Sci U S A 2000; 97: cancer reveals distinct transcriptional pro- 14145-14150. grams associated with metastatic disease. [94] Kaikkonen S, Jaaskelainen T, Karvonen U, Ryt- Cancer Res 2002; 62: 4499-4506. inki MM, Makkonen H, Gioeli D, Paschal BM [83] Qi T, Qu Q, Li G, Wang J, Zhu H, Yang Z, Sun Y, and Palvimo JJ. SUMO-specific protease 1 Lu Q and Qu J. Function and regulation of the (SENP1) reverses the hormone-augmented PEA3 subfamily of ETS transcription factors in SUMOylation of androgen receptor and modu- cancer. Am J Cancer Res 2020; 10: 3083- lates gene responses in prostate cancer cells. 3105. Mol Endocrinol 2009; 23: 292-307. [84] Degerny C, Monte D, Beaudoin C, Jaffray E, [95] Chua JP, Reddy SL, Yu Z, Giorgetti E, Montie Portois L, Hay RT, de Launoit Y and Baert JL. HL, Mukherjee S, Higgins J, McEachin RC, Rob- SUMO modification of the Ets-related - tran ins DM, Merry DE, Iniguez-Lluhi JA and Lieber- scription factor ERM inhibits its transcriptional man AP. Disrupting SUMOylation enhances activity. J Biol Chem 2005; 280: 24330- transcriptional function and ameliorates poly- 24338. glutamine androgen receptor-mediated dis- [85] Nishida T, Terashima M, Fukami K and Yamada ease. J Clin Invest 2015; 125: 831-845. Y. Repression of E1AF transcriptional activity [96] Wang Q, Xia N, Li T, Xu Y, Zou Y, Zuo Y, Fan Q, by sumoylation and PIASy. Biochem Biophys Bawa-Khalfe T, Yeh ET and Cheng J. SUMO- Res Commun 2007; 360: 226-232. specific protease 1 promotes prostate cancer [86] Bojovic BB and Hassell JA. The transactivation progression and metastasis. Oncogene 2013; function of the Pea3 subfamily Ets transcrip- 32: 2493-2498. tion factors is regulated by sumoylation. DNA [97] Xie M, Yu J, Ge S, Huang J and Fan X. SU- Cell Biol 2008; 27: 289-305. MOylation homeostasis in tumorigenesis. Can- [87] Guo B and Sharrocks AD. Extracellular signal- cer Lett 2020; 469: 301-309. regulated kinase mitogen-activated protein ki- [98] Bahnassy S, Thangavel H, Quttina M, Khan AF, nase signaling initiates a dynamic interplay Dhanyalayam D, Ritho J, Karami S, Ren J and between sumoylation and ubiquitination to Bawa-Khalfe T. Constitutively active androgen regulate the activity of the transcriptional acti- receptor supports the metastatic phenotype of vator PEA3. Mol Cell Biol 2009; 29: 3204- endocrine-resistant hormone receptor-positive 3218. breast cancer. Cell Commun Signal 2020; 18: [88] Guo B, Panagiotaki N, Warwood S and Shar- 154. rocks AD. Dynamic modification of the ETS transcription factor PEA3 by sumoylation and p300-mediated acetylation. Nucleic Acids Res 2011; 39: 6403-6413.

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