RHBDL2 promotes the proliferation, migration, and invasion of pancreatic cancer by stabilizing the N1ICD via the OTUD7B and activating the Notch signaling pathway

<jats:title>Abstract</jats:title><jats:p>Pancreatic cancer (PC) is one of the most malignant types of cancer, and is characterized by early metastasis, limited response to chemotherapeutics, and poor prognosis. Therefore, there is an urgent need to explore new therapeutic strategies for PC treatment. Human rhomboid-like 2 (RHBDL2) is differentially expressed in cervical and breast cancer. However, the correlation between RHBDL2 and PC remains unclear. We found that RHBDL2 is highly expressed in human PC cells and tissues and is significantly associated with distant metastasis and poor survival of patients with PC. Gain- and loss-of-function assays indicated that RHBDL2 could accelerate PC cell proliferation and mobility in vitro and in vivo. The RNA-Seq results suggest that RHBDL2 may be involved in the activation of Notch signaling pathway. IMR-1 could restore the proliferation and metastatic capacity of PC cells mediated by RHBDL2. RHBDL2 interacted with and cleaved Notch1, resulting in the release of N1ICD. RHBDL2 decreased the ubiquitination level of N1ICD and collaborated with Ovarian tumor domain-containing 7B (OTUD7B) to stabilize N1ICD via the ubiquitin-proteasome pathway. RHBDL2 facilitated PC cell proliferation and mobility by stabilizing the N1ICD via the OTUD7B and activating the Notch signaling pathway. Thus, targeting this novel pathway may be a potential therapeutic strategy for PC.</jats:p>

section_content: Pancreatic cancer (PC) is one of the most malignant types of cancer and is characterized by local invasion, early metastasis, and limited response to chemotherapeutics [1, 2]. These dismal characteristics lead to poor prognosis in PC patients, with a low 5-year overall survival rate (less than 5%) [3]. Hence, understanding the biological processes and underlying molecular mechanisms of PC may aid in the development of effective therapeutic methods. 

Human rhomboid-like 2 (RHBDL2) is a member of the rhomboid family of the integral membrane proteins and is associated with Drosophila rhomboid proteins [4, 5]. RHBDL2 functions as an intramembrane serine protease, which can cleave the membraneanchored DER ligand Spitz [6], the collagen receptor tyrosine kinase DDR1 [7], Ca 2+ release-activated Ca 2+ (CRAC) channels pore-forming subunit Orai1 [8], and so forth. Koch et al. identified RHBDL2 as an alternative sheddase that can efficiently induce IL-11R secretion. In addition, they determined the RHBDL2 cleavage site, which is close to the plasma membrane, between Ala-370 and Ser-371 [9]. RHBDL2 mRNA expression is markedly elevated in lowgrade breast cancer tissues compared with that in normal breast samples [10]. Cheng et al. reported that RHBDL2 is overexpressed in the breast cancer cell line MDA-MB-231 and cervical cancer cell line HeLa S3, which can cleave the EGF ligand and thereby result in the activation of EGFR signaling, finally leading to cell proliferation and reduction in cell adhesion [11]. However, to date, the impact of RHBDL2 on PC progression and its clinical and survival significance have not been clearly elucidated. 

The Notch signaling pathway modulates various biological processes including carcinogenesis [12] [13] [14]. Once the Notch receptor binds to the ligand, it is cleaved, leading to the release of the Notch intracellular domain (NICD). The NICD is translocated to the nucleus, where it binds to the transcription complex CSL/ RBPJκ. Thereafter, the transcription of downstream target genes is activated, which further accelerates cancer progression, including PC [15] [16] [17]. Notch1 is a Notch receptor and is dysregulated in a variety of cancer types [18] [19] [20] ; however, the underlying mechanism between Notch1 and PC needs to be studied further. 

Ubiquitination is a vital process involved in the progression of various cancer types, including PC [21] [22] [23]. Deubiquitinase (DUB) belongs to a large group of enzymes that can remove ubiquitin chains from specific proteins and thereby stabilize them [24]. To date, DUBs have a significant role in the regulation of DNA replication stress [25], inflammasome activation [26], and cancer progression [27] [28] [29]. Ovarian tumor domain-containing 7B (OTUD7B) is a DUB of the OTU protein superfamily and plays an important role in the development of lung cancer [30], breast cancer [31], and hepatocellular carcinoma [32]. However, whether OTUD7B can function as a DUB to modulate the Notch signaling pathway in PC remains unknown. 

The present study found that RHBDL2 is highly expressed in PC cells and tissues, and can promote the proliferation, migration, and invasion capacity of PC cells in vitro and in vivo by activating the Notch signaling pathway. RHBDL2 interacts with Notch1 and mediates the cleavage and release of Notch1 intracellular domain (N1ICD). RHBDL2 inhibits the degradation of N1ICD through the ubiquitin-proteasome pathway. OTUD7B was identified as a DUB that interacts with both RHBDL2 and N1ICD and decreases the ubiquitination level of N1ICD. RHBDL2 collaborates with OTUD7B to stabilize N1ICD. The role of RHBDL2 in the proliferation, migration, and invasion of PC cells relies on its protease-cleavage activity and OTUD7B's DUB function. The present study identified a new posttranslational modulation mechanism of N1ICD mediated by RHBDL2 and OTUD7B, which may contribute to the identification of efficient treatment strategies for PC.
section_name: INTRODUCTION

section_name: METHODS

section_content: Surgical specimens of PC and adjacent non-tumor tissues were acquired from 95 patients who underwent surgical resection for PC at the Department of Hepatobiliary Surgery, the Affiliated Hospital of Guizhou Medical University. This study was approved by the ethics committee of the Affiliated Hospital of Guizhou Medical University (2018 Lunshen No. 018), and each patient provided written informed consent.
section_name: Patients and specimens

section_content: Antibodies were obtained from Abcam (Cambridge, MA, USA), Proteintech (Wuhan, Hubei, China), and Cell Signaling Technology (Danvers, MA, USA). Human pancreatic ductal epithelial (HPDE) cells and PC cell lines (CFPAC-1, MIA PaCa-2, SW1990, AsPC-1, PANC-1, and BxPC-3) were purchased from American Type Culture Collection (Manassas, VA, USA). These cell lines were authenticated via STR profiling and confirmed to be mycoplasma-free. The cell lines were grown in Dulbecco's Modified Eagle Medium or Roswell Park Memorial Institute Medium (Gibco, Waltham, MA, USA) supplemented with 1% penicillin/streptomycin (Gibco) and 10% fetal bovine serum (Gibco) at 37 °C in a 5% CO 2 humidified atmosphere. For all of the cellular experiments, three independent repeated assays were performed. Small molecular compounds, including IMR-1 (Selleck), MG-132 (Selleck), 3-methyladenine (3-MA; Selleck), cycloheximide (CHX; Sigma), chloroquine (Sigma), and NH 4 Cl (Aladdin), were purchased from the indicated suppliers.
section_name: Cell culture and chemicals

section_content: TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to extract RNA from cells and tissues. cDNA was obtained using PrimeScript RT reagent (TaKaRa, Dalian, China) to conduct reverse transcription. A quantitative real-time polymerase chain reaction (qRT-PCR) assay was performed to investigate the gene expression levels.
section_name: RNA extraction and quantitative real-time polymerase chain reaction

section_content: Radioimmunoprecipitation assay buffer (Thermo Fisher Scientific; Waltham, MA, USA) mixed with protease inhibitors (Boster Biological Technology; Wuhan, Hubei, China) was used to extract proteins from cells and tissues. A bicinchoninic acid (BCA) assay kit (Biosharp, Hefei, Anhui, China) was used to quantify the protein. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed to separate 40 mg of total protein. Specific antibodies were used to probe blots. Three independent repeated assays were performed.
section_name: Western blot assay

section_content: Small interfering RNAs (siRNAs) targeting RHBDL2, ATXN3, OTUD7B, and USP10 were acquired from RiboBio (Guangzhou, China). Plasmids for Myc-tagged N1ICD and its truncations, Flag-tagged wild-type RHBDL2, Flagtagged RHBDL2-SA mutant, HA-tagged ubiquitin, HA-tagged OTUD7B, Flag-tagged OTUD7B, and Flag-tagged OTUD7B-C194S mutant were purchased from WZ Biosciences, Inc. (Shandong, China). The RHBDL2 and short hairpin RNA lentiviruses were purchased from GeneChem (Shanghai, China). siRNA and plasmid transfection were conducted using Lipofectamine 3000 (Invitrogen, Waltham, MA, USA) according to the manufacturer's instructions. After infecting the PC cells with lentivirus for 48 h, stable cell lines were selected for 2 weeks and cultured with puromycin (1 μM, Invitrogen).
section_name: siRNAs and plasmid transfection and lentivirus infection

section_content: Tissue sections were deparaffinized with xylene and rehydrated using an ethanol gradient. The samples were incubated with sodium citrate for antigen retrieval, and goat serum was used to block non-specific binding sites. Thereafter, primary antibodies, including Ki-67 (1:2000, Proteintech, 27309-1-AP), RHBDL2 (1:200, Proteintech, 12467-1-AP), and PCNA (1:200, Proteintech, 10205-2-AP), were incubated at 4 °C overnight, and subsequently incubated with the corresponding secondary antibodies for 2 h at room temperature. The positively stained cells and signal intensity in three randomly selected areas were examined and scored blindly by two independent observers.
section_name: Immunohistochemistry

section_content: PC cells (3000/well) were seeded in a 96-well plate and cultured for 0, 24, 48 and 72 h, respectively. Thereafter, 110 μl of mixed solution (10 μl of Cell Counting Kit-8 [CCK-8] reagent plus 100 μl of complete medium) was added to the wells in accordance with the manufacturer's instructions (Dojindo Molecular Technologies, Inc., Japan). After incubating for 2 h, a microplate reader (Tecan, Austria) was used to determine the absorbance at a wavelength of 450 nm. Five replicate wells per group and three independent repeated assays were performed.
section_name: Cell Counting Kit-8 assay

section_content: The PC cells were placed in 6-well plates (800 cells/well) and incubated for two weeks. The cells were fixed with 4% paraformaldehyde (Biosharp, Hefei, Anhui, China) for 20 min. After washing with phosphate-buffered saline (PBS), 0. 25% crystal violet solution (Biosharp, Hefei, Anhui, China) was used to stain cells for another 20 min. Finally, the culture plates were photographed.
section_name: Colony formation assay

section_content: The PC cells were seeded in 24-well culture plates covered with slides and cultured for 24 h. Subsequently, 10 μM of EdU solution was added to the cells and incubated for 24 h. The cells were fixed with 4% paraformaldehyde for 20 min and washed with PBS. After permeabilization with 0. 5% Triton X-100 for 10 min, the cells were stained with Apollo 567 and Hoechst 33342. An Olympus FSX100 microscope (Olympus, Tokyo, Japan) was used to capture the images.
section_name: EdU assay

section_content: The PC cells were seeded in 6-well plates. When the cells reached confluence, a 200-μl pipette tip was used to scratch the wound. The cells were then incubated with serum-free medium for 48 h. The images of each wound were captured using an inverted microscope (Olympus, Tokyo, Japan) at 0 h and 48 h after scraping.
section_name: Wound healing assay

section_content: The cells that underwent starvation treatment were plated in the upper chamber, while 700 μl of complete medium was added to the bottom chamber. After culturing for 24 h, the cells in the upper chamber were removed. The cells were fixed or stained with 4% paraformaldehyde and 0. 25% crystal violet solution. Matrigel was used to coat the upper membrane prior to cell seeding to analyze cell invasion. Migrated and invasive cells were imaged using a microscope.
section_name: Transwell assays

section_content: A total of 25 female 6-week-old BALB/c nude mice were randomly divided into five groups. The PC cells (2 × 10 6 /mouse) were injected subcutaneously into the right armpit of the mice. When the tumors became macroscopically visible, calipers were used to monitor them weekly. The equation (length × width 2 )/2 was used to calculate the tumor volume. After 42 days, the tumors were excised, fixed with 4% paraformaldehyde, and embedded in paraffin for IHC. For metastasis analysis, the PANC-1 cells (1 × 10 6 /mouse) were injected into the tail vein of the mice. After 56 days, the mice were euthanized to excise the lungs, and the number of pulmonary metastatic nodules was counted and further validated through HE staining. All animal studies were approved by the Animal Care Welfare Committee of Guizhou Medical University (No. 1801105).
section_name: Animal assays

section_content: The cells were cultured in 6-well plates overnight, and the plasmids were subsequently transfected into the cells using Lipofectamine 3000 reagent (Invitrogen, Waltham, MA, USA) in accordance with the manufacturer's instructions. Subsequently, the cells were lyzed, and the Dual Luciferase Reporter Assay Kit (Promega, Madison, WI, USA) was used to determine the firefly and renilla luciferase signals. 

Immunoprecipitation, silver staining, and mass spectrometry PC cells were lyzed, and proteins were harvested and incubated with the RHBDL2 primary antibody overnight at 4 °C on a rotating wheel. Subsequently, Sepharose-conjugated protein A + G beads (Beyotime, Shanghai, China) were added to the mixtures and rotated for 4 h at 4 °C. Later, the beads were collected after full centrifugation and washing, and were boiled to isolate the protein. SDS-PAGE assay was performed for further investigation, and the proteins were subjected to Western blotting, silver staining, or mass spectrometry (MS) analysis. Silver staining assay was conducted using the Silver Stain Detection Kit in accordance with the manufacturer's instructions. MS was performed at Genecreate Inc. (Wuhan, Hubei, China).
section_name: Dual Luciferase Reporter Assay

section_content: The PC cells were seeded in 24-well plates covered with slides and cultured for 24 h. Next, 4% paraformaldehyde and 0. 5% Triton X-100 solution were used to fix and permeabilize the cells, respectively. Non-specific binding sites were blocked with 5% bovine serum albumin. The cells were incubated with primary antibodies against RHBDL2 (1:200, Proteintech, 12467-1-AP) and Notch1 (1:200, Proteintech, 10062-2-AP) at 4 °C for 16 h. After washing with PBS, the cells were incubated with the corresponding fluorescent secondary antibodies in the dark for 2 h at room temperature. After washing with PBS, 4′, 6-diamidino-2-phenylindole was used to stain the cell nuclei. A fluorescence microscope was used to detect the target protein expression.
section_name: Immunofluorescence assay

section_content: The results were analyzed using SPSS (version 23. 0; IBM Corp., Armonk, NY, USA), and quantitative data were expressed as mean ± standard deviation. Student's t-test (two-tailed) was used to examine the differences between two samples, while multiple groups were evaluated using a one-way analysis of variance. The overall survival was determined using the Kaplan-Meier method. A P value of <0. 05 was considered significant.
section_name: Statistical analysis

section_content: Overexpression of RHBDL2 and its positive correlation with poor prognosis in patients with PC By analyzing the published mRNA expression profiles (GSE16515, GSE28735, and GSE15471) acquired from the Gene Expression Omnibus database, we discovered that the RHBDL2 mRNA was noticeably upregulated in PC tissues compared with that in normal/ non-tumor tissues (Fig. S1A-C ). The upregulation of RHBDL2 in PC tissues was also confirmed in the data obtained from The Cancer Genome Atlas (TCGA) database (Fig. S1D ). Furthermore, analysis of PAAD datasets from TCGA database showed that high RHBDL2 expression in PC patients resulted in a shorter survival time (Figure S1E ). Similarly, qRT-PCR and western blot assays indicated that RHBDL2 was upregulated at the mRNA and protein levels in all six human PC cell lines (CFPAC-1, MIA PaCa-2, SW1990, AsPC-1, PANC-1, and BxPC-3) compared with that in the primary normal human pancreatic duct epithelial (HPDE) cells (Fig. 1A, B ). Concordantly, the expression level of RHBDL2 was examined by conducting qRT-PCR and western blot assays of PC tissues and adjacent non-tumor tissues, which showed that the RHBDL2 was upregulated in PC tissues (Fig. 1C, D ). Furthermore, immunohistochemistry (IHC) assay also showed that the RHBDL2 expression was dramatically upregulated in PC tissues, especially in patients with distant metastasis (Fig. 1E, F ). Moreover, the Kaplan-Meier plot indicated that PC patients with high RHBDL2 expression had a short survival time, whereas PC patients with low RHBDL2 expression had better survival (Fig. 1G ). Taken together, these results suggest that RHBDL2 may have significant clinical value in PC.
section_name: RESULTS

section_content: To assess the biological function of RHBDL2 in PC cells, loss-and gain-of-function assays were performed. The results of CCK-8, colony formation, and EdU assays showed that RHBDL2overexpressing PC cells had enhanced proliferation capacity, whereas the downregulation of RHBDL2 decreased the PC cell growth (Fig. 2A-C ). As shown in the wound healing and Transwell assays, the upregulation of RHBDL2 confers the enhanced migratory and invasive abilities to PC cells. However, RHBDL2 silencing revealed a decrease in the mobility of these cells (Fig. 2D, E ). To evaluate the impact of RHBDL2 on PC cells in vivo, a nude mouse xenograft tumor model was constructed. The upregulation of RHBDL2 expression facilitated tumor growth, while RHBDL2 silencing led to the inhibition of tumor growth (Fig. 2F ). The RHBDL2 overexpression group showed higher tumor weight and volume, while the RHBDL2 knockdown groups had lower tumor weight and volume (Fig. 2G, H ). Moreover, IHC staining showed high RHBDL2, Ki-67, and PCNA expression levels in tumor tissues with RHBDL2 upregulation. However, the RHBDL2-silenced groups showed lower RHBDL2, Ki-67, and PCNA levels (Fig. 2I ). Furthermore, to verify whether RHBDL2 can accelerate metastasis in vivo, PC cells were injected in the tail vein of nude mice. The overexpression of RHBDL2 significantly increased the number of lung metastatic nodules, whereas RHBDL2 downregulation successfully weakened the degree of lung metastasis (Fig. 2J, K ). Collectively, these findings suggest that RHBDL2 facilitates PC tumorigenesis and distant metastasis.
section_name: RHBDL2 promotes the proliferation, migration, and invasion of PC cells in vitro and in vivo

section_content: To evaluate which signaling pathway may be involved in the oncogenic effect of RHBDL2 in PC cells, a RNA-seq analysis was performed. As shown in Fig. 3A, RHBDL2 expression was significantly related to the activation of Notch signaling pathway. Consistently, the results of qRT-PCR and western blot assays showed that the expression level of RHBDL2 and the wellcharacterized downstream target genes hes family bHLH transcription factor 1 (HES1), hes related family bHLH transcription factor with YRPW motif 1 (HEY1), zinc finger E-box binding homeobox 1 (ZEB1), matrix metallopeptidase 9 (MMP9), snail family transcriptional repressor 1 (SNAIL1), and twist family bHLH transcription factor 1 (TWIST1) of the Notch signaling pathway were dramatically enhanced in the RHBDL2 overexpression group but decreased in the RHBDL2-silenced groups (Fig. 3B, C ). In addition, RHBDL2 overexpression dramatically upregulated the luciferase reporter activity; however, knockdown of RHBDL2 led to the suppression of luciferase reporter activity (Fig. 3D ). Next, the influence of the Notch signaling pathway on RHBDL2′s oncogenic role in PC cells was investigated by conducting rescue assays with the application of IMR-1, a small molecule inhibitor of the Notch signaling pathway. As shown in Fig. 3E-G, the results of CCK-8, colony formation, and EdU assays indicated that the Notch signaling pathway inhibitor IMR-1 blocked the RHBDL2-mediated enhanced proliferation capacity in PC cells. Similarly, wound healing and Transwell assays also showed that the migratory and invasive abilities of PC cells strengthened by RHBDL2 were partly reversed by the IMR-1 (Fig. 3H, I ). Moreover, treatment of PC cells with IMR-1 abolished the effect of RHBDL2 on the expression level of Notch signaling pathway downstream target genes (HES1, HEY1, ZEB1, MMP9, SNAIL1, and TWIST1) (Fig. 3J, K ), and on the luciferase reporter activity (Fig. 3L ). The above results illustrate that RHBDL2 accelerates PC cell proliferation and motility by activating the Notch signaling pathway.
section_name: RHBDL2 promotes the proliferation, migration, and invasion of PC cells by activating the Notch signaling pathway

section_content: Considering that RHBDL2 functions as an intramembrane serine protease, we hypothesized that RHBDL2 could bind to and subsequently cleave specific proteins in PC cells. To identify the potential binding partners of RHBDL2, whole-cell protein lysates were immunoprecipitated with RHBDL2 antibody. A silver staining assay was performed to determine the differential protein bands in the resultant immunoprecipitates. The lysate was analyzed using the MS approach. Results showed that Notch1 was the major binding partner of RHBDL2 (Fig. 4A ). The interaction between RHBDL2 and Notch1 was determined in PC cells by IP assay (Fig. 4B, C ). Furthermore, the colocalization of RHBDL2 and Notch1 was verified by immunofluorescence (IF) staining (Fig. 4D ). IF staining also showed that the level of Notch1 increased in the nuclei of PC cells in the RHBDL2 overexpression group (Fig. 4E ). The N1ICD domain was subsequently mapped to accurately detect the N1ICD-RHBDL2 interaction region. Results of the immunoprecipitation (IP) assay revealed that the N1ICD full-length, RAM-TAD, and RAM-ANK domains could pull down the RHBDL2, but the ANK-PEST domain could not (Fig. 4F ). The upregulation of RHBDL2 led to a remarkable increase in the Notch1 and N1ICD levels, while RHBDL2 knockdown had the opposite effect. RHBDL2 enhanced the N1ICD levels in a dose-dependent manner (Figure S2 A-C ). Western blot analysis indicated that the levels of cleaved Notch1 increased in RHBDL2 wild-type (Flag-RHBDL2) PC cells. When a serine to alanine (SA) catalytic mutant of RHBDL2 (Flag-RHBDL2-SA) was constructed, the elevated expression levels of cleaved Notch1 mediated by RHBDL2 was partially rescued (Fig. 4G ). The IP assay results revealed that the N1ICD expression level was upregulated in the Flag-RHBDL2 group, while it was partly abolished in Flag-RHBDL2-SA PC cells in the input and IP groups (Fig. 4H ). Taken together, our findings demonstrate that RHBDL2 interacts with Notch1 and mediates the cleavage of Notch1 and release of N1ICD through its intramembrane serine protease cleavage activity.
section_name: RHBDL2 interacts with Notch1, mediates the cleavage of Notch1 and release of Notch1 intracellular domain

section_content: To determine whether RHBDL2 regulates N1ICD posttranslationally, the PC cells were treated with CHX to inhibit translation. N1ICD was rapidly degraded in the negative control group, while its protein half-life was prolonged in RHBDL2-overexpressing PC cells (Fig. 5A ). The co-incubation of selective lysosomal inhibitors chloroquine and NH 4 Cl, or autophagy inhibitor 3-MA in PC cells could not recover the decreased N1ICD level mediated by RHBDL2 silencing (Fig. 5B-D ). However, the co-incubation of PC cells with the proteasome inhibitor MG132 restored the decreased N1ICD level mediated by the downregulation of RHBDL2 (Fig. 5E ). Moreover, results of the ubiquitination assay showed that RHBDL2 overexpression dramatically decreased the ubiquitination level of N1ICD. By contrast, the knockdown of RHBDL2 led to a significant increase in the N1ICD ubiquitination level (Fig. 5F ). Collectively, these findings suggest that RHBDL2 inhibits the degradation of N1ICD through the ubiquitin-proteasome mechanism.
section_name: RHBDL2 inhibits the degradation of N1ICD through the ubiquitin-proteasome pathway

section_content: The RHBDL2 attenuates the ubiquitination level of N1ICD and stabilizes it via the ubiquitin-proteasome pathway, but the RHBDL2 has no DUB function. Thus, the present study identified the DUBs that could modulate N1ICD stability and interact with RHBDL2. According to a previous study [33], seven DUBs (ATXN3, BAP1, EIF3F, OTUD7B, UCHL5, USP7, and USP10) can regulate the stability of N1ICD. Furthermore, the expression levels of these seven DUBs were confirmed in the PAAD datasets from TCGA database (Fig. S3A-G ). ATXN3, OTUD7B, and USP10 were highly expressed in the PC tissues (Fig. S3A, D, G ). The N1ICD level was dramatically downregulated by OTUD7B silencing than by the knockdown of ATXN3 or USP10. Therefore, OTUD7B was selected for further analysis (Fig. 6A, B ). IP assay results revealed that OTUD7B interacted with N1ICD (Fig. 6C, D ) and RHBDL2 (Fig. 6E-F ). The HA-OTUD7B expression levels remained the same in the Flag-RHBDL2-SA and Flag-RHBDL2 cells in the input group, but the level of immunoprecipitated HA-OTUD7B significantly decreased in the Flag-RHBDL2-SA cells compared with that in the Flag-RHBDL2 cells (Fig. 6G ). The N1ICD level was profoundly upregulated in the OTUD7B wild-type (Flag-OTUD7B) group, but it was restored by the catalytically inactive mutant C194S of OTUD7B (Flag-OTUD7B-C194S) (Fig. 6H ). Concordantly, the ubiquitination level of N1ICD was dramatically attenuated in the Flag-OTUD7B group, whereas the N1ICD ubiquitination level was restored in the Flag-OTUD7B-C194S group (Fig. 6I ). Moreover, knockdown of OTUD7B in RHBDL2-overexpressing cells reversed the increase in N1ICD level and the reduction of N1ICD ubiquitination mediated by RHBDL2 (Fig. 6J, K ). In addition, the upregulation of RHBDL2 in OTUD7Boverexpressing cells led to the profound increase in the N1ICD level, which was the highest among the other groups; however, the N1ICD level in the OTUD7B-overexpressing cells was restored by the Flag-RHBDL2-SA (Fig. 6L ). Together, these findings suggest that RHBDL2 collaborates with OTUD7B to stabilize the N1ICD level through physical interactions. 

Association of the intramembrane serine protease cleavage activity of RHBDL2 and DUB function of OTUD7B with the oncogenic role of RHBDL2 in PC cells Next, we determined whether the intramembrane serine protease cleavage activity of RHBDL2 and the DUB function of OTUD7B were responsible for the RHBDL2-mediated Notch signaling pathway activation and facilitation of proliferation and mobility in PC cells. Mutation of the protease cleavage site of RHBDL2 or knockdown of OTUD7B in RHBDL2-overexpressing PC cells rescued their enhanced proliferation (Fig. 7A-C ), migration, and invasion (Fig. 7D, E ) capacity mediated by the RHBDL2. In addition, both the RHBDL2-SA and RHBDL2 plus OTUD7B-silencing groups significantly reversed the increased expression levels of the Notch signaling pathway downstream target genes (HES1, HEY1, ZEB1, MMP9, SNAIL1, and TWIST1) and cleaved Notch1 (Fig. 7F, G ), as well as the enhanced luciferase reporter activity (Fig. 7H ) mediated by the RHBDL2. These results suggest that the oncogenic role of RHBDL2 in PC cells is dependent on its intramembrane serine protease cleavage activity and the DUB function of OTUD7B.
section_name: RHBDL2 stabilizes the N1ICD through OTUD7B

section_content: The activity of the Notch signaling pathway is often altered in various human malignant tumors [34] [35] [36] including PC [37]. The Notch signaling pathway can accelerate the tumorigenesis of PC cells [17]. In the present study, the potential function and mechanism of RHBDL2 in the progression of PC were investigated, and results showed that it relied on the stabilization of N1ICD by RHBDL2 via the OTUD7B, which then activated the Notch signaling pathway. 

First, to assess the significance of RHBDL2 in the development of PC, we investigated the expression levels of RHBDL2 and discovered that RHBDL2 was overexpressed in PC cell lines and tissue samples. The Kaplan-Meier survival curve revealed that RHBDL2 upregulation was closely correlated with the shorter survival time of patients with PC, indicating the significance of RHBDL2 in affecting the prognosis of PC patients. Subsequently, the overexpression of RHBDL2 was verified to promote PC cell proliferation and motility in vitro and in vivo using functional assays. Results of the RNA-seq analysis revealed that RHBDL2 expression was significantly correlated with the Notch signaling pathway. The expression levels of downstream target genes and transcriptional activity of the Notch signaling pathway increased upon the overexpression of RHBDL2. Furthermore, the role of RHBDL2 in the development of PC and the activation of the Notch signaling pathway mediated by RHBDL2 can be partially reversed by the Notch signaling pathway inhibitor IMR-1. These findings suggest that RHBDL2 may act as an oncogene by activating the Notch signaling pathway. 

The Notch receptor binds to its ligand and subsequently undergoes a series of cleavage events induced by proteases, resulting in the production and release of the Notch intracellular domain (NICD). The translocation of NICD into the nucleus induces the activation of Notch signaling [15]. NUMB binds to N1ICD's PEST domain and then stabilizes the N1ICD [33]. In our study, we demonstrated that RHBDL2 interacts with Notch1 and that N1ICD binds to RHBDL2 through its RAM domain. Furthermore, the overexpression of RHBDL2 enhanced the expression level of Notch1 in the nuclei of PC cells and upregulated the protein expression levels of cleaved Notch1 and N1ICD. The increase in cleaved Notch1 and N1ICD levels depends on the intramembrane serine protease cleavage activity of RHBDL2. In addition, RHBDL2 suppresses the degradation of N1ICD through the ubiquitin-proteasome pathway, thereby stabilizing the N1ICD. 

As a DUB, OTUD7B removes the ubiquitin chains from proteins, thereby contributing to their stabilization [38]. OTUD7B can not only hydrolyze K11 ubiquitin chains but also decrease the K63linked GβL ubiquitination level, thus facilitating the interaction between GβL and SIN1 and promoting the formation of mTORC2 to respond to a variety of growth signals [39]. Tang et al. found that OTUD7B expression is positively correlated with the ERα levels in breast cancer and can function as an independent factor for predicting the patient's prognosis. Importantly, OTUD7B acts through a deubiquitination-dependent mechanism to stabilize the ERα [31]. OTUD7B is overexpressed in human metastatic or highgrade breast cancer; the dysregulation of OTUD7B correlates with worse survival and cancer metastasis. The OTUD7B is responsible for the deubiquitination of LSD1 at the K226/277 residues, and OTUD7B depletion increases the LSD1 K63-linked ubiquitination and p62-mediated proteolysis [40]. Zhang et al. reported that OTUD7B can bind to and deubiquitylate TRAF3, which results in the suppression of TRAF3 proteolysis and accumulation of NIK, thereby inactivating the non-canonical NF-κB signal and repressing lung cancer cell mobility induced by LCL161 [30]. Although accumulating studies have revealed that OTUD7B plays a pivotal role in tumor progression, the underlying molecular mechanism of OTUD7B in the development of PC remains unclear. In our current study, the N1ICD level is positively correlated with OTUD7B. OTUD7B interacts with N1ICD and RHBDL2 and stabilizes N1ICD through its DUB activity. The RHBDL2 collaborates with OTUD7B to block the degradation of N1ICD through their physical associations. Finally, rescue assays verified that the role of RHBDL2 in the development of PC is dependent on its intramembrane serine protease cleavage activity and the DUB function of OTUD7B. 

In summary, our study illustrates that RHBDL2 acts as an oncogene in PC, stabilizes N1ICD through the OTUD7B, and activates the Notch signaling pathway, thereby accelerating PC cell proliferation and mobility. Thus, RHBDL2 may be a novel therapeutic target for patients with PC.
section_name: DISCUSSION

section_name: ACKNOWLEDGEMENTS

section_content: We thank the Department of Hepatobiliary Surgery, The Affiliated Hospital of Guizhou Medical University for providing the pancreatic cancer tissue samples and related anonymous clinical data.
section_name: ACKNOWLEDGEMENTS

section_name: FUNDING

section_content: This work was supported by the National Natural Science Foundation of China [grant number 81860506 ], Project of Science and Technology of Guizhou Province [Grant number Qian Ke He Zhi Cheng [2021] normal 080], and Guizhou Province High-level Innovative Talents Training Plan "Ten" level talents [grant number Qian Ke He Ping Tai Ren Cai [ 2016] 5647 ].
section_name: FUNDING

section_name: DATA AVAILABILITY

section_content: The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.
section_name: DATA AVAILABILITY

section_name: AUTHOR CONTRIBUTIONS

section_content: CY and CYS designed this project. SYC, KC, and DJZ performed the experiments. Data analyses were performed by YQL and LL. ZWH assisted in harvesting the tissue samples and providing the clinical data. All authors participated in the preparation of the manuscript and approved the final submitted and published versions.
section_name: AUTHOR CONTRIBUTIONS

section_content: The authors declare no competing interests.
section_name: COMPETING INTERESTS

section_content: The use of surgical specimens of PC and adjacent non-tumor tissues in this study was approved by the ethics committee of the Affiliated Hospital of Guizhou Medical University (2018 Lunshen No. 018). All animal studies were approved by the Animal Care Welfare Committee of Guizhou Medical University (No. 1801105).
section_name: ETHICS APPROVAL AND CONSENT TO PARTICIPATE

section_content: All authors have agreed to publish this manuscript.
section_name: CONSENT FOR PUBLICATION

section_content: The online version contains supplementary material available at https://doi. org/10. 1038/s41419-022-05379-3. 

Correspondence and requests for materials should be addressed to Chao Yu. 

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section_name: ADDITIONAL INFORMATION Supplementary information