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Table of Contents
Abstract
USP25 Maintains KRAS Expression And Inhibiting The Deubiquitinase Suppresses KRAS Signaling In Human Cancer
Abstract
Introduction
Results
Discussion
Experimental Procedures
Cell Culture
RNA Isolation And RT-PCR
Western Blotting Analysis
Ubiquitination Assay
GST-Pulldown Assay
Immunoprecipitation Assay
Animal Experiments
Immunohistochemical Staining
Statistical Analysis
Figure Legend
Author Contribution Statement
Declaration Of Interest Statement
Abstract
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Journal Pre-proof USP25 Maintains KRAS Expression and Inhibiting the Deubiquitinase Suppresses KRAS Signaling in Human Cancer Huailu Ma, Huiyuan Guan, Xiao Sun, Lingzhi Wu, Mengjiao Cai, Xinghua Zhen, Xiang Shen, Suxia Han, Guangxue Liu, Jin Peng, Pumin Zhang PII: S0021-9258(25)02187-8 DOI: https://doi.org/10.1016/j.jbc.2025.110337 Reference: JBC 110337 To appear in: Journal of Biological Chemistry Received Date: 4 December 2024 Revised Date: 21 May 2025 Accepted Date: 23 May 2025 Please cite this article as: Ma H, Guan H, Sun X, Wu L, Cai M, Zhen X, Shen X, Han S, Liu G, Peng J, Zhang P, USP25 Maintains KRAS Expression and Inhibiting the Deubiquitinase Suppresses KRAS Signaling in Human Cancer, Journal of Biological Chemistry (2025), doi: https://doi.org/10.1016/ j.jbc.2025.110337. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ® 2025 THE AUTHORS. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology. 1
USP25 Maintains KRAS Expression and Inhibiting the Deubiquitinase Suppresses KRAS Signaling in Human Cancer
Huailu Ma1, 2, 3, #, Huiyuan Guan1, 2, 3, #, Xiao Sun 4, #, Lingzhi Wu1, Mengjiao Cai 4, Xinghua Zhen 1, Xiang Shen 5, Suxia Han 4, Guangxue Liu 1, 2 3, *, Jin Peng 1, 2, 3, * and Pumin Zhang 1, 2, 3 * 1 Zhejiang Provincial Key Laboratory of Pancreatic Disease, The First Affiliated Hospital of Zhejiang University, Hangzhou, Zhejiang 310003, China 2 Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310058, China 3 Cancer Center, Zhejiang University, Hangzhou, Zhejiang 310058, China 4 Department of Oncology, The First Affiliated Hospital, Xi'an Jiaotong University Medical College, Xi'an 710061, China 5 Chaser Therapeutics, Hangzhou, Zhejiang Province 310018, China Running title: USP25 is a DUB for KRAS # These authors contributed equally to this work. * Corresponding authors Address Correspondence to: Pumin Zhang, Ph.D. Provincial Key Laboratory of Pancreatic Diseases The First Affiliated Hospital of Zhejiang University Hangzhou, Zhejiang Province 310003 China E-mail: pzhangbcm@zju.edu.cn. Jo urn al Pr e-p roo f 2
Abstract
KRAS is a prominent oncogene mutated in a large number of human malignancies, particularly in pancreatic, colorectal, and lung tumors. We demonstrate here that KRAS, including its various activating mutants, is subjected to ubiquitin-mediated proteasomal degradation in cancer cells. Through a siRNA-based screening of deubiquitinases, we identified USP25 as a deubiquitinase for KRAS. Depleting USP25 expression increases ubiquitination and proteasomal degradation of KRAS, leading to the suppression of its oncogenic activity. We further show that USP25 inhibitors we have discovered are capable of destabilizing KRAS in cancer cells and are efficacious in blocking tumor xenograft growth in mice. These findings provide evidence supporting the notion that targeting the deubiquitinase USP25 can effectively, albeit indirectly, suppress KRAS and potentially aid in the treatment of tumors driven by KRAS activating mutations. Jo urn al Pr e-p roo f 3
Introduction
The ubiquitin-proteasome system (UPS) is critical in maintaining cellular protein homeostasis. Proteins destined for proteasomal degradation are usually labelled by poly-ubiquitin chains linked via the lysine 48 or the lysine 11 residues on ubiquitin (1, 2). This labelling process involves a series of enzymatic reactions mediated by three enzymes: E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3 (ubiquitin ligase), with E3s being responsible for determining substrate specificity (2-4). This process can be reversed to achieve protein homeostasis by deubiquitinases (DUBs), a class of specialized proteases that removes polyubiquitin chains from protein substrates (5-9). RAS proteins have been shown to undergo ubiquitin-mediated regulations. Three E3 ubiquitin ligases have been reported to facilitate RAS polyubiquitination and subsequent degradation, -TRCP for HRAS (10), NEDD4-1 (11) and WDR76 (12) for all three RAS homologues. An additional E3 ligase, leucine-zipper-like transcriptional regulator 1 (LZTR1), was found responsible for mono- and di-ubiquitination of RAS proteins. This mono-/di-ubiquitin modification interferes with the proper localization of RAS GTPases to the plasma membrane and disrupts RAS signaling (13, 14). Recent evidence also suggests that LZTR1 may polyubiquitinate RAS for degradation (15, 16), which is regulated by GSK and implicated in neoplastic transformation (16). Genetic studies in fruit fly as well as in mice indicate that the preferred substrate of LZTR1 is actually RIT1, a noncanonical RAS protein (17). Both LZTR1 and RIT1 are Jo urn al Pr eroo f 4 mutated in Noonan syndrome, a condition characterized by RAS overactivation (18). These results reveal that the ubiquitin modification of RAS plays important roles in regulating RAS signaling. Whether there is redundancy among the identified RAS ubiquitin ligases, or whether they are utilized differentially in varying conditions or cell types, remains elusive. Furthermore, there should be deubiquitinases which could act on RAS so that their ubiquitin modification process is kept in check to allow precise control over their abundance and activity. Thus, we launched a siRNA screen against the majority of deubiquitinases in human genome to find those important in regulating, specifically, KRAS protein abundance. The screen identified the deubiquitinase USP25. We show here that KRAS is a substrate of USP25. Depleting USP25 expression in cancer cells increases ubiquitination and proteasomal degradation of KRAS, leading to the suppression of its downstream MAP kinase pathway and cell proliferation. We further show that the inhibitor of USP25 could destabilize KRAS in cancer cells and was efficacious in blocking tumor xenograft growth in mice. These results indicate that the deubiquitinase USP25 is an indirect and yet effective target for suppressing KRAS, and its inhibition can potentially aid in the treatment of tumors driven by KRAS activating mutations. Jo ur al Pr e-p roo f 5
Results
Identification of deubiquitinases that control KRAS protein expression To identify deubiquitinases that are required to maintain KRAS protein abundance in cancer cells, we performed an siRNA screen in human colon cancer cell line HCT116 which carries a G13D mutation in KRAS. A commercial deubiquitinase siRNA library designed against 84 DUBs with each DUB targeted by 3 siRNAs was utilized. We transfected each individual siRNA into HCT116, harvested the cells 48 hrs post the transfection, and examined KRAS levels in these cells via immunoblotting (Fig. S1A). The expression levels of KRAS were quantitated and normalized to that in control cells and plotted (Fig. S1B), and the knockdown efficiency of each siRNA in the library was also assessed with qPCR analysis of the respective deubiquitinase (Fig. S1C). When at least two out of three siRNAs against a deubiquitinase could reduce KRAS expression levels more than 50%, the deubiquitinase was considered a potential regulator of KRAS expression. Based on these two criteria, USP13, USP25, USP30, OTUD4, and USP45 were chosen and those initially effective siRNAs were screened for a second time (Fig. S1C). Only USP13 and USP25 passed the secondary screen (Fig. S1D). Since the changes in KRAS protein levels could also come from changes in its mRNA levels, we analyzed KRAS mRNA expression via qPCR (Fig. S1E). It turned out that the depletion of USP13 expression decreased KRAS mRNA levels, suggesting that USP13 likely controls KRAS transcription instead of being a deubiquitinase for KRAS. On the other hand, the depletion of USP25 expression did not alter KRAS mRNA levels, making USP25 a likely KRAS deubiquitinase. Jo urn l P repro of 6 As a deubiquitinase of KRAS, we expect that depleting USP25 would increase the levels of KRAS ubiquitination. Indeed, the levels of ubiquitination on KRAS increases in HCT116 cells (Fig. 1A) as well as in HT29 cells (Fig. S1F) when the expression of USP25 was compromised. As expected, USP25 could interact with KRAS (Fig. 1B) and vice versa (Fig. 1C). To map the site in KRAS that interacts with USP25, we expressed and purified a series of truncated GST-KRAS4B proteins to pulldown His-USP25 (Fig. 1D). This GST pulldown assay implicated a 20-residue region located between residues 120 and 140 responsible for the interaction with USP25. Mutating 8 charged or polar residues to alanine in this region (KRAS-8A, Fig. 1D) abolished the interaction with USP25 in HCT116 cells (Fig. 1E). As a result, unlike its wildtype counterpart, the ubiquitination levels on 8A mutant were no longer affected by the overexpression of USP25 (Fig. 1F). Further, this 8A mutant is still functional (Fig. S2A), indicating that it can fold correctly and the effect on interaction with USP25 is not due to misfolding. It is known that KRAS is expressed as two splicing variants, KRAS4B (the main form, usually referred as KRAS) and KRAS4A (19). Since the antibodies used above (and through the rest of the work) could not differentiate the two isoforms, we wondered if USP25 regulated only a specific KRAS isoform. To that end, using KRAS4A and B-specific antibodies we found that both KRAS4A and KRAS4B require the function of USP25 to maintain their expression (Fig. S2B). Furthermore, there are two other RAS proteins, HRAS and NRAS. We wondered if they are also regulated by USP25. Indeed, depleting the expression of the deubiquitinase results in the Jo ur a Pr e-p roo f 7 downregulation of both HRAS and NRAS (Fig. S2C), suggesting USP25 likely function as a deubiquitinase for all three RAS proteins. Taken together, these data demonstrate that compromising the function of the deubiquitinase USP25 can downregulate KRAS expression and suppress KRAS signaling. Mapping the ubiquitination sites in KRAS Despite several reports of E3 ubiquitin ligases of RAS proteins (10-12, 15, 16), no lysine residues have been identified that are ubiquitinated and mediate the proteasomal degradation of RAS proteins. Four lysine residues, K104, K117, K128, and K147, were shown to be monoubiquitinated to regulate KRAS activation and signaling (20-24). Indeed, mutating these sites to arginine did not affect poly-ubiquitination of KRAS (Fig. S3A). It was shown previously that RasG, a KRAS orthologue in dictyostelium, was subjected to ubiquitin-mediated proteasomal degradation and 4 lysine residues in the C-terminal hypervariable region (HVR) were shown to be critical for this process (25). Interestingly, human KRAS4B expressed in dictyostelium was also subjected to ubiquitination and degradation (25). Given the similarities in the HVR between RasG and KRAS4B, we suspected that the lysine residue(s) responsible for KRAS4B ubiquitination and subsequent degradation might reside in the HVR (Fig. 2A). To that end, we first replaced all but 2 lysine residues in the region, generating a 9KR mutant of KRAS4B (Fig. S3B). This mutant lost almost all ubiquitination in 293T cells (Fig. S3C). From there, we generated a 4KR mutant (Fig. Jo rna l P re- pro of 8 S3B) which was unable to be poly-ubiquitinated as the 9KR mutant (Fig. S3C), suggesting that the bulk of the ubiquitination modification occur on some or all of these 4 lysine residues. Therefore, we replaced these 4 lysine residues with arginine one at a time and each replacement was assessed for its ability to be ubiquitinated. Satisfactorily, K172 was found to be responsible for all poly-ubiquitination on KRAS4B (Fig. S3D, Fig. 2A and B), and KRAS4BK172R is no longer dependent on USP25 for stable expression (Fig. 2C). Similarly, we mapped the ubiquitination sites in KRAS4A. First, we replaced all 7 lysine residues in the HVR to arginine to generate a 7KR mutant (Fig. S3E). The ubiquitination on KRAS4A7KR was nearly abolished (Fig. S3F), indicating that like KRAS4B, the ubiquitination of KRAS4A also occurs in the HVR. We subsequently generated a 4KR mutant by replacing K169, K170, K173, and K176 with arginine (Fig. S3E). Again, this 4KR mutant seemed unable to be ubiquitinated (Fig. S3F), suggesting that the ubiquitination takes place on one or more of these 4 residues. We then tested the possibility that the ubiquitination on KRAS4A takes place on one lysine residue, much like KRAS4B as shown above, by generating each individual arginine replacement. Indeed, this is the case. K169 turned out to be responsible for the ubiquitination of KRAS4A (Fig. S3G, Fig. 2A and 2D) and mutating this residue to arginine similarly stabilized KRAS4A when USP25 was depleted (Fig. 2E). To determine whether the activating mutations of KRAS would interfere with its ubiquitination, we generated a double mutant KRAS-4B in which G13 was changed to D and K172 Jo urn al Pr e-p roo f 9 to R. While KRASG13D could be ubiquitinated as its wildtype counterpart, the double mutant (KRASG13D/K172R) showed much reduced ubiquitination (Fig. S3H). USP25 is necessary for KRAS signaling and promotes cancer cell proliferation To determine if the downregulation of KRAS resulted from depleting the expression of USP25 impacts KRAS downstream signaling, we examined the phosphorylation status of MEK as well as that of ERK. As shown in Fig. 3A, the depletion of USP25 decreased the phosphorylation levels of both MEK and ERK, which could be rescued by re-expressing USP25 (Fig. 3B) or be prevented by exogenously expressed KRASG13D (Fig. 3C). The downregulation of KRAS signaling was also observable in other cancer cell lines carrying different KRAS mutations, including the pancreatic cancer cell line Capan-2 (KRASG12V) (Fig. S4A), and the lung cancer cell lines H23 (KRASG12C) and A549 (KRASG12S) (Fig. S4B). It is known that KRAS can activate AKT signaling (26). We therefore wondered if USP25 also plays a role in that regard. Indeed, depleting USP25 led to reduced levels of active (phosphorylated) AKT, which could be rescued by re-expressing KRAS (Fig. S4C). Given the well-documented importance of KRAS signaling in cell proliferation, it is expected that compromising USP25 would hamper cell proliferation. Indeed, depleting USP25 expression with two shRNAs (carrying the sequences identical to the two siRNAs identified in the initial screen, Fig. S1) suppressed the growth of HCT116 cells (Fig. 3D and Fig. S5A), and reexpressing USP25 (Fig. 3B) could bring the growth rates back (Fig. 3D and Fig. S5A). In HCT116, Jo urn al Pr e-p roo f 10 this slowdown of growth was not because the cells were dying through apoptosis (Fig. S5C), but because they were slow to enter S phase (Fig. S5D). The requirement of USP25 for optimal growth was also evident in other cancer cells (Fig. S5B). Importantly, expressing KRASG13D in USP25depleted HCT116 cells (Fig. 3C) cancelled the growth-inhibiting effect of USP25 depletion (Fig. 3E). Together, these data strongly support the notion that USP25 promotes cancer cell growth, through (at least partly) stabilizing KRAS expression. To demonstrate the role of USP25 in tumor growth in vivo, we established doxycycline (Dox)-inducible shUSP25 (and shNC as control) expressing HCT116 cells. The shNC and shUSP25 cells were inoculated in nude mice side by side. When the xenografts reached a size of ~50 mm3, the drinking water for the mice was switched to that containing doxycycline. The progression of these xenograft tumors was assessed by measuring the volume of the tumors at three-day intervals. As shown in Fig. 3F, inducing shUSP25 expression suppressed tumor growth, while the growth rates between shNC and shUSP25 tumors were similar if doxycycline was not administered. The xenograft tumors were harvested by day 12 for analyses. Consistent with their slow growth rates, shUSP25 tumors were much smaller than the controls (Fig. 3G and H). Immunoblotting of the tumor protein extracts showed that in the USP25-depleted tumors KRAS protein levels as well as the phosphorylation levels of MEK and ERK were all downregulated (Fig. 3I). Further immunohistochemical staining demonstrated much reduced proliferation (Ki67 positive) and phospho-ERK expression in shUSP25-expressing tumors (Fig. 3I). Taken together, these results demonstrate that USP25 is crucial for KRAS signaling and tumor growth. Jo urn al Pr e-p roo f 11 Pharmacological inhibition of USP25 can temper KRAS signaling We recently developed a strong inhibitor of USP25 (and its homologue USP28), CT1113 (27). To determine if pharmacological inhibition of USP25 is effective in reducing the expression levels of KRAS protein and in tempering KRAS signaling, we treated HT29 colon cancer cells (which do not harbor mutations in KRAS) with different concentrations of CT1113 (Fig. 4A). As expected, KRAS levels as well as the phosphorylation levels of MEK and ERK decreased in a dose-dependent manner. Moreover, inhibiting USP25 could temper KRAS signaling in pancreatic, lung, and colon cancer cells carrying various activating mutations in KRAS and for one of the mutants, KRASQ61H, no direct inhibitors are available (Fig. 4B). The ubiquitin level on KRAS increased as expected when USP25 was inhibited (Fig. S6A). Inhibiting USP25 caused its own downregulation in many cell lines (Fig. 4). This can be explained by USP25 being its own deubiquitinase. The downregulation also depends on cell lines. Apparently, it is not as substantial in PANC1 and SW1990 as in H460, Capan2 and A549 (Fig. 4B), which might be caused by different USP25 E3 activities in different cells. In addition, since the inhibitor inhibits both USP25 and USP28, we wanted to know if USP28 plays any role in regulating KRAS ubiquitination. We depleted USP28 expressing in HCT116 and HT29 cells with 3 different shRNAs, but detected no effects on KRAS expression levels (Fig. S6B and C). This result indicates that USP28 contribute little to KRAS ubiquitination homeostasis. Jo urn al Pr e-p roo f 12 There have been mutant KRAS inhibitors reported. We therefore compared CT1113 with KRASG12C inhibitor ARS-1620 (28) in H23 lung cancer cells which carry KRASG12C, and with KRASG12D inhibitor MRTX1133 (29, 30) in PANC1 pancreatic cancer cells which carry KRASG12D. As shown in Fig. 4C and D, CT1113 is as effective as ARS-1620 or MRTX1133 in blocking KRAS signaling, indicating that reducing the expression levels of KRAS protein can produce similar inhibitory effects on KRAS signaling as direct inhibition of the GTPase. Furthermore, stronger inhibitory effects against KRAS signaling could be achieved when CT1113 and the KRAS inhibitors were combined (Fig. 4C and D), this additive effect could be seen at even low concentrations of the inhibitors (Fig. S6D and E). As an inhibitor of USP28, CT1113 treatment also downregulated c-MYC levels as we reported previously (27), which the KRAS inhibitors are incapable of (Fig. 4C and D). Next, we applied CT1113 in vivo to determine if it is effective in blocking KRAS signaling in xenograft tumors. We inoculated nude mice with SW1990 pancreatic cancer cells (which are KRASG12C) and started to treat these mice with CT1113 (20 or 25 mg/kg, bid.) when the xenografts had grown to palpable sizes. The tumors were harvested for analysis after 2 weeks of CT1113 treatment. As expected, CT1113 treatment suppressed the growth of SW1990 xenograft tumors (Fig. 5A and B). The expression levels of KRAS as well as the phosphorylation levels of MEK and ERK were all downregulated in the tumors treated with CT1113 (Fig. 5C). Similar results were obtained in a patient-derived colon cancer xenograft model (CoY1607, from Oncocare Biotech, China) (Fig. S7). This model contains wildtype KRAS, but CT1113 was similarly effective in Jo urn al Pr e-p roo f 13 blocking KRAS signaling. In our previous report (27), CT1113 was shown to be effective in inhibiting the growth of xenograft tumors derived from HCT116 cells (which are KRASG13D) and the compound is tolerable in mice. USP25 expression correlates with RAS expression and unfavorable prognosis Having shown the importance of USP25 in maintaining KRAS expression and signaling, we next sought to determine if the expression of the deubiquitinase and KRAS are correlated in human tumors. We stained human cancer tissue arrays for USP25 and RAS (Fig. 6A and Fig. S8A). The anti-RAS antibodies were unable to distinguish K-, H-, and N-RAS and were reported previously suitable for IHC staining (31, 32). The expression of USP25 and RAS were scored and plotted (Fig. 6B). Strong correlations between USP25 expression and that of RAS were detected across lung, pancreatic, and colon cancers (Fig. 6B). In addition, USP25 expression in tumor tissues of lung and colon cancer samples is notably elevated compared to the adjacent noncancerous tissues (Fig. 6C and D). Moreover, the expression of USP25 is inversely correlated with patient survival in lung, colon, and pancreatic cancers (Fig. S8B). Jo urn al Pr e p roo f 14
Discussion
The ubiquitin-proteasome system, through the action of E3 ligases and the counteraction of deubiquitinases (DUBs), maintains protein homeostasis. Perturbing the function of E3s or DUBs can disrupt this homeostasis, which has been exploited by cancer cells. For example, the E3 ligase FBW7 is frequently mutated in human cancer due to its ability to target oncoproteins such as MYC for proteasomal degradation (33, 34). The RAS family of small GTPases are also subjected to this homeostatic regulation by a number of E3 ligases (10-12, 15, 16) and by the deubiquitinase USP25 identified here. We show here that disrupting this homeostasis through compromising USP25 is a viable approach to suppress KRAS. Genetic depletion of USP25 expression or pharmacological inhibition of the DUB, causes decreases in the expression levels of KRAS (wildtype or mutants) and in the strength of KRAS downstream signaling, resulting in reduced rates of cancer cell proliferation (Fig. 7). The development and clinical application of KRASG12C inhibitors have effectively overcome the previously perceived undruggable nature of KRAS (35, 36). Nonetheless, the emergence of drug resistance is unavoidable during the course of treatment with KRAS inhibitors (37). A side-by-side comparison of USP25 inhibitor (CT1113) with KRASG12C inhibitor (ARS1620) (Fig. 4C), or with KRASG12D inhibitor (MRTX1133) (Fig. 4D), demonstrated that the inhibition of USP25 is as effective as the direct inhibition of KRAS. These findings suggest that USP25 inhibitors may serve as viable alternatives to KRAS inhibitors for suppressing KRAS, Jo ur al Pr e-p ro f 15 particularly in cases where resistance to KRAS inhibitors has manifested. In addition, those KRAS mutants which are non-targetable currently, such as Q61X, could be suppressed by compromising USP25 function (Fig. 4B), adding more value to USP25 inhibitors as alternative KRAS blockers. USP25 was initially found to be involved in immune signaling through regulating ubiquitin modifications of TRAF proteins (38, 39). Since then, a number of proteins have been shown to require USP25 for their homeostasis, including Tankyrase (40), BCR-ABL (41), EGFR (42), HIF1 (43), and KEAP1 (44), which are all implicated in tumorigenesis one way or another. USP25 has a close homologue, USP28, and the two DUBs share 64% similarity. Due to this high degree of similarity, CT1113 can inhibit both with similar potency (27). USP28 is more implicated in cancer than US25, as it has a larger number of onco-proteins on its client list, including prominent ones like c-MYC (45), MYCN (46), LSD1 (47), etc. Now, with the identification of KRAS here as dependent on USP25 for its expression, the USP25/28 dual are gaining more importance as targets for cancer therapy, since their inhibition can now simultaneously suppress both c-MYC and KRAS (Fig. 4C and D), the two most prominent onco-drivers. There was a recent report that USP7 could deubiquitinate KRAS and maintain its expression in small cell lung cancer cells (48). In our DUB library screen, the siRNAs against USP7 did cause its mRNA levels to about 50% of the control (Fig. S1C), but we did not observe appreciable downregulation of KRAS protein levels (Fig. S1A and B), likely because there were still sufficient levels of USP7 protein translated from the remaining mRNA that were able to Jo urn al Pr e-p roo f 16 prevent excess ubiquitination of KRAS. It is not unusual that more than one deubiquitinases work on the same target just like there are more than one E3s that can ubiquitinate a target protein. Alternatively, USP7’s action on KRAS might be cell context-dependent. In any case, this reflects the complexity of protein homeostasis control.
Experimental Procedures
Cell culture
HEK293T, HCT116, HT29, NCI-H23, A549, Capan-2, SW1990, H460, PANC1, and LLC cells were purchased from ATCC. The cells were maintained in either DMEM, RPMI-1640, McCoy's 5A supplemented with 10% FBS, 1% Penicillin/Streptomycin at 37℃ with 5% CO2. siRNA library screening A siRNA library targeting 84 human deubiquitinases, and a control siRNA were purchased from RiboBio (Guangzhou, China). Each DUB is targeted with three siRNAs. HCT116 cells were reversed-transfected with 100 nM siRNAs using Lipofectamine RNAiMAX (Invitrogen, USA) for 48 h. The cells were then harvested and lysed for analysis. A secondary screening was performed similarly with the identified siRNAs in the primary screen. Cell proliferation and apoptosis assay The cells were seeded in a 96-well plate and incubated for 3-5 days. After the incubation period, the culture medium was replaced, and 100 μl of fresh medium along with 20 μl of MTS Jo urn al Pr e-p roo f 17 (Promega, USA) solution was added. The cells were then incubated for an additional 2 hours. Finally, the absorbance at 490 nm as an indicator of the number of live cells was measured using a microplate reader. For cell cycle analysis, the cells were trypsinized, collected by centrifugation, washed once in cold PBS, and fixed in 70% ice-cold alcohol over 1 hour. The cells were then spun down, washed with cold PBS, and incubated in PBS containing propidium iodide (PI, 50 μg/ml) and RNase A (50 μg/ml) for 30 min at room temperature. The PI-stained single cell suspension was analyzed on a BD LSRFortessa SORP Flow Cytometer (BD Biosciences). ModFit LT software (Verity Software House, Topsham, ME, USA) was used to analyze the DNA patterns and cell cycle stages. For apoptosis assay, the cells were trypsinized, collected by centrifugation, washed once with cold PBS, and stained with AnnexinV-FITC (Abcam, USA) and propidium iodide for 15~30 mins at room temperature. The cells were then analyzed with a flow cytometer.
RNA isolation and RT-PCR
Total RNA was extracted from cells using TRIzol (Sigma-Aldrich, USA) according to the manufacturer’s instructions. RNA was reverse-transcribed into cDNA using a reverse transcription kit (AG, China). cDNA was used for RT-qPCR reaction (AG, China). The primers used are listed in Supplementary Table 1.
Western blotting analysis
Cells or tissues were lysed with RIPA buffer (Applygen, China) supplemented with Jo ur al Pr e-p roo f 18 protease inhibitors and phosphatase inhibitors (Roche, USA), and the lysates were centrifuged at 10,000g for 10 minutes at 4°C to remove insoluble debris. The protein concentration of the resulting lysates was determined using a bicinchoninic acid (BCA) assay kit (Beyotime, China). Equal amounts of total protein were boiled for 10 minutes in 5x SDS loading buffer, separated on SDS-polyacrylamide gel, and transferred onto nitrocellulose membranes. The membranes were blocked with 5% non-fat dry milk in TBST for 1 hour at room temperature to block non-specific binding. Then, the membranes were incubated with primary antibodies overnight at 4°C. After three washes with TBST, the membranes were incubated with horseradish peroxidase (HRP)conjugated secondary antibodies for 1 hour at room temperature. The membranes were washed three times and visualized using SuperSignal™ West Pico Chemiluminescent substrate (Thermo Fisher Scientific, USA). Expression of GAPDH was commonly used as a loading control. The antibodies used are listed in Supplementary Table 2.
Ubiquitination assay
HEK293T and HCT116 cells were transfected with HA-KRAS and other relevant plasmids. 48 hours of after the transfection, the cells were treated with 10 μM MG132 for 6-8 hours, and then harvested and lysed in NETN buffer (pH 8.0 tris-HCl, 100mM NaCl, 1 mM EDTA, 0.5% Nonidetp-40) containing 1% SDS and 1% sodium deoxycholate. The lysates were vortexed vigorously for 15-30 minutes and boiled for 10 minutes. After that, 10 times the volume of NETN buffer was added to reduce the SDS content to 0.1%, and the lysates were centrifuged at 10,000g at room temperature to remove cell debris. The resulting cell lysates were incubated with Jo ur al Pr e-p roo f 19 appropriate antibody-conjugated beads followed by immunoprecipitation procedures.
GST-pulldown assay
KRAS4B fused to GST and USP25 fused to 6XHis were expressed in E. coli and purified with glutathione-agarose and nickel beads, respectively. His-USP25 was eluted out of the beads with PBS containing 250 mM imidazole and 500 mM NaCl. His-USP25 was incubated with GSTbound USP25 on the beads at 4 °C for 4 hours. The beads were then washed five times with GST buffer (1 mM EDTA, pH 8.0 Tris-HCl, 200 mM NaCl, and 1% Triton X-100), and the bound proteins were eluted from the beads using SDS gel sample buffer, separated in an SDS-PAGE gel, and analyzed with western blotting using specific antibodies.
Immunoprecipitation assay
The cells were lysed in NETN buffer for 30 minutes and centrifuged to remove cell debris. The resulting supernatant was the cell lysate. The cell lysates were then incubated with Sepharose beads (such as Flag M2 beads, Sigma) or protein A/G-conjugated Sepharose beads, which were conjugated with specific antibodies. The incubation was carried out at room temperature for 1 hour or overnight at 4°C. After the incubation, the beads were washed at least 3 times with NETN buffer. The proteins bound to the beads were eluted by boiling in SDS-gel loading buffer and analyzed by Western blotting. Plasmids and Lentiviruses Jo urn al Pr e-p roo f 20 Plasmids utilized in this work were generated through conventional cloning techniques. Specifically, the shRNAs were constructed in pLKO.1 vector using the following sequences: shNC (5’-TTCTCCGAACGTGTCACGT-3′), shUSP25-1 (5’-GCGTGAGCTGAGGTATCTATT-3’), shUSP25-2 (5’-GCACTTCTCCTGTTGACGATA-3’). Human USP25 and KRAS4A/B coding sequences were cloned into the pCDH vector with a Flag or HA tag. Lentiviruses for shRNA or gene expressing were produced in 293T cells using standard packaging plasmids and procedures. Lentiviral infection of cells was conducted using standard protocols, and the infected cells were selected with 2 μg/ml puromycin (Beyotime, China) for 2 days to establish stable cell lines.
Animal experiments
All animal experiments were approved by the Animal Care and Use Committee of the First Affiliated Hospital of Zhejiang University. Nude mice were purchased from Hangzhou Ziyuan Experimental Animal Technology Co., Ltd. To generate cell-derived xenograft tumors, Tet-on-shNC or Tet-on-shUSP25 HCT116 cells were mixed at a 1:1 ratio (volume) with matrigel (BD Biosciences, USA) and injected subcutaneously into 5-6-week-old BALB/c nude mice. After successful engraftment (when the xenograft tumors reached a size of ~50 mm3), the mice were given 0.2% doxycycline and 5% sucrose-containing drinking water and sacrificed 12 days later. The tumors were dissected out and cut into 3 equal parts, one for protein/RNA extraction, one for histology, and one for frozen storage. Jo urn al Pr e-p roo f 21 To test the efficacy of CT1113 in vivo, an established patient-derived colon cancer xenograft model (CoY1607) obtained from Oncocare Biotech, Inc. (Hangzhou, China) was used with the following procedure. A CoY1607 tumor was dissected out of its host, washed twice in PBS to remove necrotic tissue, and cut into small pieces of equal sizes and placed on ice. The tumor cubes were inoculated subcutaneously into nude mice within an hour of preparation. CT1113 treatment (20 mg/kg body weight, twice a day through an oral gavage) was started once the tumor mass became palpable. At the end of treatment, the tumor-bearing animals were sacrificed, and the tumor mass dissected out for analyses. In addition, xenograft tumors derived from SW1990 cells were used for testing CT1113. For that, 1x106 cells were mixed at a 1:1 volume ratio with matrigel (BD Biosciences, USA) and inoculated subcutaneously BALB/c nude mice. When the xenograft tumors reached approximately 50 mm³ in size, the mice were randomely divided into three groups, one group for vehicle control, and the other two for the administration of CT1113 at 20 or 25 mg/kg body weight, twice a day via oral gavage. CT1113 treatment lasted 27 days. At the conclusion of the treatment period, the tumors were excised and divided into three equal parts: one for protein/RNA extraction, one for histological examination, and one for cryogenic preservation.
Immunohistochemical staining
Tumor issues were fixed in 10% formaldehyde at 4oC overnight. Tissue processing, sectioning, and histological and immunohistochemical staining were performed by Servicebio (Wuhan, China). Each tumor sample was sectioned and the sections with largest tissue area were selected for immunohistochemical staining. For Ki67 scoring, three fields of view were randomly Jo urn al Pr e-p roo f 22 selected per section, and the ratio of Ki67-positive cells to the total number of cells within the field of view is calculated to determine the Ki67 positivity rate. The average of these rates is taken. For p-ERK scoring, again three fields of view are selected, and the ratio of the positive-staining area to the total area within these fields is calculated as the p-ERK score, with the average of these scores being determined. Human lung cancer, colon adenocarcinoma, and pancreatic cancer tissue arrays were purchased from Xiangyou Tech (Shanghai, China), and the provider also performed IHC staining of USP25 and RAS. The IHC staining strength was unbiasedly assessed using a composite positive scoring system, which is the product of staining intensity and the percentage of positive cells. The IHC staining intensity is graded on a scale from 0 to 4 as follows: 0: no observable staining; 1: very weak staining, barely perceptible; 2: weak staining, light yet clearly visible; 3: moderate staining, strong and distinct; 4: strong staining, very intense and prominent (Fig. S8A). The percentage of positive cells was scored as follows: 0: 0–5% positive cells; 1: 6–25% positive cells; 2: 26–50% positive cells; 3: 51–75% positive cells; 4: >75% positive cells. Scoring was independently conducted by three pathologists, with the final score being the average of their assessments.
Statistical analysis
Correlation analysis was performed using the Spearman correlation test to assess the relationship between variables. For paired samples, the Wilcoxon matched-pairs signed rank test Jo urn al Pr e-p roo f 23 was utilized. The significance between two groups was assessed using an unpaired two-tailed Student's t-test. Statistical analyses were conducted using GraphPad Prism 9.0. A p-value of less than 0.05 was considered statistically significant (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001). Data Availability All data are contained in this article. Supporting Information This article contains supporting information. Acknowledgement We thank the core facilities in the Central Lab of the First Affiliated Hospital of Zhejiang University and in the Zhejiang Provincial Key Laboratory of Pancreatic Disease. The authors thank people in Zhang lab for helpful discussions and suggestions throughout the work. This work was supported by grants from the National Natural Science Foundation of China (82373156), the National Key R&D Program of China (2018YFA0507500). Part of the work was supported by the R&D program of Chaser Therapeutics Inc.
Figure Legend
Figure 1. USP25 is a deubiquitinase for KRAS. A. USP25 deubiquitinates KRAS. KRAS ubiquitination levels were assessed via immunoprecipitation and western blotting following the knockdown of USP25 with two independent shRNAs in HCT116 cells. The amounts of total proteins from USP25-depleted cells were adjusted for immunoprecipitation to reflect the fact that these cells now contained less KRAS protein. B. Co-immunoprecipitation analysis of the interaction between HA-tagged KRAS and the endogenous USP25 in HEK293T cells. C. Co-immunoprecipitation analysis of the interaction between HA-tagged USP25 and the endogenous KRAS in HEK293T cells. D. GST pulldown assay with GST-KRAS (full length and truncations) and His-USP25. A diagram of KRAS protein is presented. His-USP25 was detected with western blotting (upper panel). The GST and GST fusion proteins were separated in an SDS-PAGE gel and visualized with Coomassie blue staining (lower panel). E. Co-immunoprecipitation analysis of the interaction between HA-tagged KRAS (wildtype and the 8A-mutant, D.) and the endogenous USP25 in HEK293T cells. F. Analysis of the ubiquitination levels in KRAS (wildtype and the 8A-mutant, D.) in 293T cells with or without USP25 overexpression. Figure 2. Identification of the ubiquitination sites in KRAS. A. The hypervariable regions of KRAS4A and KRAS4B. The red highlighted lysine residues are responsible for KRAS ubiquitination. B. Analysis of the ubiquitination levels in KRAS4B mutants. Flag-tagged wild-type KRAS4B (WT), KRAS4BK169R, or KRAS4BK172R were expressed in HEK293T cells and immunoprecipitated for western blotting analysis of ubiquitination levels. C. KRAS4BK172R no longer requires USP25 for stable expression. HEK293T cells were first transfected with Flag-tagged KRAS4B WT or K172R mutant and divided into two parts which were then infected with lentiviruses carrying a shUSP25-expressing cassette or shNC control cassette. Flag-tagged KRAS4B protein levels were analyzed with western blotting. D. Analysis of ubiquitination levels in KRAS4A mutants. Flag-tagged wild-type KRAS4A (WT), KRAS4AK169R, or KRAS4AK172R were expressed in HEK293T cells and immunoprecipitated for western blotting analysis of ubiquitination levels. E. KRAS4AK169R no longer requires USP25 for stable expression. HEK293T cells were first transfected with Flag-tagged KRAS4A WT or K169R mutant and divided into two parts which were then infected with lentiviruses carrying a shUSP25-expressing cassette or shNC control cassette. Flag-tagged KRAS4A protein levels were analyzed with western blotting. Jo urn al Pr e-p roo f 29 Figure 3. The proliferation of tumor cells requires USP25. A. Western blotting analysis of the expression levels of KRAS and its downstream signaling pathway proteins following USP25 knockdown in HCT116 cells. B. Western blotting analysis of the expression levels of KRAS and its downstream signaling pathway proteins following USP25 knockdown with shUSP25-1 and -2 and re-expression of USP25 in HCT116 cells. C. Western blotting analysis of the expression levels of KRAS and its downstream signaling pathway proteins following USP25 knockdown with shUSP25-2 and exogenous expression of KRASG13D in HCT116 cells. D. Growth curve analysis of HCT116 cells with USP25 expression depleted (with shUSP25-1) or depleted plus re-expression of USP25. E. Growth curve analysis of HCT116 cells with USP25 expression depleted (with shUSP25-2) or depleted plus exogenous expression of KRASG13D. F. The growth of the xenograft tumors derived from HCT116 cells carrying doxycycline-inducible shUSP25 expression cassette (Tet-on-shUSP25) or a negative control (Tet-on-shNC). G. Photographic representation of tumors excised from the nude mice. H. The weight of tumors in (C). I. Western blotting analysis for KRAS and its downstream signaling proteins in the tumor samples. J. Immunohistochemical (IHC) analysis of the expression of proliferation marker Ki67 and phosphor-ERK (p-ERK) in tumor tissue sections along with quantitation of the IHC staining. One section from each one of the 5 tumor samples was stained and quantitated. Data are presented as mean ± SD. **, p < 0.01; ***, p < 0.001. Figure 4. Pharmacological inhibition of USP25 downregulates KRAS expression and its downstream signaling. A. Analysis of KRAS signaling pathway proteins with western blotting analysis in HT29 cells treated with different concentrations of CT1113 for 72 hrs. B. Western blotting analysis of KRAS signaling pathway proteins across multiple cell lines treated with 800 nM CT1113 for 72 hrs. C. Examination of KRAS signaling pathway proteins with western blotting analysis in H23 cells treated with CT1113 and ARS-1620 individually or in combination for 72 hours. Since CT1113 also inhibits USP28, the expression levels of USP28 and its substrate c-MYC were also examined. D. Examination of KRAS signaling pathway proteins with western blotting analysis in PANC1 cells treated with CT1113 and MRTX1133 individually or in combination for 72 hours. The expression levels of USP28 and its substrate c-MYC were also examined. Figure 5. Pharmacological inhibition of USP25 suppresses tumor growth. A. Photographs of the xenograft tumors derived from human pancreatic cancer cell line SW1990. Jo urn al P e-p roo f 30 1x106 SW1990 cells were inoculated in each nude mouse. When the xenografts grew to palpable sizes, the mice were given CT1113 orally (20 or 25 mg/kg body weight) twice a day for 2 weeks. B. The weight of the tumors in A. C. Western blotting analysis of KRAS signaling pathway proteins in vehicle control and CT1113 (25 mg/kg) treated tumors. Data are presented as mean ± SD. ****, p < 0.0001. Figure 6. The expression of USP25 correlates with RAS in human cancer. A. Representative immunohistochemical staining images from tissue microarrays (TMA) for USP25 and RAS proteins. The human lung and colon adenocarcinoma TMAs consist of 80 pairs of cancerous and para-cancerous tissues. The human pancreatic cancer TMA consists of cancerous tissues only. Scale bar, 100 µm. B. The correlation between USP25 and RAS expression in human cancers. The expression scores of the two proteins in TMAs were plotted against each other. C. Enhanced expression of USP25 and RAS in human cancer specimens relative to adjacent nontumor tissues. Shown are representative images of IHC staining of USP25 and RAS in cancerous and para-cancerous tissues of the lung cancer and colon adenocarcinoma samples. No paracancerous tissues are available in the pancreatic cancer samples for comparison. D. Pair-wise comparison of USP25 and RAS expression in cancerous and para-cancerous tissues from C. Figure 7. A schematic illustration of the regulation of KRAS by USP25. The expression of KRAS proteins depends on USP25. Compromising USP25 function can lead to suppression of KRAS. Jo urn al Pr e-p roo f A B Figure 1 D His-USP25 GS T GS T 1 -10 0 GS T 1 -12 0 GS T 1 -14 0 GS T 1 -18 8 1 188166 G-domain HVR LPSRTVDTKQAQDLARSYGIP KRAS4B KRAS4B-8A 120 140 LPAAAVDTAQAAALAAAYGIP 120 140 C GAPDH KRAS HA HA KRAS Co ntr ol US P2 5-H A IP: HA Input 293T GAPDH KRAS USP25 KRAS Ub IP: KRAS Input HCT116 sh NC sh US P2 5- 1 sh US P2 5- 2 -70 -55 -180 -15 -130 -15 -35 kDa GAPDH USP25 HA HA USP25 Co ntr ol HA -K RA S 293T IP: HA Input -130 -25 -25 -130 -37 kDa -130 -25 -130 -25 -35 kDa kDa -130 -25 -35 -55 -40 Jo urn al Pr e-p roo f Figure 1 F 35- 55- 130- 130- 70- 55- 70- 130- 100- 170- 35- (kDa) Ub Ub FLAG-KRAS FLAG-KRAS USP25-HA Tubulin IP : F LA G In pu t FL AG -K RA S FL AG -K RA S 8A FL AG -K RA S FL AG -K RA S 8A pC DH -F LA G USP25-HA 293T E USP25 HA USP25 GAPDH HA GF P KR AS -H A KR AS 8A -H A IP : H A In pu t 293T -130 -25 -25 -35 -130 kDa Jo urn al Pr e-p roo f -35 -25 -130 YRLKKISKEEKTPGCVKIKKCIIM HKEKM-SKDGKKKKKKSKTKCVIM KRAS4A KRAS4B 166 189 Hypervariable Region (HVR) CAAX A B 166 188 GAPDH FLAG-KRAS4B HA-Ub FLAG-KRAS4B HA-Ub HA-Ub FLAG-KRAS4B W T + + K1 69 R + IP : F LA G In pu t K1 72 R + - GAPDH FLAG-KRAS4A HA-Ub FLAG-KRAS4A HA-Ub HA-Ub FLAG-KRAS4A W T + + K1 69 R + IP : F LA G In pu t K1 70 R + - D 169 172 C E Figure 2 GAPDH USP25 FLAG FLAG-KRAS4B (WT) FLAG-KRAS4B (K172R) shN C shU SP 25 shN C shU SP 25 -35 -25 -130 GAPDH USP25 FLAG shN C shU SP 25 shN C shU SP 25 FLAG-KRAS4A (WT) FLAG-KRAS4A (K169R) kDakDa 1.0 0.66 1.07 0.99 1.0 0.46 1.17 1.29 -130 -25 -25 -35 -70 -70 -130 kDa kDa -130 -25 -25 -130 -35 Jo urn al Pr e-p roo f Figure 3 A B C USP25 KRAS p-MEK MEK ERK GAPDH sh NC sh US P2 5-1 sh US P2 5-2 HCT116 p-ERK -130 kDa -15 -40 -40 -40 -40 -35 GAPDH KRAS p-ERK p-MEK USP25 ERK sh NC sh US P2 5-2 sh US P2 5-2 +U SP 25 sh NC +U SP 25 HCT116 MEK sh US P2 5-1 +U SP 25 sh US P2 5-1 kDa -130 -15 -40 -40 -40 -40 -35 GAPDH KRAS p-ERK p-MEK USP25 ERK MEK HCT116 sh NC sh US P2 5-2 sh US P2 5-2 +G 13 D sh NC +G 13 D kDa -130 -15 -40 -40 -40 -40 -35 D O D (4 90 n m ) HCT1160.8 0.6 0.4 0.2 0 0 1 2 3 4 Time (day) 5 E shNC shUSP25-2 shNC+KRASG13D shUSP25-2+KRASG13D Tet-on-shNC Tet-on-shUSP25 - Dox + Dox500 400 300 200 100 0 Tu m or v ol um e (m m 3 ) 0 5 10 Days Tet-on-shNC+Dox Tet-on-shUSP25+Dox Tet-on-shUSP25 Tet-on-shNC ** ns 0 1 2 3 4 Time (day) 5 0.8 O D (4 90 n m ) 0.6 0.4 0.2 0 HCT116 *** shNC shUSP25-1+USP25 shNC+USP25 shUSP25-1 ns F G *** ns 1 2 3 4 51 2 3 Jo urn al Pr e-p roo f Figure 3 GAPDH KRAS pERK USP25 ERK pMEK MEK Tet-on shNC + Dox Tet-on shUSP25 + Dox 1 2 3 4 5 1 2 3 4 5 kDa I Tu m or w ei gh t ( g) 0.0 0.2 0.4 0.6 Te t-o n-s hU SP 25 +D OX Te t-o n-s hU SP 25 -2 Te t-o n-s hN C+ DO X Te t-o n-s hN C *** *** ns ns 60 80 40 20 0 Te t-o n-s hN C +D OX Te t-o ns hU SP 25 +D OX KI 67 p os iti ve c el ls (% ) *** H J Ki67 p-ERK Te t-o nsh N C +D O X Te t-o nsh U SP 25 +D O X 20 30 10 0p -E R K po si tiv e ar ea (% ) *** Te t-o n-s hN C +D OX Te t-o ns hU SP 25 +D OX -130 -15 -40 -40 -40 -40 -35 Jo urn al Pr e-p roo f Figure 4 C A B USP25 KRAS p-MEK MEK p-ERK ERK GAPDH HT29 (KRASWT) DM SO 20 0n M 40 0n M 60 0n M 80 0n M CT1113 kDa -130 -15 -40 -40 -40 -40 -35 D GAPDH KRAS ERK p-ERK p-MEK MEK USP25 PANC1 SW1990 Capan2 A549 CT1113 (800 nM) G12VG12D G12D G12S Pancreas Pancreas Pancreas Lung - + - + - + - + - + KRAS: Tissue Origin: Cell Line: -130 -25 -40 -40 -40 -40 -35 kDa H460 Q61H Lung USP25 KRAS p-ERK ERK p-MEK MEK GAPDH CT1113 (nM) ARS-1620 (nM) H23 (G12C) kDa -130 -15 -40 -40 -40 -40 -35 10005000 10005000 1000500010005000 0 0 0 0 0 0 USP28 c-MYC -130 -55 CT1113 (nM) MRTX1133 (nM) kDa 5000 5000 50005000 0 0 0 0 GAPDH p-ERK p-MEK USP25 ERK MEK KRAS PANC1(G12D) -130 -15 -40 -40 -40 -40 -35 c-Myc USP28 -130 -55 Jo urn al Pr e-p roo f USP25 KRAS ERK p-ERK GAPDH p-MEK MEK 130 15 40 40 40 40 40 Vehicle CT1113 (25 mg/kg) 1 2 3 4 5 kDa1 2 3 4 5 CT 11 13 (2 0m g/k g) CT 11 13 (2 5m g/k g) **** **** 1.8 1.5 1.2 0.9 0.6 0.3 0 Ve hic le Tu m or w ei gh t ( g) Ve hic le CT 11 13 (2 0m g/k g) CT 11 13 (2 5m g/k g) 1 2 3 4 5 6 7 8 A C B Figure 5 Jo urn al Pr e-p roo f A B C Para-carcinoma TissueTumor Lu ng c an ce r C ol on ad en oc ar ci no m a Figure 6 15 R=0.6341 P<0.0001 15 10 5 0 0 5 10 U SP 25 S co re RAS Score Lung cancer 15 10 5 0 0 5 10 15 RAS Score R=0.6339 P<0.0001 Pancreatic cancer 15 R=0.2522 P=0.0014 10 4 2 0 0 5 10 RAS Score Colon adenocarcinoma 8 6 15 10 5 0 Tumor Normal U SP 25 S co re p＜0.0001 15 10 5 0 Tumor Normal R AS S co re Lung cancer p＜0.0001 10 2 0 Tumor Normal U SP 25 S co re p=0.0443 4 6 8 15 10 5 0 Tumor Normal R AS S co re Colon adenocarcinoma p＜0.0001 D R AS U SP 25 Pancreatic cancerLung cancer Colon adenocarcinoma Jo urn al Pr e-p roo f KRAS USP25 KRAS Ub Ub Ub Ub CT1113 Cancer Cell Proliferation Proteasomal Degradation Figure 7 Jo urn al Pr e-p roo f
Author Contribution Statement
Huailu Ma: Conceptualization and Experimenting. Huiyuan Guan: Conceptualization and Experimenting. Xiao Sun: Conceptualization and Experimenting. Lingzhi Wu: Experimenting. Mengjiao Cai: Experimenting. Xinghua Zhen: Experimenting. Xiang Shen: Experimenting. Suxia Han: Supervision. Jin Peng: Conceptualization and Experimenting. Guangxue Liu: Conceptualization and Experimenting. Pumin Zhang: Conceptualization, Supervision, and Drafting manuscript. Jo urn al Pr e-p roo f
Declaration of Interest Statement
☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐ The author is an Editorial Board Member/Editor-in-Chief/Associate Editor/Guest Editor for this journal and was not involved in the editorial review or the decision to publish this article. ☐ The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jo urn al Pr e-p roo f
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